http://2014.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=100&target=Slowe&year=&month=2014.igem.org - User contributions [en]2024-03-28T14:23:23ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/File:Melbourne_iGEM_Information.pdfFile:Melbourne iGEM Information.pdf2015-08-11T12:21:31Z<p>Slowe: </p>
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<div></div>Slowehttp://2014.igem.org/File:Melbourne_iGEM_-_Information_for_Prospective_Applicants.pdfFile:Melbourne iGEM - Information for Prospective Applicants.pdf2015-08-11T12:08:13Z<p>Slowe: uploaded a new version of &quot;File:Melbourne iGEM - Information for Prospective Applicants.pdf&quot;</p>
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<div></div>Slowehttp://2014.igem.org/File:Melbourne_iGEM_-_Information_for_Prospective_Applicants.pdfFile:Melbourne iGEM - Information for Prospective Applicants.pdf2015-08-10T14:03:52Z<p>Slowe: </p>
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<div></div>Slowehttp://2014.igem.org/Team:Melbourne/RecruitmentTeam:Melbourne/Recruitment2015-05-27T18:50:17Z<p>Slowe: </p>
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width="46"></a> </td><br />
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<big><br><br />
Thank<br />
you for your interest in the<br />
2015-16 Melbourne University iGEM Team! </big><br><br />
<br><br />
<big>Please<br />
read the information below to find out what iGEM is all about. We are<br />
currently looking for members of the leadership team, and after the<br />
completion of Semester 1 exams, there will be a general recruitment<br />
round. Please <a href="#subscribe">sign<br />
up to the Melbourne iGEM mailing list</a>, below,<br />
to be alerted to the latest developments.</big><br><br />
<br><br />
<p><big><big><big><big><span<br />
style="font-weight: bold;">What is iGEM?</span></big></big></big></big></p><br />
<div style="text-align: center;"><big><big><big><big><span<br />
style="font-weight: bold;"><img<br />
style="width: 274px; height: 209px;" alt="IGEM logo"<br />
src="https://static.igem.org/mediawiki/2014/d/d8/Team_Berlin_igem_logo.png"></span></big></big></big></big><o:p></o:p></div><br />
<big><span class="GramE"><span class="usercontent"><br><br />
iGEM</span></span><span class="usercontent"><br />
is a unique opportunity to get involved<br />
in an enterprising<br />
student research group and make a scientific impact.</span><span<br />
class="GramE"><span class="textexposedshow"></span></span></big><br />
<br><br />
<br><br />
<big><span class="GramE"><span<br />
class="textexposedshow">iGEM</span></span><span<br />
class="textexposedshow"> is short for the International<br />
Genetically Engineering<br />
Machine competition. <span class="GramE">iGEM</span><br />
is an undergraduate science<br />
competition held each September in Boston. In the period leading up to<br />
iGEM,<br />
university teams use the latest tools from synthetic<br />
biology to<br />
create a novel single celled organism or “biological machine”. They can<br />
also develop synthetic biology computing and lab tools, engage in<br />
scientific outreach, and even develop biotech businesses.<br><br />
</span><span data-ft="{&quot;tn&quot;:&quot;K&quot;}" class="userContent"><span<br />
class="text_exposed_show"></span></span></big><br><br />
<big><span data-ft="{&quot;tn&quot;:&quot;K&quot;}" class="userContent"><span<br />
class="text_exposed_show">Each year, students from around<br />
the globe form<br />
teams at their respective universities with the goal of building a<br />
biological system or tool, which they later present at the iGEM global<br />
conference. Students in the past have designed bacteria that<br />
produce new types of drugs and biofuels, act as biosensors of toxic<br />
pollutants, and&nbsp;serve as biological computation<br />
platforms&nbsp; (for more examples of past<br />
projects, see <a href="https://igem.org/About"<br />
rel="nofollow nofollow" target="_blank"<br />
onmouseover='LinkshimAsyncLink.swap(this, "http:\/\/igem.org\/About");'<br />
onclick='LinkshimAsyncLink.swap(this, "http:\/\/l.facebook.com\/l.php?u=http\u00253A\u00252F\u00252Figem.org\u00252FAbout&h=zAQFmUzcK&enc=AZNGkoGC-HF5q40u8IHnH2vOwvgHdyy0ClVop-mb6cjZGHojVOVS9IRhqf-Bi3W3tjr6Zt8No6t5_Rcx8yzlpP7oLfAxZyO28QciO52K7yqag82-VE7nwRVRmBKK-HgbxqVsq9pAa5Lv-OJPoe-BAhfl&s=1");'>https://igem.org/About</a>)</span></span>.<span<br />
class="textexposedshow"> </span><o:p></o:p></big><br />
<br><br />
<br><br />
<big><span class="GramE"><span<br />
class="textexposedshow">iGEM</span></span><span<br />
class="textexposedshow"> teams manage everything from the<br />
conception to the<br />
execution of the iGEM project, with the aid of faculty supervisors. </span><br><br />
<br><br />
<span class="textexposedshow">iGEM allows students to:</span><br><br />
</big><br />
<ul><br />
<li><big><span class="textexposedshow">Get<br />
valuable scientific and<br />
leadership experience</span></big></li><br />
<li><big><span class="textexposedshow">Develop<br />
a useful, novel<br />
biotechnology and gain experience<br />
in the latest in interdisciplinary biological research<o:p></o:p></span></big><br />
</li><br />
<li><big><span class="textexposedshow">Make<br />
an impact</span></big></li><br />
<li><big><span class="textexposedshow">Have<br />
fun!</span></big></li><br />
</ul><br />
<big><span class="textexposedshow"><br><br />
</span></big><br><br />
<p><big><big><big><big><span<br />
style="font-weight: bold;">Project</span></big></big></big></big></p><br />
<big><big><big><big><span<br />
style="font-weight: bold;"></span></big></big></big>Students<br />
in the iGEM<br />
teams have complete control over the teams’<br />
project and scope. There are many possible projects and types of teams.<br />
There are 15 tracks in the iGEM competition. They include 8 standard<br />
tracks and several special tracks<br />
(e.g. entrepreneurship, software, social policy, etc.). Two examples of<br />
possible iGEM teams would include:<br><br />
</big><br />
<br><br />
<big>1. Standard tracks: In the standard tracks, teams<br />
develop iGEM<br />
projects for research purposes and to advance science on a foundational<br />
or applied level. Last year, the Melbourne iGEM<br />
team competed in the Manufacturing track, working on a project to<br />
develop bacteria capable of producing new types of antibiotics.<br />
However, there is enormous variety in the types of problems teams can<br />
work on (see <a<br />
href="https://igem.org/Results?year=2013&amp;region=Championship&amp;division=igem">past<br />
team Wiki's</a> for examples).</big><br />
<big><br><br />
<br><br />
2. Special tracks: the iGEM competition has expanded<br />
significantly since its inception, with the addition of several new<br />
tracks to capture the enormous growth of synthetic biology.<br />
For example, teams can now compete in an entrepreneurship track where<br />
they produce not only a scientific advancement, but also a business<br />
plan to<br />
commercialise the work. The&nbsp;track is aimed at giving students<br />
a conducive and educational environment to start a synthetic biology<br />
company, and is inspired by the recent boom in the synthetic biology<br />
startups<br />
(e.g. see <a href="http://eu.indie.bio/">Indie Bio</a>).<br><br />
<br><br />
</big><br />
<p><big><big><big><big><span<br />
style="font-weight: bold;">Timeline</span></big></big></big></big></p><br />
<big><br><br />
</big><big>The next<br />
iGEM team will run from Semester 2, 2015 to Semester 2, 2016. In<br />
Semester 2, 2015, the team<br />
will be organised, the project selected, and the lab set up. During<br />
this period, team members will need to work to&nbsp;ensure that the<br />
foundations are in place to start significant lab/project work during<br />
the summer. A large portion of the practical work will then take<br />
place during the summer of 2015-16. Therefore, to participate in the<br />
team you will need to be available during the upcoming summer. Finally,<br />
additional work will be carried out as needed during the lead up to the<br />
2016 iGEM conference (the iGEM Giant Jamboree) in September of next<br />
year.</big><br><br />
&nbsp;<br><br />
<p class="MsoNormal"><big><big><big><big><span<br />
style="font-weight: bold;">What is involved and what is<br />
needed?</span></big></big></big></big></p><br />
<big><big><big><big><span<br />
style="font-weight: bold;"><br><br />
</span></big></big></big></big><br />
<p style="text-align: center;" class="MsoNormal"><big><img<br />
style="width: 264px; height: 197px;" alt="DNA image"<br />
src="https://static.igem.org/mediawiki/2014/9/91/DNA_v1_by_bak16.jpg"></big></p><br />
<big>We are searching for<br />
enthusiastic<br />
students with<br />
an interest in biotechnology and science.<o:p>&nbsp;</o:p></big><br />
<br><br />
<br><br />
<big>As<br />
part of the iGEM team, you would help with the<br />
following tasks:</big><br />
<ul><br />
<li><big>Research on the iGEM project ideas. The ideas<br />
for the iGEM project are student driven, and team members often need to<br />
answer specific research question to design new experiments or come up<br />
with new ideas. This will typically involve doing&nbsp; searches of<br />
the literature using Google Scholar or other tools and reading<br />
scientific articles.</big></li><br />
<li><big>Help with developing experimental methods. The<br />
2014 team has built up a library of protocols and experimental methods.<br />
However, the project for 2015 will likely require new methods. You will<br />
need to look up protocols in the literature and adapt them to the<br />
project requirements.</big></li><br />
<li><big>Wet lab work, modelling/computation or<br />
engineering design. By joining the iGEM team, you will have an<br />
opportunity to participate in the lab and learn many standard<br />
techniques in molecular biology. Alternatively, many iGEM teams make<br />
use of the skills of engineers, computer scientists, and other<br />
non-biological science disciplines.&nbsp; For example, this may<br />
take the form of modeling a biological system using software like<br />
Matlab, designing a microfluidic device with biological applications or<br />
designing an electrical device which interfaces with a biological<br />
system. If you have an interest in interdisciplinary research between<br />
your field and biology, it is likely iGEM will be able to accommodate<br />
it.</big></li><br />
<li><big>The scope of iGEM also extends into<br />
non-traditional science areas, including biotechnology<br />
entrepreneurship, biotechnology ethics and the law, and science<br />
outreach, and so students with business, law, design, and arts<br />
backgrounds are also welcome to participate. You could, for example,<br />
create a business plan for a iGEM-created company, examine bioethics<br />
within synthetic biology, or design educational/outreach program for<br />
high schoolers.</big></li><br />
</ul><br />
<ul><br />
<br><br />
</ul><br />
<p class="MsoNormal"><big><o:p></o:p></big><big><big><big><big><span<br />
style="font-weight: bold;">Recruitment</span></big></big></big></big></p><br />
<big>We are currently seeking<br />
members of<br />
the executive (see below), witha&nbsp; general<br />
recruitment round after<br />
the exam period. Recruitment will be open to all undergraduate and<br />
masters students who are available<br />
during Semester 2 2015 and the summer of 2015-16. Please sign up below<br />
to be notified when the general recruitment opens (also follow<br />
us on <a href="https://www.facebook.com/MelbourneUniIGem">Facebook</a>).<br />
If you have specific questions about iGEM, please direct them to<br />
Sean at melbourneuniigem@gmail.com.&nbsp;</big><br />
<p class="MsoNormal"><!-- Begin MailChimp Signup Form --></p><br />
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<h2><a name="subscribe"></a>Sign up for<br />
Melbourne iGEM recruitment updates</h2><br />
<div class="indicates-required"><span class="asterisk">*</span><br />
indicates required</div><br />
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<big>If you are particularly<br />
keen on iGEM, and would like to play a leadership role, we currently<br />
require additional members of the executive/leadership team. As part<br />
of the team's leadership, you<br />
will be in a unique position to mold the team. Duties include:</big><br />
<ol><br />
<li><big>Helping to set up the team and determining the<br />
teams' goals and iGEM track.</big></li><br />
<li><big>Serving as a spokesperson&nbsp;to<br />
external parties and academics. This will include recruiting additional<br />
academic and graduate student supervisors.<br><br />
</big></li><br />
<li><big>Helping to fundraise for the team, liaising with<br />
university and external sponsors.</big></li><br />
<li><big>Helping to recruit new members</big></li><br />
</ol><br />
<big><br><br />
Being a founding member of the next iGEM team provides a<br />
great opportunity to play a driving role in Melbourne Uni's most<br />
exciting student based group. The key requirements for the role is a<br />
strong commitment to the team, as well as good communication and<br />
leadership skills. If you are interested in<br />
playing this role, please email<br />
Sean&nbsp;at MelbourneUniIGem@gmail.com to express your interest. <o:p></o:p></big><br><br />
<br><br />
<p class="MsoNormal"><big><o:p></o:p></big><big><big><big><big><span<br />
style="font-weight: bold;">Further information</span></big></big></big></big></p><br />
<big>Learn more about iGEM in<br />
general at: <a<br />
href="http://www.facebook.com/l.php?u=http%3A%2F%2Figem.org%2FAbout&amp;h=HAQEjYmHL&amp;s=1"<br />
target="_blank">https://igem.org/About</a><br<br />
style=""><br />
</big><br><br />
<big>Learn about last year’s<br />
team by<br />
clicking on the links above.<o:p></o:p></big><br />
<br><br />
<big><br><br />
<span class="textexposedshow">Also browse the previous<br />
Melbourne <span class="SpellE">Uni</span> iGEM team<br />
at<span class="GramE">:</span></span><br><br />
<a href="http://openwetware.org/wiki/IGEM:Melbourne/2008"<br />
target="_blank">http://openwetware.org/wiki/IGEM:Melbourne/2008</a><br><br />
<a<br />
href="http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-melbourne-university-te"<br />
target="_blank">http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-<span<br />
class="SpellE">melbourne</span>-university-<span<br />
class="SpellE">te</span></a></big><br><br />
<br><br />
<br><br />
<big>Also, follow us on Facebook:</big><br />
<br><br />
<div style="text-align: center;"><a<br />
href="https://www.facebook.com/MelbourneUniIGem"><img<br />
style="border: 0px solid ; width: 102px; height: 102px;"<br />
alt="Facebook logo"<br />
src="https://static.igem.org/mediawiki/2014/8/88/OxigemIconfacebook.png"></a><a<br />
href="http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-melbourne-university-te"<br />
target="_blank"><span class="SpellE"></span></a><span<br />
class="textexposedshow"><o:p></o:p></span><br><br />
</div><br />
<!-- END EDIT HERE HERE --><br />
</body><br />
</html></div>Slowehttp://2014.igem.org/Team:Melbourne/RecruitmentTeam:Melbourne/Recruitment2015-05-26T09:09:09Z<p>Slowe: </p>
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<body><br />
<img src="https://static.igem.org/mediawiki/2014/9/9b/Melbourne_Banner.png"<br />
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style="color: rgb(0, 0, 0);">Notebook</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Protocols"<br />
style="color: rgb(0, 0, 0);"> Protocols</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Safety"<br />
style="color: rgb(0, 0, 0);">Safety</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Sponsors"<br />
style="color: rgb(0, 0, 0);">Sponsors</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Attributions"<br />
style="color: rgb(0, 0, 0);">Attributions</a></td><br />
<td align="right"> <a<br />
href="https://2014.igem.org/Main_Page"> <img<br />
src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png"<br />
width="46"></a> </td><br />
</tr><br />
</tbody><br />
</table><br />
<big><br><br />
Thank<br />
you for your interest in the<br />
2015-16 Melbourne University iGEM Team! </big><br><br />
<br><br />
<big>Please<br />
read the information below to find out what iGEM is all about. We are<br />
currently looking for members of the leadership team, and after the<br />
completion of Semester 1 exams, there will be a general recruitment<br />
round. Please <a href="#subscribe">sign<br />
up to the Melbourne iGEM mailing list</a>, below,<br />
to be alerted to the latest developments.</big><br><br />
<br><br />
<p><big><big><big><big><span<br />
style="font-weight: bold;">What is iGEM?</span></big></big></big></big></p><br />
<div style="text-align: center;"><big><big><big><big><span<br />
style="font-weight: bold;"><img<br />
style="width: 274px; height: 209px;" alt="IGEM logo"<br />
src="https://static.igem.org/mediawiki/2014/d/d8/Team_Berlin_igem_logo.png"></span></big></big></big></big><o:p></o:p></div><br />
<big><span class="GramE"><span class="usercontent"><br><br />
iGEM</span></span><span class="usercontent"><br />
is a unique opportunity to get involved<br />
in an enterprising<br />
student research group and make a scientific impact.</span><span<br />
class="GramE"><span class="textexposedshow"></span></span></big><br />
<br><br />
<br><br />
<big><span class="GramE"><span<br />
class="textexposedshow">iGEM</span></span><span<br />
class="textexposedshow"> is short for the International<br />
Genetically Engineering<br />
Machine competition. <span class="GramE">iGEM</span><br />
is an undergraduate science<br />
competition held each September in Boston. In the period leading up to<br />
iGEM,<br />
university teams use the latest tools from synthetic<br />
biology to<br />
create a novel single celled organism or “biological machine”. They can<br />
also develop synthetic biology computing and lab tools, engage in<br />
scientific outreach, and even develop biotech businesses.<br><br />
</span><span data-ft="{&quot;tn&quot;:&quot;K&quot;}" class="userContent"><span<br />
class="text_exposed_show"></span></span></big><br><br />
<big><span data-ft="{&quot;tn&quot;:&quot;K&quot;}" class="userContent"><span<br />
class="text_exposed_show">Each year, students from around<br />
the globe form<br />
teams at their respective universities with the goal of building a<br />
biological system or tool, which they later present at the iGEM global<br />
conference. Students in the past have designed bacteria that<br />
produce new types of drugs and biofuels, act as biosensors of toxic<br />
pollutants, and&nbsp;serve as biological computation<br />
platforms&nbsp; (for more examples of past<br />
projects, see <a href="https://igem.org/About"<br />
rel="nofollow nofollow" target="_blank"<br />
onmouseover='LinkshimAsyncLink.swap(this, "http:\/\/igem.org\/About");'<br />
onclick='LinkshimAsyncLink.swap(this, "http:\/\/l.facebook.com\/l.php?u=http\u00253A\u00252F\u00252Figem.org\u00252FAbout&h=zAQFmUzcK&enc=AZNGkoGC-HF5q40u8IHnH2vOwvgHdyy0ClVop-mb6cjZGHojVOVS9IRhqf-Bi3W3tjr6Zt8No6t5_Rcx8yzlpP7oLfAxZyO28QciO52K7yqag82-VE7nwRVRmBKK-HgbxqVsq9pAa5Lv-OJPoe-BAhfl&s=1");'>https://igem.org/About</a>)</span></span>.<span<br />
class="textexposedshow"> </span><o:p></o:p></big><br />
<br><br />
<br><br />
<big><span class="GramE"><span<br />
class="textexposedshow">iGEM</span></span><span<br />
class="textexposedshow"> teams manage everything from the<br />
conception to the<br />
execution of the iGEM project, with the aid of faculty supervisors. </span><br><br />
<br><br />
<span class="textexposedshow">iGEM allows students to:</span><br><br />
</big><br />
<ul><br />
<li><big><span class="textexposedshow">Get<br />
valuable scientific and<br />
leadership experience</span></big></li><br />
<li><big><span class="textexposedshow">Develop<br />
a useful, novel<br />
biotechnology and gain experience<br />
in the latest in interdisciplinary biological research<o:p></o:p></span></big><br />
</li><br />
<li><big><span class="textexposedshow">Make<br />
an impact</span></big></li><br />
<li><big><span class="textexposedshow">Have<br />
fun!</span></big></li><br />
</ul><br />
<big><span class="textexposedshow"><br><br />
</span></big><br><br />
<p><big><big><big><big><span<br />
style="font-weight: bold;">Project</span></big></big></big></big></p><br />
<big><big><big><big><span<br />
style="font-weight: bold;"></span></big></big></big>Students<br />
in the iGEM<br />
teams have complete control over the teams’<br />
project and scope. There are many possible projects and types of teams.<br />
There are 15 tracks in the iGEM competition. They include 8 standard<br />
tracks and several special tracks<br />
(e.g. entrepreneurship, software, social policy, etc.). Two examples of<br />
possible iGEM teams would include:<br><br />
</big><br />
<br><br />
<big>1. Standard tracks: In the standard tracks, teams<br />
develop iGEM<br />
projects for research purposes and to advance science on a foundational<br />
or applied level. Last year, the Melbourne iGEM<br />
team competed in the Manufacturing track, working on a project to<br />
develop bacteria capable of producing new types of antibiotics.<br />
However, there is enormous variety in the types of problems teams can<br />
work on (see <a<br />
href="https://igem.org/Results?year=2013&amp;region=Championship&amp;division=igem">past<br />
team Wiki's</a> for examples).</big><br />
<big><br><br />
<br><br />
2. Special tracks: the iGEM competition has expanded<br />
significantly since its inception, with the addition of several new<br />
tracks to capture the enormous growth of synthetic biology.<br />
For example, teams can now compete in an entrepreneurship track where<br />
they produce not only a scientific advancement, but also a business<br />
plan to<br />
commercialise the work. The&nbsp;track is aimed at giving students<br />
a conducive and educational environment to start a synthetic biology<br />
company, and is inspired by the recent boom in the synthetic biology<br />
startups<br />
(e.g. see <a href="http://eu.indie.bio/">Indie Bio</a>).<br><br />
<br><br />
</big><br />
<p><big><big><big><big><span<br />
style="font-weight: bold;">Timeline</span></big></big></big></big></p><br />
<big><br><br />
</big><big>The next<br />
iGEM team will run from Semester 2, 2015 to Semester 2, 2016. In<br />
Semester 2, 2015, the team<br />
will be organised, the project selected, and the lab set up. During<br />
this period, team members will need to work to&nbsp;ensure that the<br />
foundations are in place to start significant lab/project work during<br />
the summer. A large portion of the practical work will then take<br />
place during the summer of 2015-16. Therefore, to participate in the<br />
team you will need to be available during the upcoming summer. Finally,<br />
additional work will be carried out as needed during the lead up to the<br />
2016 iGEM conference (the iGEM Giant Jamboree) in September of next<br />
year.</big><br><br />
&nbsp;<br><br />
<p class="MsoNormal"><big><big><big><big><span<br />
style="font-weight: bold;">What is involved and what is<br />
needed?</span></big></big></big></big></p><br />
<big><big><big><big><span<br />
style="font-weight: bold;"><br><br />
</span></big></big></big></big><br />
<p style="text-align: center;" class="MsoNormal"><big><img<br />
style="width: 264px; height: 197px;" alt="DNA image"<br />
src="https://static.igem.org/mediawiki/2014/9/91/DNA_v1_by_bak16.jpg"></big></p><br />
<big>We are searching for<br />
enthusiastic<br />
students with<br />
an interest in biotechnology and science.<o:p>&nbsp;</o:p></big><br />
<br><br />
<br><br />
<big>As<br />
part of the iGEM team, you would help with the<br />
following tasks:</big><br />
<ul><br />
<li><big>Research on the iGEM project ideas. The ideas<br />
for the iGEM project are student driven, and team members often need to<br />
answer specific research question to design new experiments or come up<br />
with new ideas. This will typically involve doing&nbsp; searches of<br />
the literature using Google Scholar or other tools and reading<br />
scientific articles.</big></li><br />
<li><big>Help with developing experimental methods. The<br />
2014 team has built up a library of protocols and experimental methods.<br />
However, the project for 2015 will likely require new methods. You will<br />
need to look up protocols in the literature and adapt them to the<br />
project requirements.</big></li><br />
<li><big>Wet lab work, modelling/computation or<br />
engineering design. By joining the iGEM team, you will have an<br />
opportunity to participate in the lab and learn many standard<br />
techniques in molecular biology. Alternatively, many iGEM teams make<br />
use of the skills of engineers, computer scientists, and other<br />
non-biological science disciplines.&nbsp; For example, this may<br />
take the form of modeling a biological system using software like<br />
Matlab, designing a microfluidic device with biological applications or<br />
designing an electrical device which interfaces with a biological<br />
system. If you have an interest in interdisciplinary research between<br />
your field and biology, it is likely iGEM will be able to accommodate<br />
it.</big></li><br />
<li><big>The scope of iGEM also extends into<br />
non-traditional science areas, including biotechnology<br />
entrepreneurship, biotechnology ethics and the law, and science<br />
outreach, and so students with business, law, design, and arts<br />
backgrounds are also welcome to participate. You could, for example,<br />
create a business plan for a iGEM-created company, examine bioethics<br />
within synthetic biology, or design educational/outreach program for<br />
high schoolers.</big></li><br />
</ul><br />
<ul><br />
<br><br />
</ul><br />
<p class="MsoNormal"><big><o:p></o:p></big><big><big><big><big><span<br />
style="font-weight: bold;">Recruitment</span></big></big></big></big></p><br />
<big>We are currently seeking<br />
members of<br />
the executive (see below), witha&nbsp; general<br />
recruitment round after<br />
the exam period. Recruitment will be open to all undergraduate and<br />
masters students who are available<br />
during Semester 2 2015 and the summer of 2015-16. Please sign up below<br />
to be notified when the general recruitment opens (also follow<br />
us on <a href="https://www.facebook.com/MelbourneUniIGem">Facebook</a>).<br />
If you have specific questions about iGEM, please direct them to<br />
Sean at melbourneuniigem@gmail.com.&nbsp;</big><br />
<p class="MsoNormal"><!-- Begin MailChimp Signup Form --></p><br />
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<h2><a name="subscribe"></a>Sign up for<br />
Melbourne iGEM recruitment updates</h2><br />
<div class="indicates-required"><span class="asterisk">*</span><br />
indicates required</div><br />
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<big>If you are particularly<br />
keen on iGEM, and would like to play a leadership role, we currently<br />
require additional members of the executive/leadership team. As part<br />
of the team's leadership, you<br />
will be in a unique position to mold the team. Duties include:</big><br />
<ol><br />
<li><big>Helping to set up the team and determining the<br />
teams' goals and iGEM track.</big></li><br />
<li><big>Serving as a spokesperson&nbsp;to<br />
external parties and academics. This will include recruiting additional<br />
academic and graduate student supervisors.<br><br />
</big></li><br />
<li><big>Helping to fundraise for the team, liaising with<br />
university and external sponsors.</big></li><br />
<li><big>Helping to recruit new members</big></li><br />
</ol><br />
<big><br><br />
Being a founding member of the next iGEM team provides a<br />
great opportunity to play a driving role in Melbourne Uni's most<br />
exciting student based group. The key requirements for the role is a<br />
strong commitment to the team, as well as good communication and<br />
leadership skills. If you are interested in<br />
playing this role, please email<br />
Sean&nbsp;at MelbourneUniIGem@gmail.com to express your interest. <o:p></o:p></big><br><br />
<br><br />
<p class="MsoNormal"><big><o:p></o:p></big><big><big><big><big><span<br />
style="font-weight: bold;">Further information</span></big></big></big></big></p><br />
<big>Learn more about iGEM in<br />
general at: <a<br />
href="http://www.facebook.com/l.php?u=http%3A%2F%2Figem.org%2FAbout&amp;h=HAQEjYmHL&amp;s=1"<br />
target="_blank">https://igem.org/About</a><br<br />
style=""><br />
</big><br><br />
<big>Learn about last year’s<br />
team by<br />
clicking on the links above.<o:p></o:p></big><br />
<br><br />
<big><br><br />
<span class="textexposedshow">Also browse the previous<br />
Melbourne <span class="SpellE">Uni</span> iGEM team<br />
at<span class="GramE">:</span></span><br><br />
<a href="http://openwetware.org/wiki/IGEM:Melbourne/2008"<br />
target="_blank">http://openwetware.org/wiki/IGEM:Melbourne/2008</a><br><br />
<a<br />
href="http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-melbourne-university-te"<br />
target="_blank">http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-<span<br />
class="SpellE">melbourne</span>-university-<span<br />
class="SpellE">te</span></a></big><br><br />
<br><br />
<br><br />
<big>Follow us on Facebook:</big><br />
<br><br />
<div style="text-align: center;"><a<br />
href="https://www.facebook.com/MelbourneUniIGem"><img<br />
style="border: 0px solid ; width: 102px; height: 102px;"<br />
alt="Facebook logo"<br />
src="https://static.igem.org/mediawiki/2014/8/88/OxigemIconfacebook.png"></a><a<br />
href="http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-melbourne-university-te"<br />
target="_blank"><span class="SpellE"></span></a><span<br />
class="textexposedshow"><o:p></o:p></span><br><br />
</div><br />
<!-- END EDIT HERE HERE --><br />
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</html></div>Slowehttp://2014.igem.org/Team:Melbourne/RecruitmentTeam:Melbourne/Recruitment2015-05-21T17:49:33Z<p>Slowe: </p>
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<big><br><br />
Thank<br />
you for your interest in the<br />
2015-16 Melbourne University iGEM Team! </big><br><br />
<br><br />
<big>Please<br />
read the information below to find out what iGEM is all about. We are<br />
currently looking for members of the leadership team, and after the<br />
completion of Semester 1 exams, there will be a general recruitment<br />
round. Please <a href="#subscribe">sign<br />
up to the Melbourne iGEM mailing list</a>, below,<br />
to be alerted to this recruitment.</big><br><br />
<br><br />
<p><big><big><big><big><span<br />
style="font-weight: bold;">What is iGEM?</span></big></big></big></big></p><br />
<div style="text-align: center;"><big><big><big><big><span<br />
style="font-weight: bold;"><img<br />
style="width: 274px; height: 209px;" alt="IGEM logo"<br />
src="https://static.igem.org/mediawiki/2014/d/d8/Team_Berlin_igem_logo.png"></span></big></big></big></big><o:p></o:p></div><br />
<big><span class="GramE"><span class="usercontent"><br><br />
iGEM</span></span><span class="usercontent"><br />
is a unique opportunity to get involved<br />
in an enterprising<br />
student research group and make a scientific impact.</span><span<br />
class="GramE"><span class="textexposedshow"></span></span></big><br />
<br><br />
<br><br />
<big><span class="GramE"><span<br />
class="textexposedshow">iGEM</span></span><span<br />
class="textexposedshow"> is short for the International<br />
Genetically Engineering<br />
Machine competition. <span class="GramE">iGEM</span><br />
is an undergraduate science<br />
competition held each September in Boston. In the period leading up to<br />
iGEM,<br />
university teams use the latest tools from synthetic<br />
biology to<br />
create a novel single celled organism or “biological machine”. They can<br />
also develop synthetic biology computing and lab tools, engage in<br />
scientific outreach, and even develop biotech businesses.<br><br />
</span><span data-ft="{&quot;tn&quot;:&quot;K&quot;}" class="userContent"><span<br />
class="text_exposed_show"></span></span></big><br><br />
<big><span data-ft="{&quot;tn&quot;:&quot;K&quot;}" class="userContent"><span<br />
class="text_exposed_show">Each year, students from around<br />
the globe form<br />
teams at their respective universities with the goal of building a<br />
biological system or tool, which they later present at the iGEM global<br />
conference. Students in the past have designed bacteria that<br />
produce new types of drugs and biofuels, act as biosensors of toxic<br />
pollutants, and&nbsp;serve as biological computation<br />
platforms&nbsp; (for more examples of past<br />
projects, see <a href="https://igem.org/About"<br />
rel="nofollow nofollow" target="_blank"<br />
onmouseover='LinkshimAsyncLink.swap(this, "http:\/\/igem.org\/About");'<br />
onclick='LinkshimAsyncLink.swap(this, "http:\/\/l.facebook.com\/l.php?u=http\u00253A\u00252F\u00252Figem.org\u00252FAbout&h=zAQFmUzcK&enc=AZNGkoGC-HF5q40u8IHnH2vOwvgHdyy0ClVop-mb6cjZGHojVOVS9IRhqf-Bi3W3tjr6Zt8No6t5_Rcx8yzlpP7oLfAxZyO28QciO52K7yqag82-VE7nwRVRmBKK-HgbxqVsq9pAa5Lv-OJPoe-BAhfl&s=1");'>https://igem.org/About</a>)</span></span>.<span<br />
class="textexposedshow"> </span><o:p></o:p></big><br />
<br><br />
<br><br />
<big><span class="GramE"><span<br />
class="textexposedshow">iGEM</span></span><span<br />
class="textexposedshow"> teams manage everything from the<br />
conception to the<br />
execution of the iGEM project, with the aid of faculty supervisors. </span><br><br />
<br><br />
<span class="textexposedshow">iGEM allows students to:</span><br><br />
</big><br />
<ul><br />
<li><big><span class="textexposedshow">Get<br />
valuable scientific and<br />
leadership experience</span></big></li><br />
<li><big><span class="textexposedshow">Develop<br />
a useful, novel<br />
biotechnology and gain experience<br />
in the latest in interdisciplinary biological research<o:p></o:p></span></big><br />
</li><br />
<li><big><span class="textexposedshow">Make<br />
an impact</span></big></li><br />
<li><big><span class="textexposedshow">Have<br />
fun!</span></big></li><br />
</ul><br />
<big><span class="textexposedshow"><br><br />
</span></big><br><br />
<p><big><big><big><big><span<br />
style="font-weight: bold;">Project</span></big></big></big></big></p><br />
<big><big><big><big><span<br />
style="font-weight: bold;"></span></big></big></big>Students<br />
in the iGEM<br />
teams have complete control over the teams’<br />
project and scope. There are many possible projects and types of teams.<br />
There are 15 tracks in the iGEM competition. They include 8 standard<br />
tracks and several special tracks<br />
(e.g. entrepreneurship, software, social policy, etc.). Two examples of<br />
possible iGEM teams would include:<br><br />
</big><br />
<br><br />
<big>1. Standard tracks: In the standard tracks, teams<br />
develop iGEM<br />
projects for research purposes and to advance science on a foundational<br />
or applied level. Last year, the Melbourne iGEM<br />
team competed in the Manufacturing track, working on a project to<br />
develop bacteria capable of producing new types of antibiotics.<br />
However, there is enormous variety in the types of problems teams can<br />
work on (see <a<br />
href="https://igem.org/Results?year=2013&amp;region=Championship&amp;division=igem">past<br />
team Wiki's</a> for examples).</big><br />
<big><br><br />
<br><br />
2. Special tracks: the iGEM competition has expanded<br />
significantly since its inception, with the addition of several new<br />
tracks to capture the enormous growth of synthetic biology.<br />
For example, teams can now compete in an entrepreneurship track where<br />
they produce not only a scientific advancement, but also a business<br />
plan to<br />
commercialise the work. The&nbsp;track is aimed at giving students<br />
a conducive and educational environment to start a synthetic biology<br />
company, and is inspired by the recent boom in the synthetic biology<br />
startups<br />
(e.g. see <a href="http://eu.indie.bio/">Indie Bio</a>).<br><br />
<br><br />
</big><br />
<p><big><big><big><big><span<br />
style="font-weight: bold;">Timeline</span></big></big></big></big></p><br />
<big><br><br />
</big><big>The next<br />
iGEM team will run from Semester 2, 2015 to Semester 2, 2016. In<br />
Semester 2, 2015, the team<br />
will be organised, the project selected, and the lab set up. During<br />
this period, team members will need to work to&nbsp;ensure that the<br />
foundations are in place to start significant lab/project work during<br />
the summer. A large portion of the practical work will then take<br />
place during the summer of 2015-16. Therefore, to participate in the<br />
team you will need to be available during the upcoming summer. Finally,<br />
additional work will be carried out as needed during the lead up to the<br />
2016 iGEM conference (the iGEM Giant Jamboree) in September of next<br />
year.</big><br><br />
&nbsp;<br><br />
<p class="MsoNormal"><big><big><big><big><span<br />
style="font-weight: bold;">What is involved and what is<br />
needed?</span></big></big></big></big></p><br />
<big><big><big><big><span<br />
style="font-weight: bold;"><br><br />
</span></big></big></big></big><br />
<p style="text-align: center;" class="MsoNormal"><big><img<br />
style="width: 264px; height: 197px;" alt="DNA image"<br />
src="https://static.igem.org/mediawiki/2014/9/91/DNA_v1_by_bak16.jpg"></big></p><br />
<big>We are searching for<br />
enthusiastic<br />
students with<br />
an interest in biotechnology and science.<o:p>&nbsp;</o:p></big><br />
<br><br />
<br><br />
<big>As<br />
part of the iGEM team, you would help with the<br />
following tasks:</big><br />
<ul><br />
<li><big>Research on the iGEM project ideas. The ideas<br />
for the iGEM project are student driven, and team members often need to<br />
answer specific research question to design new experiments or come up<br />
with new ideas. This will typically involve doing&nbsp; searches of<br />
the literature using Google Scholar or other tools and reading<br />
scientific articles.</big></li><br />
<li><big>Help with developing experimental methods. The<br />
2014 team has built up a library of protocols and experimental methods.<br />
However, the project for 2015 will likely require new methods. You will<br />
need to look up protocols in the literature and adapt them to the<br />
project requirements.</big></li><br />
<li><big>Wet lab work, modelling/computation or<br />
engineering design. By joining the iGEM team, you will have an<br />
opportunity to participate in the lab and learn many standard<br />
techniques in molecular biology. Alternatively, many iGEM teams make<br />
use of the skills of engineers, computer scientists, and other<br />
non-biological science disciplines.&nbsp; For example, this may<br />
take the form of modeling a biological system using software like<br />
Matlab, designing a microfluidic device with biological applications or<br />
designing an electrical device which interfaces with a biological<br />
system. If you have an interest in interdisciplinary research between<br />
your field and biology, it is likely iGEM will be able to accommodate<br />
it.</big></li><br />
<li><big>The scope of iGEM also extends into<br />
non-traditional science areas, including biotechnology<br />
entrepreneurship, biotechnology ethics and the law, and science<br />
outreach, and so students with business, law, design, and arts<br />
backgrounds are also welcome to participate. You could, for example,<br />
create a business plan for a iGEM-created company, examine bioethics<br />
within synthetic biology, or design educational/outreach program for<br />
high schoolers.</big></li><br />
</ul><br />
<ul><br />
<br><br />
</ul><br />
<p class="MsoNormal"><big><o:p></o:p></big><big><big><big><big><span<br />
style="font-weight: bold;">Recruitment</span></big></big></big></big></p><br />
<big>We are currently seeking<br />
members of<br />
the executive (see below), witha&nbsp; general<br />
recruitment round after<br />
the exam period. Recruitment will be open to all undergraduate and<br />
masters students who are available<br />
during Semester 2 2015 and the summer of 2015-16. Please sign up below<br />
to be notified when the general recruitment opens (also follow<br />
us on <a href="https://www.facebook.com/MelbourneUniIGem">Facebook</a>).<br />
If you have specific questions about iGEM, please direct them to<br />
Sean at melbourneuniigem@gmail.com.&nbsp;</big><br />
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<big>If you are particularly<br />
keen on iGEM, and would like to play a leadership role, we currently<br />
require additional members of the executive/leadership team. As part<br />
of the team's leadership, you<br />
will be in a unique position to mold the team. Duties include:</big><br />
<ol><br />
<li><big>Helping to set up the team and determining the<br />
teams' goals and iGEM track.</big></li><br />
<li><big>Serving as a spokesperson&nbsp;to<br />
external parties and academics. This will include recruiting additional<br />
academic and graduate student supervisors.<br><br />
</big></li><br />
<li><big>Helping to fundraise for the team, liaising with<br />
university and external sponsors.</big></li><br />
<li><big>Helping to recruit new members</big></li><br />
</ol><br />
<big><br><br />
Being a founding member of the next iGEM team provides a<br />
great opportunity to play a driving role in Melbourne Uni's most<br />
exciting student based group. The key requirements for the role is a<br />
strong commitment to the team, as well as good communication and<br />
leadership skills. If you are interested in<br />
playing this role, please email<br />
Sean&nbsp;at MelbourneUniIGem@gmail.com to express your interest. <o:p></o:p></big><br><br />
<br><br />
<p class="MsoNormal"><big><o:p></o:p></big><big><big><big><big><span<br />
style="font-weight: bold;">Further information</span></big></big></big></big></p><br />
<big>Learn more about iGEM in<br />
general at: <a<br />
href="http://www.facebook.com/l.php?u=http%3A%2F%2Figem.org%2FAbout&amp;h=HAQEjYmHL&amp;s=1"<br />
target="_blank">https://igem.org/About</a><br<br />
style=""><br />
</big><br><br />
<big>Learn about last year’s<br />
team by<br />
clicking on the links above.<o:p></o:p></big><br />
<br><br />
<big><br><br />
<span class="textexposedshow">Also browse the previous<br />
Melbourne <span class="SpellE">Uni</span> iGEM team<br />
at<span class="GramE">:</span></span><br><br />
<a href="http://openwetware.org/wiki/IGEM:Melbourne/2008"<br />
target="_blank">http://openwetware.org/wiki/IGEM:Melbourne/2008</a><br><br />
<a<br />
href="http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-melbourne-university-te"<br />
target="_blank">http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-<span<br />
class="SpellE">melbourne</span>-university-<span<br />
class="SpellE">te</span></a></big><br><br />
<br><br />
<br><br />
<big>Follow us on Facebook:</big><br />
<br><br />
<div style="text-align: center;"><a<br />
href="https://www.facebook.com/MelbourneUniIGem"><img<br />
style="border: 0px solid ; width: 102px; height: 102px;"<br />
alt="Facebook logo"<br />
src="https://static.igem.org/mediawiki/2014/8/88/OxigemIconfacebook.png"></a><a<br />
href="http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-melbourne-university-te"<br />
target="_blank"><span class="SpellE"></span></a><span<br />
class="textexposedshow"><o:p></o:p></span><br><br />
</div><br />
<!-- END EDIT HERE HERE --><br />
</body><br />
</html></div>Slowehttp://2014.igem.org/Team:Melbourne/RecruitmentTeam:Melbourne/Recruitment2015-05-21T17:46:21Z<p>Slowe: </p>
<hr />
<div><html><br />
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</style><br />
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rel="stylesheet" type="text/css"><br />
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</head><br />
<body><br />
<img src="https://static.igem.org/mediawiki/2014/9/9b/Melbourne_Banner.png"<br />
alt="Banner" height="225" width="1000"><br />
<table<br />
style="text-transform: uppercase; font-family: 'Lato',sans-serif;"<br />
width="100%"><br />
<tbody><br />
<tr height="10"><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne"<br />
style="color: rgb(0, 0, 0);">Home</a> </td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Team"<br />
style="color: rgb(0, 0, 0);">Team</a> </td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Project"<br />
style="color: rgb(0, 0, 0);">Project</a> </td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Human_Practices"<br />
style="color: rgb(0, 0, 0);"> Human Practices</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Achievements"<br />
style="color: rgb(0, 0, 0);">Achievements</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Notebook"<br />
style="color: rgb(0, 0, 0);">Notebook</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Protocols"<br />
style="color: rgb(0, 0, 0);"> Protocols</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Safety"<br />
style="color: rgb(0, 0, 0);">Safety</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Sponsors"<br />
style="color: rgb(0, 0, 0);">Sponsors</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Attributions"<br />
style="color: rgb(0, 0, 0);">Attributions</a></td><br />
<td align="right"> <a<br />
href="https://2014.igem.org/Main_Page"> <img<br />
src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png"<br />
width="46"></a> </td><br />
</tr><br />
</tbody><br />
</table><br />
<big><br><br />
Thank<br />
you for your interest in the<br />
2015-16 Melbourne University iGEM Team! </big><br><br />
<br><br />
<big>Please<br />
read the information below to find out what iGEM is all about. We are<br />
currently looking for members of the leadership team, and after the<br />
completion of Semester 1 exams, there will be a general recruitment<br />
round. Please <a href="#subscribe">sign<br />
up to the Melbourne iGEM mailing list</a>, below,<br />
to be alerted to this recruitment.</big><br><br />
<br><br />
<p><big><big><big><big><span<br />
style="font-weight: bold;">What is iGEM?</span></big></big></big></big></p><br />
<div style="text-align: center;"><big><big><big><big><span<br />
style="font-weight: bold;"><img<br />
style="width: 274px; height: 209px;" alt="IGEM logo"<br />
src="https://static.igem.org/mediawiki/2014/d/d8/Team_Berlin_igem_logo.png"></span></big></big></big></big><o:p></o:p></div><br />
<big><span class="GramE"><span class="usercontent"><br><br />
iGEM</span></span><span class="usercontent"><br />
is a unique opportunity to get involved<br />
in an enterprising<br />
student research group and make a scientific impact.</span><span<br />
class="GramE"><span class="textexposedshow"></span></span></big><br />
<br><br />
<br><br />
<big><span class="GramE"><span<br />
class="textexposedshow">iGEM</span></span><span<br />
class="textexposedshow"> is short for the International<br />
Genetically Engineering<br />
Machine competition. <span class="GramE">iGEM</span><br />
is an undergraduate science<br />
competition held each September in Boston. In the period leading up to<br />
iGEM,<br />
university teams use the latest tools from synthetic<br />
biology to<br />
create a novel single celled organism or “biological machine”. They can<br />
also develop synthetic biology computing and lab tools, engage in<br />
scientific outreach, and even develop biotech businesses.<br><br />
</span><span data-ft="{&quot;tn&quot;:&quot;K&quot;}" class="userContent"><span<br />
class="text_exposed_show"></span></span></big><br><br />
<big><span data-ft="{&quot;tn&quot;:&quot;K&quot;}" class="userContent"><span<br />
class="text_exposed_show">Each year, students from around<br />
the globe form<br />
teams at their respective universities with the goal of building a<br />
biological system or tool, which they later present at the iGEM global<br />
conference. Students in the past have designed bacteria that<br />
produce new types of drugs and biofuels, act as biosensors of toxic<br />
pollutants, and&nbsp;serve as biological computation<br />
platforms&nbsp; (for more examples of past<br />
projects, see <a href="https://igem.org/About"<br />
rel="nofollow nofollow" target="_blank"<br />
onmouseover='LinkshimAsyncLink.swap(this, "http:\/\/igem.org\/About");'<br />
onclick='LinkshimAsyncLink.swap(this, "http:\/\/l.facebook.com\/l.php?u=http\u00253A\u00252F\u00252Figem.org\u00252FAbout&h=zAQFmUzcK&enc=AZNGkoGC-HF5q40u8IHnH2vOwvgHdyy0ClVop-mb6cjZGHojVOVS9IRhqf-Bi3W3tjr6Zt8No6t5_Rcx8yzlpP7oLfAxZyO28QciO52K7yqag82-VE7nwRVRmBKK-HgbxqVsq9pAa5Lv-OJPoe-BAhfl&s=1");'>https://igem.org/About</a>)</span></span>.<span<br />
class="textexposedshow"> </span><o:p></o:p></big><br />
<br><br />
<br><br />
<big><span class="GramE"><span<br />
class="textexposedshow">iGEM</span></span><span<br />
class="textexposedshow"> teams manage everything from the<br />
conception to the<br />
execution of the iGEM project, with the aid of faculty supervisors. </span><br><br />
<br><br />
<span class="textexposedshow">iGEM allows students to:</span><br><br />
</big><br />
<ul><br />
<li><big><span class="textexposedshow">Get<br />
valuable scientific and<br />
leadership experience</span></big></li><br />
<li><big><span class="textexposedshow">Develop<br />
a useful, novel<br />
biotechnology and gain experience<br />
in the latest in interdisciplinary biological research<o:p></o:p></span></big><br />
</li><br />
<li><big><span class="textexposedshow">Make<br />
an impact</span></big></li><br />
<li><big><span class="textexposedshow">Have<br />
fun!</span></big></li><br />
</ul><br />
<big><span class="textexposedshow"><br><br />
</span></big><br><br />
<p><big><big><big><big><span<br />
style="font-weight: bold;">Project</span></big></big></big></big></p><br />
<big><big><big><big><span<br />
style="font-weight: bold;"></span></big></big></big>Students<br />
in the iGEM<br />
teams have complete control over the teams’<br />
project and scope. There are many possible projects and types of teams.<br />
There are 15 tracks in the iGEM competition. They include 8 standard<br />
tracks and several special tracks<br />
(e.g. entrepreneurship, software, social policy, etc.). Two examples of<br />
possible iGEM teams would include:<br><br />
</big><br />
<br><br />
<big>1. Standard tracks: In the standard tracks, teams<br />
develop iGEM<br />
projects for research purposes and to advance science on a foundational<br />
or applied level. Last year, the Melbourne iGEM<br />
team competed in the Manufacturing track, working on a project to<br />
develop bacteria capable of producing new types of antibiotics.<br />
However, there is enormous variety in the types of problems teams can<br />
work on (see <a<br />
href="https://igem.org/Results?year=2013&amp;region=Championship&amp;division=igem">past<br />
team Wiki's</a> for examples).</big><br />
<big><br><br />
<br><br />
2. Special tracks: the iGEM competition has expanded<br />
significantly since its inception, with the addition of several new<br />
tracks to capture the enormous growth of synthetic biology.<br />
For example, teams can now compete in an entrepreneurship track where<br />
they produce not only a scientific advancement, but also a business<br />
plan to<br />
commercialise the work. The&nbsp;track is aimed at giving students<br />
a conducive and educational environment to start a synthetic biology<br />
company, and is inspired by the recent boom in the synthetic biology<br />
startups<br />
(e.g. see <a href="http://eu.indie.bio/">Indie Bio</a>).<br><br />
<br><br />
</big><br />
<p><big><big><big><big><span<br />
style="font-weight: bold;">Timeline</span></big></big></big></big></p><br />
<big><br><br />
</big><big>The next<br />
iGEM team will run from Semester 2, 2015 to Semester 2, 2016. In<br />
Semester 2, 2015, the team<br />
will be organised, the project selected, and the lab set up. During<br />
this period, team members will need to work to&nbsp;ensure that the<br />
foundations are in place to start significant lab/project work during<br />
the summer. A large portion of the practical work will then take<br />
place during the summer of 2015-16. Therefore, to participate in the<br />
team you will need to be available during the upcoming summer. Finally,<br />
additional work will be carried out as needed during the lead up to the<br />
2016 iGEM conference (the iGEM Giant Jamboree) in September of next<br />
year.</big><br><br />
&nbsp;<br><br />
<p class="MsoNormal"><big><big><big><big><span<br />
style="font-weight: bold;">What is involved in what is<br />
needed?</span></big></big></big></big></p><br />
<big><big><big><big><span<br />
style="font-weight: bold;"><br><br />
</span></big></big></big></big><br />
<p style="text-align: center;" class="MsoNormal"><big><img<br />
style="width: 264px; height: 197px;" alt="DNA image"<br />
src="https://static.igem.org/mediawiki/2014/9/91/DNA_v1_by_bak16.jpg"></big></p><br />
<big>We are searching for<br />
enthusiastic<br />
students with<br />
an interest in biotechnology and science.<o:p>&nbsp;</o:p></big><br />
<br><br />
<br><br />
<big>As<br />
part of the iGEM team, you would help with the<br />
following tasks:</big><br />
<ul><br />
<li><big>Research on the iGEM project ideas. The ideas<br />
for the iGEM project are student driven, and team members often need to<br />
answer specific research question to design new experiments or come up<br />
with new ideas. This will typically involve doing&nbsp; searches of<br />
the literature using Google Scholar or other tools and reading<br />
scientific articles.</big></li><br />
<li><big>Help with developing experimental methods. The<br />
2014 team has built up a library of protocols and experimental methods.<br />
However, the project for 2015 will likely require new methods. You will<br />
need to look up protocols in the literature and adapt them to the<br />
project requirements.</big></li><br />
<li><big>Wet lab work, modelling/computation or<br />
engineering design. By joining the iGEM team, you will have an<br />
opportunity to participate in the lab and learn many standard<br />
techniques in molecular biology. Alternatively, many iGEM teams make<br />
use of the skills of engineers, computer scientists, and other<br />
non-biological science disciplines.&nbsp; For example, this may<br />
take the form of modeling a biological system using software like<br />
Matlab, designing a microfluidic device with biological applications or<br />
designing an electrical device which interfaces with a biological<br />
system. If you have an interest in interdisciplinary research between<br />
your field and biology, it is likely iGEM will be able to accommodate<br />
it.</big></li><br />
<li><big>The scope of iGEM also extends into<br />
non-traditional science areas, including biotechnology<br />
entrepreneurship, biotechnology ethics and the law, and science<br />
outreach, and so students with business, law, design, and arts<br />
backgrounds are also welcome to participate. You could, for example,<br />
create a business plan for a iGEM-created company, examine bioethics<br />
within synthetic biology, or design educational/outreach program for<br />
high schoolers.</big></li><br />
</ul><br />
<ul><br />
<br><br />
</ul><br />
<p class="MsoNormal"><big><o:p></o:p></big><big><big><big><big><span<br />
style="font-weight: bold;">Recruitment</span></big></big></big></big></p><br />
<big>We are currently seeking<br />
members of<br />
the executive (see below), witha&nbsp; general<br />
recruitment round after<br />
the exam period. Recruitment will be open to all undergraduate and<br />
masters students who are available<br />
during Semester 2 2015 and the summer of 2015-16. Please sign up below<br />
to be alerted when the general recruitment opens (also follow<br />
us on <a href="https://www.facebook.com/MelbourneUniIGem">Facebook</a>).<br />
If you have specific questions about iGEM, please direct them to<br />
Sean at melbourneuniigem@gmail.com.&nbsp;</big><br />
<p class="MsoNormal"><!-- Begin MailChimp Signup Form --></p><br />
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Melbourne iGEM recruitment updates</h2><br />
<div class="indicates-required"><span class="asterisk">*</span><br />
indicates required</div><br />
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<big>If you are particularly<br />
keen on iGEM, and would like to play a leadership role, we currently<br />
require additional members of the executive/leadership team. As part<br />
of the team's leadership, you<br />
will be in a unique position to mold the team. Duties include:</big><br />
<ol><br />
<li><big>Helping to set up the team and determining the<br />
teams' goals and iGEM track.</big></li><br />
<li><big>Serving as a spokesperson&nbsp;to<br />
external parties and academics. This will include recruiting additional<br />
academic and graduate student supervisors.<br><br />
</big></li><br />
<li><big>Helping to fundraise for the team, liaising with<br />
university and external sponsors.</big></li><br />
<li><big>Helping to recruit new members</big></li><br />
</ol><br />
<big><br><br />
Being a founding member of the next iGEM team provides a<br />
great opportunity to play a driving role in Melbourne Uni's most<br />
exciting student based group. The key requirements for the role is a<br />
strong commitment to the team, as well as good communication and<br />
leadership skills. If you are interested in<br />
playing this role, please email<br />
Sean&nbsp;at MelbourneUniIGem@gmail.com to express your interest. <o:p></o:p></big><br><br />
<br><br />
<p class="MsoNormal"><big><o:p></o:p></big><big><big><big><big><span<br />
style="font-weight: bold;">Further information</span></big></big></big></big></p><br />
<big>Learn more about iGEM in<br />
general at: <a<br />
href="http://www.facebook.com/l.php?u=http%3A%2F%2Figem.org%2FAbout&amp;h=HAQEjYmHL&amp;s=1"<br />
target="_blank">https://igem.org/About</a><br<br />
style=""><br />
</big><br><br />
<big>Learn about last year’s<br />
team by<br />
clicking on the links above.<o:p></o:p></big><br />
<br><br />
<big><br><br />
<span class="textexposedshow">Also browse the previous<br />
Melbourne <span class="SpellE">Uni</span> iGEM team<br />
at<span class="GramE">:</span></span><br><br />
<a href="http://openwetware.org/wiki/IGEM:Melbourne/2008"<br />
target="_blank">http://openwetware.org/wiki/IGEM:Melbourne/2008</a><br><br />
<a<br />
href="http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-melbourne-university-te"<br />
target="_blank">http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-<span<br />
class="SpellE">melbourne</span>-university-<span<br />
class="SpellE">te</span></a></big><br><br />
<br><br />
<br><br />
<big>Follow us on Facebook:</big><br />
<br><br />
<div style="text-align: center;"><a<br />
href="https://www.facebook.com/MelbourneUniIGem"><img<br />
style="border: 0px solid ; width: 102px; height: 102px;"<br />
alt="Facebook logo"<br />
src="https://static.igem.org/mediawiki/2014/8/88/OxigemIconfacebook.png"></a><a<br />
href="http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-melbourne-university-te"<br />
target="_blank"><span class="SpellE"></span></a><span<br />
class="textexposedshow"><o:p></o:p></span><br><br />
</div><br />
<!-- END EDIT HERE HERE --><br />
</body><br />
</html></div>Slowehttp://2014.igem.org/File:DNA_v1_by_bak16.jpgFile:DNA v1 by bak16.jpg2015-05-21T13:05:40Z<p>Slowe: </p>
<hr />
<div></div>Slowehttp://2014.igem.org/Team:Melbourne/Recruitment2Team:Melbourne/Recruitment22015-05-21T10:41:53Z<p>Slowe: </p>
<hr />
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<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne"<br />
style="color: rgb(0, 0, 0);">Home</a> </td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Team"<br />
style="color: rgb(0, 0, 0);">Team</a> </td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Project"<br />
style="color: rgb(0, 0, 0);">Project</a> </td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Human_Practices"<br />
style="color: rgb(0, 0, 0);"> Human Practices</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Achievements"<br />
style="color: rgb(0, 0, 0);">Achievements</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Notebook"<br />
style="color: rgb(0, 0, 0);">Notebook</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Protocols"<br />
style="color: rgb(0, 0, 0);"> Protocols</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Safety"<br />
style="color: rgb(0, 0, 0);">Safety</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Sponsors"<br />
style="color: rgb(0, 0, 0);">Sponsors</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Attributions"<br />
style="color: rgb(0, 0, 0);">Attributions</a></td><br />
<td align="right"> <a<br />
href="https://2014.igem.org/Main_Page"> <img<br />
src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png"<br />
width="46"></a> </td><br />
</tr><br />
</tbody><br />
</table><br />
<p class="MsoNormal" style="margin-top: 12pt;"><big>Thank<br />
you for your interest in the<br />
2015-16 Melbourne University iGEM Team!&nbsp;</big></p><br />
<p class="MsoNormal" style="margin-top: 12pt;">Please<br />
read the information below to find out what iGEM is all about. We are<br />
currently looking for members of the leadership team. After the<br />
completion of Semester 1 exams, there will be a general recruitment<br />
round open. Please sign up to the Melbourne iGEM mailing list, below,<br />
to be alerted of this recruitment round.</p><br />
<big><big><big><big><span<br />
style="font-weight: bold;">What is iGEM?</span></big></big></big></big><o:p></o:p><br />
<p class="MsoNormal"><big><span class="GramE"><span<br />
class="usercontent">iGEM</span></span><span<br />
class="usercontent"> is a unique opportunity to get involved<br />
in an enterprising<br />
student research group and make a scientific impact.</span><br><br />
<br><br />
<span class="GramE"><span class="textexposedshow">iGEM</span></span><span<br />
class="textexposedshow"> is short for the International<br />
Genetically Engineering<br />
Machine competition. <span class="GramE">iGEM</span><br />
is an undergraduate science<br />
competition held each September in Boston. In the . leading up to<br />
iGEM,<br />
university teams use the latest tools from synthetic<br />
biology/biotechnology to<br />
create a novel single celled organism or “biological machine”. They can<br />
also develop synthetic biology computing and lab tools, engage in<br />
scientific outreach, and even develop bio-based businesses.</span></big></p><br />
<p class="MsoNormal"><big><span<br />
data-ft="{&quot;tn&quot;:&quot;K&quot;}" class="userContent"><span<br />
class="text_exposed_show">Each<br />
year, students from around the globe form<br />
teams at their respective universities with the goal of building a<br />
biological system or tool, which they present at the iGEM global<br />
conference. Students in the past have designed bacteria that<br />
produce new types of drugs and biofuels, act as biosensors of toxic<br />
pollutants, and even serve&nbsp; as biological computation<br />
platforms&nbsp; (for examples of past<br />
projects, see <a href="https://igem.org/About"<br />
rel="nofollow nofollow" target="_blank"<br />
onmouseover='LinkshimAsyncLink.swap(this, "http:\/\/igem.org\/About");'<br />
onclick='LinkshimAsyncLink.swap(this, "http:\/\/l.facebook.com\/l.php?u=http\u00253A\u00252F\u00252Figem.org\u00252FAbout&h=zAQFmUzcK&enc=AZNGkoGC-HF5q40u8IHnH2vOwvgHdyy0ClVop-mb6cjZGHojVOVS9IRhqf-Bi3W3tjr6Zt8No6t5_Rcx8yzlpP7oLfAxZyO28QciO52K7yqag82-VE7nwRVRmBKK-HgbxqVsq9pAa5Lv-OJPoe-BAhfl&s=1");'>https://igem.org/About</a>)</span></span>.<span<br />
class="textexposedshow"> </span><o:p></o:p></big></p><br />
<p class="MsoNormal"><big><span class="GramE"><span<br />
class="textexposedshow">iGEM</span></span><span<br />
class="textexposedshow"> teams manage everything from the<br />
conception to the<br />
execution of the iGEM project, with the aid of faculty supervisors. </span><br><br />
<br><br />
<span class="textexposedshow">The benefits to<br />
participating in iGEM include:</span><br><br />
<span class="textexposedshow">-Get valuable scientific and<br />
leadership experience</span><br><br />
<span class="textexposedshow">-Develop a useful, novel<br />
biotechnology and gain experience<br />
in the latest in interdisciplinary biological research<o:p></o:p></span></big></p><br />
<p class="MsoNormal"><big><span<br />
class="textexposedshow">-Make<br />
an impact</span><br><br />
<span class="textexposedshow">-Have fun!</span></big></p><br />
<big><big><big><big><span<br />
style="font-weight: bold;">Project and timeline<br><br />
</span></big></big></big>The next<br />
iGEM team will start during Semester 2, 2015. In Semester 2, the team<br />
will be organised, the project selected, and the lab set up. During<br />
this period, you will need to work with fellow team members on a weekly<br />
basis to build the team and ensure that it is ready for lab work during<br />
the summer. A large portion of the lab/project work will then take<br />
place during the summer of 2015-16. Therefore, to participate in the<br />
team you will need to be available during the upcoming summer. Finally,<br />
additional work will be carried out as needed during the lead up to the<br />
2016 iGEM conference (the iGEM Giant Jamboree) in September of next<br />
year.<br><br />
<br><br />
Students in the iGEM teams have complete control over the teams’<br />
project and scope. There are many possible projects and types of teams.<br />
There are 15 tracks in the iGEM competition. They include 8 standard<br />
tracks (foundational advancements, applied advancements in<br />
energy/environment/health.etc), and several special tracks<br />
(entrepreneurship, software, social policy, etc.). Two examples of<br />
possible iGEM teams would include:<br><br />
</big><br />
<ol><br />
<li><big>Standard track: In this standard tracks, teams<br />
develop iGEM<br />
projects purely for research purposes. Last year, the Melbourne iGEM<br />
team competed in the Manufacturing track, working on a project to<br />
develop bacteria capable of producing new types of antibiotics.<br />
However, there is enormous variety in the types of problems teams can<br />
work on (see past teams' Wiki's for examples).</big></li><br />
<li><big>Special track: the iGEM competition has expanded<br />
significantly since its inception, with the addition of several new<br />
tracks to capture the enormous growth in scope of synthetic biology.<br />
For example, teams can now compete in an entrepreneurship track. Here,<br />
teams produce not only a scientific advancement, but a business plan to<br />
commercialise the work. The entrepreneurship track thus gives students<br />
a conducive and educational environment to start a synthetic biology<br />
company, joining the recent boom in the synthetic biology startup scene<br />
(e.g. see <a href="http://eu.indie.bio/">Indie Bio</a>).</big><br />
</li><br />
</ol><br />
<br><br />
<p class="MsoNormal"><big><big><big><big><span<br />
style="font-weight: bold;">What is involved in what is<br />
needed?</span></big></big></big></big><br />
</p><br />
<p class="MsoNormal"><big>We are searching for<br />
enthusiastic<br />
students with<br />
an interest in biotechnology and science.<o:p>&nbsp;</o:p></big></p><br />
<p class="MsoNormal" style="page-break-after: avoid;"><big>As<br />
part of the general iGEM team, you would help with the<br />
following tasks:<o:p></o:p></big></p><br />
<p class="MsoListParagraphCxSpFirst"<br />
style="text-indent: -18pt; page-break-after: avoid;"><big><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->Research<br />
on the iGEM project ideas. The ideas<br />
for the iGEM project are student driven, and team members often need to<br />
answer<br />
specific research question to design new experiments or come up with<br />
new ideas.<br />
This will typically involve doing&nbsp; searches of the literature<br />
using Google<br />
Scholar or other tools and reading scientific articles.<o:p></o:p></big></p><br />
<p class="MsoListParagraphCxSpMiddle"<br />
style="text-indent: -18pt; page-break-after: avoid;"><big><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->Help<br />
with developing experimental methods. The<br />
2014 team has built up a library of protocols and experimental methods.<br />
However, the project for 2015 will likely require new methods. You will<br />
need to<br />
look up protocols in the literature and adapt them to the project<br />
requirements.<o:p></o:p></big></p><br />
<p class="MsoListParagraphCxSpMiddle"<br />
style="text-indent: -18pt;"><big><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->Wet<br />
lab work,&nbsp;modelling/computation or engineering<br />
design. By joining the iGEM team, you will have an opportunity to<br />
participate in<br />
the lab and learn many standard techniques in molecular biology.<br />
Alternatively, many iGEM teams make use of the skills of<br />
engineers,<br />
computer scientists, and other non-biological science disciplines.<br />
&nbsp;For example, this<br />
may<br />
take the form of <span class="SpellE">modeling</span><br />
a biological<br />
system using software like <span class="SpellE">Matlab</span>,<br />
designing a microfluidic device with biological applications or<br />
designing an electrical device which interfaces with a biological<br />
system. If<br />
you have an interest in interdisciplinary research between your field<br />
and biology,<br />
it is likely iGEM will be able to accommodate it.<o:p></o:p></big></p><br />
<p class="MsoListParagraphCxSpLast"<br />
style="text-indent: -18pt;"><big><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->The<br />
scope of iGEM also extends into<br />
non-traditional science areas, including biotechnology<br />
entrepreneurship,<br />
biotechnology ethics and the law, and science outreach, and so we are<br />
actively<br />
seeking students with business, law, design, and arts backgrounds. You<br />
could,<br />
for example, create a business plan for <span class="GramE">a</span><br />
iGEM-created<br />
company, examine bioethics within synthetic biology, or design<br />
educational/outreach program for high schoolers.<o:p></o:p></big></p><br />
<p class="MsoNormal"><big><o:p></o:p></big><big><big><big><big><span<br />
style="font-weight: bold;">Recruitment</span></big></big></big></big></p><br />
<p class="MsoNormal">We are currently seeking members of<br />
the executive (see below), but the general recruitment will open after<br />
the exam period. Please sign up below for alerts on the latest<br />
recruitment activities:</p><br />
<p class="MsoNormal"><br />
<br />
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<h2>Sign up for Melbourne iGEM recruitment updates</h2><br />
<div class="indicates-required"><span class="asterisk">*</span> indicates required</div><br />
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<input type="text" value="" name="FNAME" class="required" id="mce-FNAME"><br />
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<div class="clear"><input type="submit" value="Subscribe" name="subscribe" id="mc-embedded-subscribe" class="button"></div><br />
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<script type='text/javascript' src='//s3.amazonaws.com/downloads.mailchimp.com/js/mc-validate.js'></script><script type='text/javascript'>(function($) {window.fnames = new Array(); window.ftypes = new Array();fnames[0]='EMAIL';ftypes[0]='email';fnames[1]='FNAME';ftypes[1]='text';fnames[2]='LNAME';ftypes[2]='text';fnames[4]='DEGREE';ftypes[4]='text';}(jQuery));var $mcj = jQuery.noConflict(true);</script><br />
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<br />
<br />
<p class="MsoNormal">If you are interested in being a part<br />
of iGEM, please email<br />
Sean Lowe at MelbourneUniIGem@gmail.com for more information. <o:p></o:p></p><br />
<p class="MsoNormal">Further information about iGEM and<br />
updates about the<br />
recruitment process will also be made available at <a<br />
href="https://www.facebook.com/MelbourneUniIGem">https://www.facebook.com/MelbourneUniIGem</a>.<o:p></o:p></p><br />
<br><br />
<h1>Further information</h1><br />
<p class="MsoNormal">Learn more about iGEM in<br />
general at: <a<br />
href="http://www.facebook.com/l.php?u=http%3A%2F%2Figem.org%2FAbout&amp;h=HAQEjYmHL&amp;s=1"<br />
target="_blank">https://igem.org/About</a><br<br />
style=""><br />
</p><br />
<!--[if !supportLineBreakNewLine]--><br style=""><br />
<!--[endif]--><o:p></o:p><br />
<p class="MsoNormal">Learn about this year’s team by<br />
clicking on the links above.<o:p></o:p></p><br />
<p class="MsoNormal"><br><br />
<span class="textexposedshow">Also browse the previous<br />
Melbourne <span class="SpellE">Uni</span> iGEM team<br />
at<span class="GramE">:</span></span><br><br />
<a href="http://openwetware.org/wiki/IGEM:Melbourne/2008"<br />
target="_blank">http://openwetware.org/wiki/IGEM:Melbourne/2008</a><br><br />
<a<br />
href="http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-melbourne-university-te"<br />
target="_blank">http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-<span<br />
class="SpellE">melbourne</span>-university-<span<br />
class="SpellE">te</span></a><span<br />
class="textexposedshow"><o:p></o:p></span></p><br />
<!-- END EDIT HERE HERE --><br />
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<p class="MsoNormal" style="margin-top: 12pt;"><big>Thank<br />
you for your interest in the<br />
2015-16 Melbourne University iGEM Team!&nbsp;</big></p><br />
<p class="MsoNormal" style="margin-top: 12pt;">Please<br />
read the information below to find out what iGEM is all about. We are<br />
currently looking for members of the leadership team. After the<br />
completion of Semester 1 exams, there will be a general recruitment<br />
round open. Please sign up to the Melbourne iGEM mailing list, below,<br />
to be alerted of this recruitment round.</p><br />
<big><big><big><big><span<br />
style="font-weight: bold;">What is iGEM?</span></big></big></big></big><o:p></o:p><br />
<p class="MsoNormal"><big><span class="GramE"><span<br />
class="usercontent">iGEM</span></span><span<br />
class="usercontent"> is a unique opportunity to get involved<br />
in an enterprising<br />
student research group and make a scientific impact.</span><br><br />
<br><br />
<span class="GramE"><span class="textexposedshow">iGEM</span></span><span<br />
class="textexposedshow"> is short for the International<br />
Genetically Engineering<br />
Machine competition. <span class="GramE">iGEM</span><br />
is an undergraduate science<br />
competition held each September in Boston. In the . leading up to<br />
iGEM,<br />
university teams use the latest tools from synthetic<br />
biology/biotechnology to<br />
create a novel single celled organism or “biological machine”. They can<br />
also develop synthetic biology computing and lab tools, engage in<br />
scientific outreach, and even develop bio-based businesses.</span></big></p><br />
<p class="MsoNormal"><big><span<br />
data-ft="{&quot;tn&quot;:&quot;K&quot;}" class="userContent"><span<br />
class="text_exposed_show">Each<br />
year, students from around the globe form<br />
teams at their respective universities with the goal of building a<br />
biological system or tool, which they present at the iGEM global<br />
conference. Students in the past have designed bacteria that<br />
produce new types of drugs and biofuels, act as biosensors of toxic<br />
pollutants, and even serve&nbsp; as biological computation<br />
platforms&nbsp; (for examples of past<br />
projects, see <a href="https://igem.org/About"<br />
rel="nofollow nofollow" target="_blank"<br />
onmouseover='LinkshimAsyncLink.swap(this, "http:\/\/igem.org\/About");'<br />
onclick='LinkshimAsyncLink.swap(this, "http:\/\/l.facebook.com\/l.php?u=http\u00253A\u00252F\u00252Figem.org\u00252FAbout&h=zAQFmUzcK&enc=AZNGkoGC-HF5q40u8IHnH2vOwvgHdyy0ClVop-mb6cjZGHojVOVS9IRhqf-Bi3W3tjr6Zt8No6t5_Rcx8yzlpP7oLfAxZyO28QciO52K7yqag82-VE7nwRVRmBKK-HgbxqVsq9pAa5Lv-OJPoe-BAhfl&s=1");'>https://igem.org/About</a>)</span></span>.<span<br />
class="textexposedshow"> </span><o:p></o:p></big></p><br />
<p class="MsoNormal"><big><span class="GramE"><span<br />
class="textexposedshow">iGEM</span></span><span<br />
class="textexposedshow"> teams manage everything from the<br />
conception to the<br />
execution of the iGEM project, with the aid of faculty supervisors. </span><br><br />
<br><br />
<span class="textexposedshow">The benefits to<br />
participating in iGEM include:</span><br><br />
<span class="textexposedshow">-Get valuable scientific and<br />
leadership experience</span><br><br />
<span class="textexposedshow">-Develop a useful, novel<br />
biotechnology and gain experience<br />
in the latest in interdisciplinary biological research<o:p></o:p></span></big></p><br />
<p class="MsoNormal"><big><span<br />
class="textexposedshow">-Make<br />
an impact</span><br><br />
<span class="textexposedshow">-Have fun!</span></big></p><br />
<big><big><big><big><span<br />
style="font-weight: bold;">Project and timeline<br><br />
</span></big></big></big>The next<br />
iGEM team will start during Semester 2, 2015. In Semester 2, the team<br />
will be organised, the project selected, and the lab set up. During<br />
this period, you will need to work with fellow team members on a weekly<br />
basis to build the team and ensure that it is ready for lab work during<br />
the summer. A large portion of the lab/project work will then take<br />
place during the summer of 2015-16. Therefore, to participate in the<br />
team you will need to be available during the upcoming summer. Finally,<br />
additional work will be carried out as needed during the lead up to the<br />
2016 iGEM conference (the iGEM Giant Jamboree) in September of next<br />
year.<br><br />
<br><br />
Students in the iGEM teams have complete control over the teams’<br />
project and scope. There are many possible projects and types of teams.<br />
There are 15 tracks in the iGEM competition. They include 8 standard<br />
tracks (foundational advancements, applied advancements in<br />
energy/environment/health.etc), and several special tracks<br />
(entrepreneurship, software, social policy, etc.). Two examples of<br />
possible iGEM teams would include:<br><br />
</big><br />
<ol><br />
<li><big>Standard track: In this standard tracks, teams<br />
develop iGEM<br />
projects purely for research purposes. Last year, the Melbourne iGEM<br />
team competed in the Manufacturing track, working on a project to<br />
develop bacteria capable of producing new types of antibiotics.<br />
However, there is enormous variety in the types of problems teams can<br />
work on (see past teams' Wiki's for examples).</big></li><br />
<li><big>Special track: the iGEM competition has expanded<br />
significantly since its inception, with the addition of several new<br />
tracks to capture the enormous growth in scope of synthetic biology.<br />
For example, teams can now compete in an entrepreneurship track. Here,<br />
teams produce not only a scientific advancement, but a business plan to<br />
commercialise the work. The entrepreneurship track thus gives students<br />
a conducive and educational environment to start a synthetic biology<br />
company, joining the recent boom in the synthetic biology startup scene<br />
(e.g. see <a href="http://eu.indie.bio/">Indie Bio</a>).</big><br />
</li><br />
</ol><br />
<br><br />
<p class="MsoNormal"><big><big><big><big><span<br />
style="font-weight: bold;">What is involved in what is<br />
needed?</span></big></big></big></big><br />
</p><br />
<p class="MsoNormal"><big>We are searching for<br />
enthusiastic<br />
students with<br />
an interest in biotechnology and science.<o:p>&nbsp;</o:p></big></p><br />
<p class="MsoNormal" style="page-break-after: avoid;"><big>As<br />
part of the general iGEM team, you would help with the<br />
following tasks:<o:p></o:p></big></p><br />
<p class="MsoListParagraphCxSpFirst"<br />
style="text-indent: -18pt; page-break-after: avoid;"><big><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->Research<br />
on the iGEM project ideas. The ideas<br />
for the iGEM project are student driven, and team members often need to<br />
answer<br />
specific research question to design new experiments or come up with<br />
new ideas.<br />
This will typically involve doing&nbsp; searches of the literature<br />
using Google<br />
Scholar or other tools and reading scientific articles.<o:p></o:p></big></p><br />
<p class="MsoListParagraphCxSpMiddle"<br />
style="text-indent: -18pt; page-break-after: avoid;"><big><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->Help<br />
with developing experimental methods. The<br />
2014 team has built up a library of protocols and experimental methods.<br />
However, the project for 2015 will likely require new methods. You will<br />
need to<br />
look up protocols in the literature and adapt them to the project<br />
requirements.<o:p></o:p></big></p><br />
<p class="MsoListParagraphCxSpMiddle"<br />
style="text-indent: -18pt;"><big><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->Wet<br />
lab work,&nbsp;modelling/computation or engineering<br />
design. By joining the iGEM team, you will have an opportunity to<br />
participate in<br />
the lab and learn many standard techniques in molecular biology.<br />
Alternatively, many iGEM teams make use of the skills of<br />
engineers,<br />
computer scientists, and other non-biological science disciplines.<br />
&nbsp;For example, this<br />
may<br />
take the form of <span class="SpellE">modeling</span><br />
a biological<br />
system using software like <span class="SpellE">Matlab</span>,<br />
designing a microfluidic device with biological applications or<br />
designing an electrical device which interfaces with a biological<br />
system. If<br />
you have an interest in interdisciplinary research between your field<br />
and biology,<br />
it is likely iGEM will be able to accommodate it.<o:p></o:p></big></p><br />
<p class="MsoListParagraphCxSpLast"<br />
style="text-indent: -18pt;"><big><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->The<br />
scope of iGEM also extends into<br />
non-traditional science areas, including biotechnology<br />
entrepreneurship,<br />
biotechnology ethics and the law, and science outreach, and so we are<br />
actively<br />
seeking students with business, law, design, and arts backgrounds. You<br />
could,<br />
for example, create a business plan for <span class="GramE">a</span><br />
iGEM-created<br />
company, examine bioethics within synthetic biology, or design<br />
educational/outreach program for high schoolers.<o:p></o:p></big></p><br />
<p class="MsoNormal"><big><o:p></o:p></big><big><big><big><big><span<br />
style="font-weight: bold;">Recruitment</span></big></big></big></big></p><br />
<p class="MsoNormal">We are currently seeking members of<br />
the executive (see below), but the general recruitment will open after<br />
the exam period. Please sign up below for alerts on the latest<br />
recruitment activities:</p><br />
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<label for="mce-FNAME">First Name <span<br />
class="asterisk">*</span><br />
</label><input value="" name="FNAME"<br />
class="required" id="mce-FNAME" type="text"><br />
</div><br />
<div class="mc-field-group"><br />
<label for="mce-LNAME">Last Name </label><br />
<input value="" name="LNAME" class=""<br />
id="mce-LNAME" type="text"></div><br />
<div class="mc-field-group"><br />
<label for="mce-DEGREE">Degree (Major) </label><br />
<input value="" name="DEGREE" class=""<br />
id="mce-DEGREE" type="text"></div><br />
<div id="mce-responses" class="clear"><br />
<div class="response" id="mce-error-response"<br />
style="display: none;"></div><br />
<div class="response" id="mce-success-response"<br />
style="display: none;"></div><br />
</div><br />
<!-- real people should not fill this in and expect good things - do not remove this or risk form bot signups--><br />
<div style="position: absolute; left: -5000px;"><input<br />
name="b_47561ab8e57f78fb52279255a_1e678a25cd" tabindex="0"<br />
value="" type="text"></div><br />
<div class="clear"><input value="Subscribe"<br />
name="subscribe" id="mc-embedded-subscribe"<br />
class="button" type="submit"></div><br />
</div><br />
</form><br />
</div><br />
<script type="text/javascript"<br />
src="//s3.amazonaws.com/downloads.mailchimp.com/js/mc-validate.js"></script><br />
<script type="text/javascript">(function($) {window.fnames = new Array(); window.ftypes = new Array();fnames[0]='EMAIL';ftypes[0]='email';fnames[1]='FNAME';ftypes[1]='text';fnames[2]='LNAME';ftypes[2]='text';fnames[4]='DEGREE';ftypes[4]='text';}(jQuery));var $mcj = jQuery.noConflict(true);</script><!--End mc_embed_signup--><br />
<script type="text/javascript"<br />
src="//s3.amazonaws.com/downloads.mailchimp.com/js/mc-validate.js"></script><br />
<script type="text/javascript">(function($) {window.fnames = new Array(); window.ftypes = new Array();fnames[0]='EMAIL';ftypes[0]='email';fnames[1]='FNAME';ftypes[1]='text';fnames[2]='LNAME';ftypes[2]='text';fnames[4]='DEGREE';ftypes[4]='text';}(jQuery));var $mcj = jQuery.noConflict(true);</script><!--End mc_embed_signup--><big><big><big><big><span<br />
style="font-weight: bold;"></span></big></big></big></big><o:p></o:p><br />
<h1><br><br />
</h1><br />
<p class="MsoNormal">If you are interested in being a part<br />
of iGEM, please email<br />
Sean Lowe at MelbourneUniIGem@gmail.com for more information. <o:p></o:p></p><br />
<p class="MsoNormal">Further information about iGEM and<br />
updates about the<br />
recruitment process will also be made available at <a<br />
href="https://www.facebook.com/MelbourneUniIGem">https://www.facebook.com/MelbourneUniIGem</a>.<o:p></o:p></p><br />
<br><br />
<h1>Further information</h1><br />
<p class="MsoNormal">Learn more about iGEM in<br />
general at: <a<br />
href="http://www.facebook.com/l.php?u=http%3A%2F%2Figem.org%2FAbout&amp;h=HAQEjYmHL&amp;s=1"<br />
target="_blank">https://igem.org/About</a><br<br />
style=""><br />
</p><br />
<!--[if !supportLineBreakNewLine]--><br style=""><br />
<!--[endif]--><o:p></o:p><br />
<p class="MsoNormal">Learn about this year’s team by<br />
clicking on the links above.<o:p></o:p></p><br />
<p class="MsoNormal"><br><br />
<span class="textexposedshow">Also browse the previous<br />
Melbourne <span class="SpellE">Uni</span> iGEM team<br />
at<span class="GramE">:</span></span><br><br />
<a href="http://openwetware.org/wiki/IGEM:Melbourne/2008"<br />
target="_blank">http://openwetware.org/wiki/IGEM:Melbourne/2008</a><br><br />
<a<br />
href="http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-melbourne-university-te"<br />
target="_blank">http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-<span<br />
class="SpellE">melbourne</span>-university-<span<br />
class="SpellE">te</span></a><span<br />
class="textexposedshow"><o:p></o:p></span></p><br />
<!-- END EDIT HERE HERE --><br />
</body><br />
</html></div>Slowehttp://2014.igem.org/Team:Melbourne/RecruitmentTeam:Melbourne/Recruitment2015-03-09T15:23:42Z<p>Slowe: </p>
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textarea {<br />
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/**<br />
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table {<br />
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}<br />
html, body {<br />
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width: 100%;<br />
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margin: 0;<br />
padding: 0;<br />
display:block;<br />
line-height:normal;<br />
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h1 {<br />
font-weight:bold;<br />
font-size:52px;<br />
margin:0;<br />
text-transform:uppercase;<br />
font-family: 'Lato', sans-serif;<br />
font-weight:900;<br />
text-align:center;<br />
/*font-family: "Courier New", Courier, "Lucida Sans Typewriter", "Lucida Typewriter", monospace;*/<br />
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h2 {<br />
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font-size:24px;<br />
text-transform:uppercase;<br />
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</style><br />
<img src="https://static.igem.org/mediawiki/2014/9/9b/Melbourne_Banner.png"<br />
alt="Banner" height="225" width="1000"><br />
<table<br />
style="text-transform: uppercase; font-family: 'Lato',sans-serif;"<br />
width="100%"><br />
<tbody><br />
<tr height="10"><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne"<br />
style="color: rgb(0, 0, 0);">Home</a> </td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Team"<br />
style="color: rgb(0, 0, 0);">Team</a> </td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Project"<br />
style="color: rgb(0, 0, 0);">Project</a> </td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Human_Practices"<br />
style="color: rgb(0, 0, 0);"> Human Practices</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Achievements"<br />
style="color: rgb(0, 0, 0);">Achievements</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Notebook"<br />
style="color: rgb(0, 0, 0);">Notebook</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Protocols"<br />
style="color: rgb(0, 0, 0);"> Protocols</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Safety"<br />
style="color: rgb(0, 0, 0);">Safety</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Sponsors"<br />
style="color: rgb(0, 0, 0);">Sponsors</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Attributions"<br />
style="color: rgb(0, 0, 0);">Attributions</a></td><br />
<td align="right"> <a<br />
href="https://2014.igem.org/Main_Page"> <img<br />
src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png"<br />
width="46"></a> </td><br />
</tr><br />
</tbody><br />
</table><br />
<p class="MsoNormal" style="margin-top: 12pt;">Thank<br />
you for your interest in the<br />
2015-16 Melbourne University iGEM Team! &nbsp;We will be recruiting<br />
for the next iGEM team in semester 1 2015. To be notified by email of<br />
recruitment developments, please express your interest at<br />
melbourneuniigem@gmail.com.</p><br />
<h1>What is iGEM?<o:p></o:p></h1><br />
<p class="MsoNormal"><span class="GramE"><span<br />
class="usercontent">iGEM</span></span><span<br />
class="usercontent"> is a unique opportunity to get involved<br />
in an enterprising<br />
student research group and make a scientific impact.</span><br><br />
<br><br />
<span class="GramE"><span class="textexposedshow">iGEM</span></span><span<br />
class="textexposedshow"> is short for the International<br />
Genetically Engineering<br />
Machine competition. <span class="GramE">iGEM</span><br />
is an undergraduate science<br />
competition held each November in Boston. In the months leading up to<br />
iGEM,<br />
university teams use the latest tools from synthetic<br />
biology/biotechnology to<br />
create a novel single celled organism or “biological machine”.</span></p><br />
<p class="MsoNormal"><span data-ft="{&quot;tn&quot;:&quot;K&quot;}"<br />
class="userContent"><span class="text_exposed_show">Each<br />
year, students from around the globe form<br />
teams at their respective universities with the goal of building a<br />
biological system or tool, which they present at the iGEM global<br />
conference. Students in the past have designed bacteria that<br />
produce new types of drugs and biofuels, act as biosensors of toxic<br />
pollutants, and even serve&nbsp; as biological computation<br />
platforms&nbsp; (for examples of past<br />
projects, see <a href="https://igem.org/About"<br />
rel="nofollow nofollow" target="_blank"<br />
onmouseover='LinkshimAsyncLink.swap(this, "http:\/\/igem.org\/About");'<br />
onclick='LinkshimAsyncLink.swap(this, "http:\/\/l.facebook.com\/l.php?u=http\u00253A\u00252F\u00252Figem.org\u00252FAbout&h=zAQFmUzcK&enc=AZNGkoGC-HF5q40u8IHnH2vOwvgHdyy0ClVop-mb6cjZGHojVOVS9IRhqf-Bi3W3tjr6Zt8No6t5_Rcx8yzlpP7oLfAxZyO28QciO52K7yqag82-VE7nwRVRmBKK-HgbxqVsq9pAa5Lv-OJPoe-BAhfl&s=1");'>https://igem.org/About</a>)</span></span>.<span<br />
class="textexposedshow"> </span><o:p></o:p></p><br />
<p class="MsoNormal"><span class="GramE"><span<br />
class="textexposedshow">iGEM</span></span><span<br />
class="textexposedshow"> teams manage everything from the<br />
conception to the<br />
execution of the iGEM project, with the aid of faculty supervisors. </span><br><br />
<br><br />
<span class="textexposedshow">The benefits to<br />
participating in iGEM include:</span><br><br />
<span class="textexposedshow">-Get valuable scientific and<br />
leadership experience</span><br><br />
<span class="textexposedshow">-Develop a useful, novel<br />
biotechnology and gain experience<br />
in the latest in interdisciplinary biological research<o:p></o:p></span></p><br />
<p class="MsoNormal"><span class="textexposedshow">-Make<br />
an impact</span><br><br />
<span class="textexposedshow">-Have fun!</span><br><br />
<br><br />
</p><br />
<h1><span class="GramE"></span>What is<br />
involved and what is needed</h1><br />
<p class="MsoNormal">We are searching for enthusiastic<br />
students with<br />
an interest in science.<o:p></o:p></p><br />
<p class="MsoNormal"><o:p>&nbsp;</o:p></p><br />
<p class="MsoNormal" style="page-break-after: avoid;">As<br />
part of the iGEM team, you would help with the<br />
following tasks:<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpFirst"<br />
style="text-indent: -18pt; page-break-after: avoid;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->Research<br />
on the iGEM project ideas. The ideas<br />
for the iGEM project are student driven, and team members often need to<br />
answer<br />
specific research question to design new experiments or come up with<br />
new ideas.<br />
This will typically involve doing&nbsp; searches of the literature<br />
using Google<br />
Scholar or other tools and reading scientific articles.<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpMiddle"<br />
style="text-indent: -18pt; page-break-after: avoid;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->Help<br />
with developing experimental methods. The<br />
2014 team has built up a library of protocols and experimental methods.<br />
However, the project for 2015 will likely require new methods. You will<br />
need to<br />
look up protocols in the literature and adapt them to the project<br />
requirements.<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpMiddle"<br />
style="text-indent: -18pt;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->Wet<br />
lab work OR modelling/computation OR engineering<br />
design. By joining the iGEM team, you will have an opportunity to<br />
participate in<br />
the lab and learn many standard techniques in molecular biology.<br />
Alternatively, many iGEM teams make use of the skills of<br />
engineers,<br />
computer scientists, and other non-biological science disciplines.<br />
&nbsp;For example, this<br />
may<br />
take the form of <span class="SpellE">modeling</span><br />
a biological<br />
system using software like <span class="SpellE">Matlab</span>,<br />
designing a microfluidic device with biological applications or<br />
designing an electrical device which interfaces with a biological<br />
system. If<br />
you have an interest in interdisciplinary research between your field<br />
and biology,<br />
it is likely iGEM will be able to accommodate it.<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpLast"<br />
style="text-indent: -18pt;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->The<br />
scope of iGEM also extends into<br />
non-traditional science areas, including biotechnology<br />
entrepreneurship,<br />
biotechnology ethics and the law, and science outreach, and so we are<br />
actively<br />
seeking students with business, law, design, and arts backgrounds. You<br />
could,<br />
for example, create a business plan for <span class="GramE">a</span><br />
iGEM-created<br />
company, examine bioethics within synthetic biology, or even explore<br />
the realm&nbsp; of biologically-inspired art.<o:p></o:p></p><br />
<p class="MsoNormal"><o:p>&nbsp;</o:p></p><br />
<p class="MsoNormal">There are several traits needed on<br />
the iGEM team:<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpFirst"<br />
style="text-indent: -18pt;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]--><span<br />
class="GramE">Research skills. IGEM</span> is an<br />
exciting<br />
opportunity to undertake self-directed research in synthetic biology.<br />
We will<br />
therefore need students who are keenly interested and adept in<br />
research. To<br />
participate, you will need to have the capacity to quickly get up to<br />
speed in<br />
the field of biotechnology and to eventually excel in a lab with<br />
limited<br />
instruction.<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpMiddle"<br />
style="text-indent: -18pt;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->We’re<br />
looking for team members from a range of<br />
backgrounds. iGEM is about cross-disciplinary research, so in addition<br />
to<br />
biological and biomedical science students, we welcome students from<br />
engineering (electrical, mechanical, chemical, software etc.), computer<br />
science, maths, physics, chemistry, and other physical sciences. Also,<br />
students<br />
from non-science backgrounds are very welcome to get in touch to<br />
explore how they<br />
can contribute to the team.<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpLast"<br />
style="text-indent: -18pt;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->Participation<br />
would be most suited to students<br />
with a high level of academic maturity. Typically, this includes third<br />
year,<br />
honours, or Masters <span class="GramE">students</span>,<br />
<span class="SpellE">but students</span><br />
from all levels who can demonstrate an aptitude for research or<br />
leadership are welcome<br />
to apply.<o:p></o:p></p><br />
<p class="MsoNormal">A biological science background is<br />
helpful, but not<br />
required. Team members will need to use knowledge from second-level<br />
biology subjects.<br />
However, we have had team members without a biological science<br />
background who<br />
have excelled. <o:p></o:p></p><br />
<h1>Recruitment</h1><br />
<p class="MsoNormal">If you are interested in being a part<br />
of iGEM, please email<br />
Sean Lowe at MelbourneUniIGem@gmail.com for more information. <o:p></o:p></p><br />
<p class="MsoNormal">Further information about iGEM and<br />
updates about the<br />
recruitment process will also be made available at <a<br />
href="https://www.facebook.com/MelbourneUniIGem">https://www.facebook.com/MelbourneUniIGem</a>.<o:p></o:p></p><br />
<br><br />
<h1>Further information</h1><br />
<p class="MsoNormal">Learn more about iGEM in<br />
general at: <a<br />
href="http://www.facebook.com/l.php?u=http%3A%2F%2Figem.org%2FAbout&amp;h=HAQEjYmHL&amp;s=1"<br />
target="_blank">https://igem.org/About</a><br<br />
style=""><br />
</p><br />
<!--[if !supportLineBreakNewLine]--><br style=""><br />
<!--[endif]--><o:p></o:p><br />
<p class="MsoNormal">Learn about this year’s team by<br />
clicking on the links above.<o:p></o:p></p><br />
<p class="MsoNormal"><br><br />
<span class="textexposedshow">Also browse the previous<br />
Melbourne <span class="SpellE">Uni</span> iGEM team<br />
at<span class="GramE">:</span></span><br><br />
<a href="http://openwetware.org/wiki/IGEM:Melbourne/2008"<br />
target="_blank">http://openwetware.org/wiki/IGEM:Melbourne/2008</a><br><br />
<a<br />
href="http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-melbourne-university-te"<br />
target="_blank">http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-<span<br />
class="SpellE">melbourne</span>-university-<span<br />
class="SpellE">te</span></a><span<br />
class="textexposedshow"><o:p></o:p></span></p><br />
<!-- END EDIT HERE HERE --><br />
</body><br />
</html></div>Slowehttp://2014.igem.org/Team:Melbourne/ProjectTeam:Melbourne/Project2014-12-11T13:47:25Z<p>Slowe: </p>
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style="color: rgb(0, 0, 0);">Sponsors</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Attributions"<br />
style="color: rgb(0, 0, 0);">Attributions</a></td><br />
<td align="right"> <a<br />
href="https://2014.igem.org/Main_Page"> <img<br />
src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png"<br />
width="46"></a> </td><br />
</tr><br />
</tbody><br />
</table><br />
<h1>Project and results</h1><br />
<p>Read about our experimental work below, or jump to our<br />
theoretical collaboration with the <a href="#Oxford">University<br />
of Oxford</a><br />
</p><br />
<h2>Introduction and theory</h2><br />
<p>Synthetic peptide chemists have long produced peptide-based<br />
materials <em>in vitro</em>. Star-shaped peptides are a<br />
promising type of biomaterial being explored in the field of<br />
nanomedicine (Sulistio et al., 2012). Star peptides can have several<br />
biomedical uses, such as acting as drug delivery vehicles (Sulistio et<br />
al., 2011) or linkers for other biomacromolecules. Star peptides<br />
generally take the form of several linear peptide arms linked together<br />
in a central core. One way of linking these linear peptide arms<br />
together is to used covalent bonds such as those in disulfides.<br />
Typically, disulfide bonds are formed synthetically by taking several<br />
linear arms and treating them with an oxidant <em>in vitro</em>.<br />
Here, we introduce a new approach to forming star peptides using <em>E.<br />
coli</em> and synthetic biology. Thus, we aimed to show how the<br />
peptides synthesis and disulfide bond forming machinery of <em>E.<br />
coli</em> can be used to form disulfide linked star peptides and<br />
key star peptide precursors.</p><br />
<p>&nbsp;</p><br />
<h2>Synthesis approach</h2><br />
<p><em>E. coli</em> naturally possesses the capacity<br />
to form disulfide bonds. In native strains, disulfide bonds are<br />
naturally formed by an array of enzymes which are part of the Dsb<br />
family (e.g. DsbA and DsbC) (Kadokura et al., 2003, Kadokura and<br />
Beckwith, 2009). Normally, these enzymes are found in the oxidizing<br />
periplasm of the cell. However, several new strains of <em>E.<br />
coli</em> have recently been engineered which contain an<br />
oxidizing cytoplasm conducive to disulfide bond formation. One example<br />
of this is the SHuffle cell line (Lobstein et al., 2012). The cell line<br />
contains mutations to key enzymes responsible for the reducing nature<br />
of the cytoplasm, namely thioredoxin reductase (<em>trxB</em>)<br />
and glutathione reductase (<em>gor</em>). Furthermore, the<br />
Shuffle cell line over expresses the disulfide bond isomerase DsbC to<br />
the cytoplasm. Together, these mutations allow SHuffle to fold<br />
disulfide-bonded proteins in the cytoplasm at a higher success rate<br />
compared to non-mutants. We aimed to take advantage of the disulfide<br />
bond forming capabilities of this strain of <em>E. coli</em><br />
to synthesize star peptides in cells. As shown in the figure below, the<br />
synthesis steps may proceed as follows:</p><br />
<p>&nbsp;</p><br />
<ol><br />
<li> Express a short peptide containing two cysteine residues<br />
either to the <em>E. coli</em> periplasm or the cytoplasm<br />
of a <em>trxB gor </em>mutant.</li><br />
<li><em>E. coli</em> disulfide bond forming enzymes<br />
fold the peptide into a hairpin loop structure.<br />
</li><br />
<li>Cut the loop at a protease recognition site engineered into<br />
the peptide. This may be done by extracting the folded peptide from the<br />
cell and treating it with the protease in vitro. Alternatively, the<br />
protease may be co-expressed in the cell to allow for in vivo cleavage.<br />
</li><br />
</ol><br />
<p>&nbsp;</p><br />
<table align="center"><br />
<tbody><br />
<tr><br />
<td align="center"><br />
<img<br />
src="https://static.igem.org/mediawiki/2014/9/9d/Project_concept-v2.png"<br />
alt="" height="600" width="480"></td><br />
</tr><br />
</tbody><br />
</table><br />
<p>&nbsp;</p><br />
<p>This synthesis approach has several benefits over purely <em>in<br />
vitro</em> approaches. Firstly, the exact peptide sequence can be<br />
precisely programmed into <em>E. coli</em> using<br />
recombinant DNA synthesis. Secondly, by performing the disulfide bond<br />
formation in cells and optionally the proteolytic cleavage, several<br />
synthesis steps which would need to be performed <em>in vitro</em><br />
are eliminated. From a scale up perspective, this would eliminate<br />
entire unit operations which would otherwise be required to produce<br />
this product. Given these benefits, in the current study, we aimed to<br />
express a star peptide precursor to the cytoplasm of SHuffle cells,<br />
which would later be extracted and externally digested with the<br />
star-forming protease. In order to achieve this, we first designed<br />
several star peptides which might be amenable to this synthetic<br />
strategy. These peptides are described below.</p><br />
<p>&nbsp;</p><br />
<h2>Rationally designed peptides</h2><br />
<p>There are two approaches to functionalising star peptides. In<br />
the first, the identity of the arms can be chosen to be bioactive<br />
peptide molecules. This is the simplest approach to producing a<br />
biologically-relevant star. In the second, the arms can be<br />
functionalised by ligating molecules to them at any point. We used both<br />
of these approaches to design two separate peptides.</p><br />
<h3>Magainin 1 star and linear peptides</h3><br />
<p>Our first strategy was to make a star peptide using<br />
antimicrobial peptides(AMPs) as building blocks. AMPs are small,<br />
approximately 50 residue peptides secreted by some bacterial and<br />
eukaryotic cells which selectively kill microbial cells. It is thought<br />
that AMPs work by forming pores in the membrane of prokaryotic cells<br />
(Brogden, 2005). AMPs have been recombinantly expressed in a number of<br />
organisms, including <em>E. coli</em> (for a review, see<br />
Li, 2011) and <em>B. Subtilis</em> (Chen et al., 2009, Yu<br />
et al., 2013).</p><br />
<p>Our concept was to design a star peptide with antimicrobial<br />
peptide arms. Wiradharma et al. (2012) first showed that placing linear<br />
antimicrobial peptides in a star configuration could lead to enhanced<br />
antimicrobial activity and decreased hemolytic activity. Although it is<br />
unclear why this is the case, it may be due to the ability of<br />
neighbouring antimicrobial peptide arms to interact with each other to<br />
synergistically rupture the membrane.<br><br />
While Wiradharma used a synthetic peptide sequence, we designed a<br />
peptide using the naturally occurring AMP, Magainin 1 (Zasloff, 1987).<br />
Magainin 1 peptides will be placed to the ends of each star arm.</p><br />
<p>Magainin 1 was chosen because the Magainins are one of the<br />
major classes of antimicrobial peptides, being well studied and<br />
characterised. In addition, we were concerned that tethering the<br />
antimicrobial peptide to the star peptide might interfere with its<br />
antimicrobial activity. Magainin 1, however, has previously been<br />
tethered to surfaces, where it has imparted the surfaces with<br />
microbicidal properties (Glinel et al., 2008; Humblot et al., 2009). We<br />
surmised that if Magainin 1 maintained its activity while anchored to<br />
surfaces, it may also maintain its activity while anchored to a star<br />
peptide.</p><br />
<p>The sequence for Magainin 1 is: GIGKFLHSAGKFGKAFVGEIMKS.</p><br />
<p>When attached to a star peptide it will have the following<br />
structure:</p><br />
<img<br />
src="https://static.igem.org/mediawiki/2014/2/27/MAGAININ_1_peptide.png"<br />
alt="" height="300" width="720"><br />
<p>There are several design elements to note:</p><br />
<ul><br />
<li><br />
The antimicrobial peptide star will be expressed with a SUMO fusion<br />
protein. This is because without the fusion, it is likely that the<br />
peptide would be toxic to the host cell.</li><br />
<li>The peptide includes a Factor X cutting site between the<br />
two cysteines for eventual proteolytic cleavage and formation of the<br />
star peptide.</li><br />
</ul><br />
<p>In addition to the star peptide Magainin 1, we synthesised a<br />
gene for a linear Magainin 1 peptide as well. This is identical to the<br />
construct above, except that there is only one Magainin 1 peptide<br />
attached to the SUMO fusion.</p><br />
<h3>Unstructured Peptide (USP) Construct</h3><br />
<p>In the second approach, we designed a peptide which can be<br />
functionalised using chemical approaches. This peptide was designed to<br />
have flexible, unstructured arms and was termed the USP construct.<br />
Unlike the Magainin 1 star, the arms of this peptide serve not as<br />
active peptides themselves, but as inert structural linkers.</p><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/USPI_Peptide.png"<br />
alt="" height="216" width="720"><br />
<p>The arms were designed with the following elements in mind:</p><br />
<ul><br />
<li><br />
<b>Lack of structure.</b> The arms were designed with a<br />
bioinspired approach, using the FxFG motif of nucleoporins (where x is<br />
a variable amino acid residue). Such segments naturally repeat in<br />
nucleoporins and are thought to lead to disorder/lack of stable<br />
secondary structure. Nucleoporins are found in mammalian cells, serving<br />
as flexible brushes around nuclear pores (Ader et al., 2010).<br />
</li><br />
<li><b>Water-soluble.</b> The arms were designed<br />
with several charged amino acids to improve solubility.<br />
</li><br />
<li><b>Designed to form a disulfide bond.</b><br />
Although it is difficult to rationally ensure that the disulfide bond<br />
will form between two cysteines in our peptide, we incorporated a beta<br />
turn between the two cysteines which may encourage the peptide to fold<br />
at the apex of the hairpin loop. This may bring the cysteines into<br />
closer proximity, providing more probable bond formation.</li><br />
</ul><br />
<p>The ultimate utility of this peptide lies in its ability to be<br />
functionalised with other biomacromolecules. For example, the technique<br />
of Native Chemical Ligation(NCL) can be used to join peptides,<br />
proteins, and other ligands to the arms (Dawson et al., 1994). The idea<br />
of attaching enzymes to the star peptide was explored by the University<br />
of Oxford iGEM 2014 team in a collaborative effort between our two<br />
teams (See Supplementary Project Work at the end of this page). </p><br />
<p>&nbsp;</p><br />
<h2>Expression system</h2><br />
<p>In order to successfully express our constructs, we designed<br />
our protein expression vectors to include a fusion protein. The fusion<br />
protein was necessary for two reasons. First, some of our constructs<br />
are very small (e.g. the non-star Magainin 1), and expression levels of<br />
very small peptides can be difficult without a fusion partner. Second,<br />
two of our constructs code for antimicrobial peptides. Without a fusion<br />
partner, it is likely that these genes would be toxic to their hosts<br />
upon induction.</p><br />
<p>To find a suitable expression system, we looked towards the<br />
Registry of Standard Parts. We used the SUMO protein expression system<br />
designed by TU Delft 2014 (for example, see <a target="_blank"<br />
href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1022101">http://parts.igem.org/wiki/index.php?title=Part:BBa_K1022101</a>).<br />
This system essentially consists of a N-terminal HIS-tag followed by<br />
the SUMO protein (also known as UlpI).</p><br />
<p><br />
The strategy of expressing toxic AMPs using SUMO has been successfully<br />
reported in the literature (Bommarius et al., 2010). We surmise that<br />
the SUMO protein could inhibit the antimicrobial activity of single,<br />
linear peptides, and that it may also inhibit the activity of our star<br />
antimicrobial peptide.</p><br />
<p><br />
SUMO as a fusion protein also has the benefit of leaving no residues at<br />
the C-terminal end of the cleavage site. This means that upon cleavage<br />
with the SUMO protease, the native protein can be recovered. In our<br />
case, this means that one of the arms of the star can be designed<br />
without the need to take into account the addition of any amino acid<br />
residues left behind by the protease.<br><br />
We used the SUMO peptide sequence reported by TU Delft. However, our<br />
construct contained the following unique features:</p><br />
<ol><br />
<li> Standardisation of the biobrick by substituting the T7<br />
promoter and RBS (the origins of which are both not specified in the<br />
Delft documentation) with the standard T7 promoter and RBS BioBrick <a<br />
target="_blank" href="http://parts.igem.org/Part:BBa_K525998">BBa_K525998</a>.<br />
In addition to supporting the principle of standardization, using the<br />
well-characterized promoter BioBrick should help assure expression<br />
levels.<br><br />
</li><br />
<li>Biobrick BBa_K1022101 lacks a terminator sequence (this was<br />
presumably because the part was meant to be integrated into a larger<br />
genetic construct with a terminator). A terminator from the registry of<br />
standard parts was added (specifically, the wild type terminator from<br />
T7 bacteriophage,&nbsp;<a target="_blank"<br />
href="http://parts.igem.org/Part:BBa_K731721">BBa_K731721</a>).<br />
</li><br />
<li>The original biobrick BBa_K1022101 codes for three amino<br />
acid residues before the HIS-tag (ASM), which appeared to be redundant.<br />
Correspondence with the 2013 TU Delft team suggested that these<br />
residues were unnecessary and appear to be cleaved within the cell as<br />
part of the cells post-translational modifications. However, their<br />
presence complicates the addition of additional tags at the N-terminus<br />
of the protein (e.g. periplasmic export tags), and therefore were not<br />
included.<br><br />
</li><br />
<li>The SUMO sequence was codon optimised for E. coli during<br />
synthesis. As the Delft documentation did not specify whether the gene<br />
was codon optimal, codon optomisation was undertaken to potentially<br />
improve expression levels. The linear Magainin 1 construct, however,<br />
was not codon optimised in the SUMO region in order to provide a<br />
control condition.</li><br />
</ol><br />
<p>To summarise, the protein expression devices used in our<br />
project to the following form:</p><br />
<img<br />
src="https://static.igem.org/mediawiki/2014/d/d0/Gene_circuit_export.png"<br />
alt="" height="91" width="720"><br />
<p>The protein coding region consisted of a 6x-HIS tag followed<br />
by the SUMO protein and the relevant star or linear peptide.</p><br />
<p>&nbsp;</p><br />
<h2>Plasmid preparation: Cloning and acquisition of the genetic<br />
constructs</h2><br />
<h3>Magainin 1 Star Peptide</h3><br />
<p>This construct was synthesised in the standard shipping<br />
plasmid from Life Technologies- plasmid pMK. We had originally planned<br />
to express our protein to the periplasm of E. coli, and therefore had<br />
included in the synthesis a periplasmic export tag, the TorTss signal<br />
sequence (Steiner et al., 2006).</p><br />
<p><br />
Cytoplasmic and periplasmic expression may both allow for disulfide<br />
bond formation in the cell. We decided, however, to focus on<br />
cytoplasmic expression. There are distinct advantages to cytoplasmic<br />
expression (e.g. the absence of several periplasmic proteases and<br />
potentially higher expression levels (Baneyx, 1999)).</p><br />
<p><br />
Another reason the cytoplasm was chosen was to allow us to test the<br />
effects of an oxidising versus reducing intracellular environment on<br />
disulfide bond formation. We planned to express the construct in both<br />
SHuffle T7 cells (oxidising cytoplasm) and BL21(DE3) (reducing<br />
cytoplasm) to probe whether there was a difference in disulfide bond<br />
formation. Therefore, we needed to remove the periplasmic export tag<br />
from the gene.</p><br />
<p><br />
To do this, we use the following cloning strategy (noting that the top<br />
row represents the gene initially synthesised in plasmid pMK):</p><br />
<img<br />
src="https://static.igem.org/mediawiki/2014/2/2c/Melbourne_cloning_strategy_diagram.jpg"<br />
alt="" height="437" width="900"><br />
<p>The gene was inserted into plasmid pSB1C3 containing the T7<br />
promoter and ribosome binding site, BBa_K525998. It was inserted after<br />
the promoter and before the BioBrick suffix. This was accomplished by<br />
digesting the destination vector with SpeI and PstI. At the same time,<br />
PCR was used to amplify the segment of the gene containing the SUMO<br />
fusion and the Magainin 1 Star peptide, adding XbaI and keeping the<br />
PstI site existing in the gene.</p><br />
<p><br />
Digestion of the PCR product then allowed for ligation of the insert<br />
and destination vector using the PstI sites and XbaI and SpeI (XbaI and<br />
SpeI have compatible sticky ends). Note that after the ligation, there<br />
will be a scar in the gene where the XbaI and the SpeI sites were<br />
ligated.</p><br />
<p><br />
The ligation appeared to be successful. The ligation mixture was<br />
transformed to DH5α competent cells and plated onto<br />
chloramphenicol-containing agar plates.</p><br />
<p><br />
Plasmid from 5 colonies (C1, C2, C3, C4, and C5) was cultured,<br />
extracted, and then digested with EcoRI and PstI.</p><br />
<p><br />
As evident in the figure below, at least 4 of the colonies appeared to<br />
contain the insert. The empty, linearised pSB1C3 backbone ran at<br />
approximately the correct size (2070 bps), as did the insert (740 bps).</p><br />
<center><img<br />
src="https://static.igem.org/mediawiki/2014/9/95/Magainin_1_subcloning.png"<br />
alt="" height="400" width="353"></center><br />
<p>N.B. MW marker is the 100 bp Ladder from Axygen. * indicates<br />
the colony picked for sequencing and eventual transformation to the<br />
expression cell lines.</p><br />
<p>The DNA from Colony C2 was confirmed using Sanger sequencing<br />
at the Australian Genome Research Facility. This DNA appears in the<br />
registry of standard parts as BioBrick <a target="_blank"<br />
href="http://parts.igem.org/Part:BBa_K1394000">BBa_K1394000</a>.</p><br />
<p><br />
For the USP peptide and the linear Magainin 1 peptide, the genes were<br />
synthesised by GenScript and delivered in pSB1C3. The expression<br />
vectors had identical gene regulatory elements to that used for the<br />
Magainin 1 Star peptide. They only differed in the codon optimisation<br />
used, and they also lacked the assembly scar described above.</p><br />
<h2><br />
Protein expression and characterisation<br />
</h2><br />
<p><br />
After acquiring our genes, the project moved to protein expression and<br />
purification. We expressed both star peptides (Magainin 1 Star Peptide<br />
and the USP peptide) in both the SHuffle T7 and BL21(DE3) cell lines,<br />
both sourced from New England Biolabs. As the Linear Magainin 1 peptide<br />
does not have any special disulfide bonding<br />
requirements, we expressed it in BL21(DE3).<br />
</p><br />
<p><br />
The plasmids were transformed to the expression cells, and a single<br />
colony was cultured and induced overnight at 17 °C. A whole cell sample<br />
both before<br />
IPTG induction (-IPTG) and after the induction period (+IPTG) were<br />
boiled in SDS-PAGE sample buffer and loaded on a 15% tris-glycine gel.<br />
The whole cell<br />
Coomassie stained gel is shown below alongside the NEB P7712S molecular<br />
weight marker.<br />
</p><br />
<br><br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/2/24/Whole_cell_CS.png"<br />
height="330" width="439"></p><br />
<br><br />
<p><br />
From theoretical prediction of the peptide masses, we would expect the<br />
following distribution of molecular weights:<br />
</p><br />
<br><br />
<table align="center" border="1" cellpadding="0"<br />
cellspacing="0"><br />
<tbody><br />
<tr><br />
<td width="156"><br />
<p align="center">Magainin 1 Star Peptide (Mag1<br />
Star)<br />
</p><br />
</td><br />
<td width="145"><br />
<p align="center">USP<br />
</p><br />
</td><br />
<td width="156"><br />
<p align="center">Linear Magainin 1 (Linear Mag1)<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="156"><br />
<p align="center">22.15 kDa<br />
</p><br />
</td><br />
<td width="145"><br />
<p align="center">29.3 kDa<br />
</p><br />
</td><br />
<td width="156"><br />
<p align="center">14.32 kDa<br />
</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<br><br />
<p><br />
We looked for bands corresponding to overexpression of the proteins. We<br />
observed a thick band, post IPTG around 32 kDa in the BL21(DE3) cells<br />
carrying the<br />
USP plasmid. However, we saw this same band appearing in the Linear<br />
Magainin 1 lane but the Linear Magainin 1 protein should have a much<br />
lower molecular<br />
weight than what was observed. Therefore, we did not assume that we had<br />
overexpression overexpression of the USP 1 protein.<br />
</p><br />
<p><br />
We also noted bands in all post-IPTG lanes around 17 kDa. This would be<br />
roughly consistent with both the Linear Magainin 1 and Magainin 1 Star<br />
Peptide,<br />
noting that small proteins may not run at their expected molecular<br />
weight. Again, the analysis is complicated by the fact that the band<br />
also appears in the<br />
lane corresponding to the larger molecular weight protein, USP I. Given<br />
this ambiguity, we decided to purify all the protein in the sample<br />
using Ni-NTA<br />
purification.<br />
</p><br />
<h3><br />
Purification and further characterisation<br />
</h3><br />
<p><br />
A small-scale purification was carried out according to our <a<br />
href="https://static.igem.org/mediawiki/2014/e/e1/MELBOURNE_D1V1_Column_Purification_of_His-Tagged_Proteins.pdf">protocol</a>.<br />
The cells were lysed with iodoacetamide, an alkylating agent, added to<br />
the lysis buffer. Iodoacetamide blocks all free cysteines on proteins<br />
with a short alkyl group. This was added<br />
because we wanted to determine if a disulphide bond had formed inside<br />
the cell. If we did not block free cysteines, then any bond that had<br />
formed could be<br />
attributed to spontaneous/air oxidation of disulfides outside the cell.<br />
</p><br />
<p><br />
After purification, we ran a concurrent Western Blot and Coomassie<br />
stain on both the pre-induction samples from above and the purified<br />
protein (namely, the<br />
first elution from the batch purification).<br />
</p><br />
<p><br />
The Western Blot used mouse monoclonal antibodies against the<br />
N-terminal HIS-tag as primary antibodies and anti-mouse secondary<br />
antibodies:<br />
</p><br />
<p align="center"><br />
<img<br />
src="https://static.igem.org/mediawiki/2014/5/54/Western_Blot_Melb.png"<br />
border="0" height="400" width="460"></p><br />
<p><br />
The corresponding Coomassie stain is show below:<br />
</p><br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f0/Purified_CS.png"<br />
border="0" height="365" width="486"></p><br />
<p><br />
There are several interesting aspects of the Western Blot. Firstly,<br />
there was a strong band between 25 and 32 kDa in most lanes. However,<br />
it<br />
appears in all of the pre-induction controls, suggesting non-specific<br />
binding of the anti-HIS antibodies.<br />
</p><br />
<p><br />
Secondly, we observed a band in most of the lanes around 17 kDa (with<br />
the exception being the USP protein in SHuffle cells). As these bands<br />
were not<br />
present in the pre-induction controls, we suspected they were a result<br />
of the induction.<br />
</p><br />
<h3><br />
Mass spectrometry<br />
</h3><br />
<p><br />
In order to assess the identity of the bands, we performed an in-gel<br />
tryptic digestion on select bands. We digested bands in the Coomassie<br />
stain which<br />
corresponded to the HIS-tagged bands in the Western Blot. This was<br />
followed by mass spec analysis (LC MS/MS). We focused our analysis on<br />
the Magainin 1<br />
Star Peptide bands and the Linear Magainin 1 band. Our USP peptide was,<br />
by design, highly rich in basic residues, greatly reducing the<br />
likelihood that<br />
tryptic fragments would be detected by the mass spec.<br />
</p><br />
<p><br />
We found that the Linear Magainin 1 peptide appeared to be present in<br />
the approximately 17 kDa band, with the following detected tryptic<br />
fragments in the<br />
sequence below (bold; basic residues underlined):<br />
</p><br />
<p><br />
<img<br />
src="https://static.igem.org/mediawiki/2014/1/1b/Tryptic_digest_linear_mag1.PNG"<br />
border="0" height="63" width="601"></p><br />
<p><br />
Note that spaces have been added in the sequence to emphasise distinct<br />
domains in the protein (in this case, the fusion protein versus the<br />
Magainin 1<br />
peptide). Together with the fact that the protein runs close to the<br />
expected molecular weight, this seems to provide good evidence that the<br />
protein is<br />
being expressed.<br />
</p><br />
<p><br />
We then examined the bands corresponding to the Magainin 1 Star<br />
Peptide.<br />
</p><br />
<p><br />
Again, we digested bands in the Coomassie stained gel corresponding to<br />
the prominent HIS-tagged bands on the <em>Western Blot</em><br />
(near 17 kDa).<br />
</p><br />
<p><br />
The mass spec coverage for the Magainin 1 Star Peptide expressed in<br />
SHuffle cells was as follows:<br />
</p><br />
<p><br />
<img<br />
src="https://static.igem.org/mediawiki/2014/6/63/Tryptic_digest_Sample_A_Star_Mag_Shuffle.PNG"<br />
border="0" height="90" width="601"></p><br />
<p><br />
The coverage for the same peptide expressed in BL21(DE3) was found to<br />
be:<br />
</p><br />
<p><br />
<img<br />
src="https://static.igem.org/mediawiki/2014/d/db/Tryptic_digest_Sample_A_Star_Mag_BL21%28DE3%29.PNG"<br />
border="0" height="85" width="602"></p><br />
<p><br />
Again, we can see that tryptic peptides from the expressed protein<br />
appear to present in the gel band under analysis.<br />
</p><br />
<p><br />
There is a relatively lower sequence coverage. This could be a<br />
limitation of our procedure: for example, improper destaining during<br />
the in gel digestion<br />
procedure can inhibit tryptic digestion.<br />
</p><br />
<p><br />
However, even if the digestion was complete, the mass spec will only<br />
detect fragments which ionize well. The Magainin 1 Star peptide<br />
consists of four<br />
repeats of the Magainin 1 sequence (corresponding to the arms of the<br />
star). If the two tryptic fragments within Magainin 1 do not ionize<br />
well, then indeed<br />
the entire peptide would not be read.<br />
</p><br />
<p><br />
Finally, it is possible that the protein has been cleaved or degraded.<br />
This would account for the slightly lower-than-expected mass on the SDS<br />
page gel.<br />
</p><br />
<p><br />
Nonetheless, it is a possibility that the peptide fragments are present<br />
but not detected. Replication of the in-gel mass spec would be useful<br />
in clarifying this point, or provided the purity of the sample is<br />
acceptable, it may be possible to run an intact mass spec. Digestion<br />
with alternative enzymes may also provide a more robust sequence<br />
coverage, particularly for the USP peptide.<br />
</p><br />
<h2><br />
Conclusions<br />
</h2><br />
<p><br />
We designed several novel star peptides based on several rational<br />
design criteria. Further, we began the optimisation process required to<br />
express these peptides in <em>E. coli</em>.<br />
</p><br />
<p><br />
To this end, we constructed an expression system based on the SUMO<br />
protein and standard BioBrick parts. The function of this system was<br />
confirmed, as it<br />
appears it does produce HIS-tagged SUMO fusion protein in <em>E.<br />
coli</em>, as expected. Nonetheless, we hope to continue to<br />
characterise our parts, likely through further purification and<br />
measurement of the mass. </p><br />
<p>Given the success of SUMO in the protein expression community,<br />
we hope our BioBrick<br />
will encourage further use of the fusion domain within iGEM. In<br />
addition, we hope that our ideas and concepts will inspire future<br />
iGEM efforts to explore the applications of synthetic biology for<br />
material science. We continue to believe that bacteria have great<br />
promise for the <em>in vivo</em><br />
construction of unique biomaterials, and we look forward to seeing how<br />
the synthetic biology community will develop in this area.<br />
</p><br />
<h2>References</h2><br />
<p>ADER, C., FREY, S., MAAS, W., SCHMIDT, H. B., GÖRLICH, D.<br />
&amp; BALDUS, M. 2010. Amyloid-like interactions within nucleoporin<br />
FG hydrogels. <em>Proceedings of the National Academy of<br />
Sciences,</em> 107<strong>,</strong> 6281-6285.</p><br />
<p><br />
BANEYX, F. 1999. Recombinant protein expression in Escherichia coli. <em>Current<br />
Opinion in Biotechnology,</em> 10<strong>,</strong><br />
411-421.</p><br />
<p><br />
BOMMARIUS, B., JENSSEN, H., ELLIOTT, M., KINDRACHUK, J., PASUPULETI,<br />
M., GIEREN, H., JAEGER, K. E., HANCOCK, R. E. W. &amp; KALMAN, D.<br />
2010. Cost-effective expression and purification of antimicrobial and<br />
host defense peptides in Escherichia coli. <em>Peptides,</em><br />
31<strong>,</strong> 1957-1965.</p><br />
<p><br />
BROGDEN, K. A. 2005. Antimicrobial peptides: Pore formers or metabolic<br />
inhibitors in bacteria? <em>Nature Reviews Microbiology,</em><br />
3<strong>,</strong> 238-250.</p><br />
<p><br />
CHANG, C., CHHOR, G., CLANCY, S. &amp; JOACHIMIAK, A. 2014. Crystal<br />
Structure of Glutathione S-transferase Domain Protein From Haliangium<br />
Ochraceum DSM 14365 [Online]. ''NCBI. Available:<br />
http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=4w66'<br />
[Accessed September 9 2014].</p><br />
<p><br />
CHEN, X., ZHU, F., CAO, Y. &amp; QIAO, S. 2009. Novel expression<br />
vector for secretion of cecropin AD in Bacillus subtilis with enhanced<br />
antimicrobial activity. <em>Antimicrobial agents and<br />
chemotherapy,</em> 53<strong>,</strong> 3683-3689.</p><br />
<p><br />
GLINEL, K., JONAS, A. M., JOUENNE, T., LEPRINCE, J., GALAS, L.<br />
&amp; HUCK, W. T. 2008. Antibacterial and antifouling polymer<br />
brushes incorporating antimicrobial peptide. <em>Bioconjug Chem,</em><br />
20<strong>,</strong> 71-77.</p><br />
<p><br />
HUMBLOT, V., YALA, J.-F., THEBAULT, P., BOUKERMA, K., HÉQUET, A.,<br />
BERJEAUD, J.-M. &amp; PRADIER, C.-M. 2009. The antibacterial<br />
activity of Magainin I immobilized onto mixed thiols self-assembled<br />
monolayers. <em>Biomaterials,</em> 30<strong>,</strong><br />
3503-3512.</p><br />
<p><br />
DAWSON, P. E., MUIR, T. W., CLARK-LEWIS, I. &amp; KENT, S. 1994.<br />
Synthesis of proteins by native chemical ligation. <em>Science,</em><br />
266<strong>,</strong> 776-779.</p><br />
<p><br />
KADOKURA, H. &amp; BECKWITH, J. 2009. Detecting folding<br />
intermediates of a protein as it passes through the bacterial<br />
translocation channel. <em>Cell,</em> 138<strong>,</strong><br />
1164-1173.</p><br />
<p><br />
KADOKURA, H., KATZEN, F. &amp; BECKWITH, J. 2003. Protein disulfide<br />
bond formation in prokaryotes. <em>Annual review of biochemistry,</em><br />
72<strong>,</strong> 111-135.</p><br />
<p><br />
LI, Y. 2011. Recombinant production of antimicrobial peptides<br />
in&lt; i&gt; Escherichia coli&lt;/i&gt;: A review. <em>Protein<br />
Expression and Purification,</em> 80<strong>,</strong><br />
260-267.</p><br />
<p><br />
LOBSTEIN, J., EMRICH, C. A., JEANS, C., FAULKNER, M., RIGGS, P.<br />
&amp; BERKMEN, M. 2012. SHuffle, a novel Escherichia coli protein<br />
expression strain capable of correctly folding disulfide bonded<br />
proteins in its cytoplasm. <em>Microb Cell Fact,</em> 11<strong>,</strong><br />
56-56.</p><br />
<p><br />
PROTEIN MODEL PORTAL. Query result, GST and DcmA [Online]. Available:<br />
http://www.proteinmodelportal.org/query/uniprot/P21161 [Accessed<br />
September 9 2014].</p><br />
<p><br />
STEINER, D., FORRER, P., STUMPP, M. T. &amp; PLUCKTHUN, A. 2006.<br />
Signal sequences directing cotranslational translocation expand the<br />
range of proteins amenable to phage display. <em>Nat Biotech,</em><br />
24<strong>,</strong> 823-831.</p><br />
<p><br />
SULISTIO, A., GURR, P. A., BLENCOWE, A. &amp; QIAO, G. G. 2012.<br />
Peptide-Based Star Polymers: The Rising Star in Functional Polymers. <em>Australian<br />
Journal of Chemistry,</em> 65<strong>,</strong><br />
978-984.</p><br />
<p><br />
SULISTIO, A., LOWENTHAL, J., BLENCOWE, A., BONGIOVANNI, M. N., ONG, L.,<br />
GRAS, S. L., ZHANG, X. &amp; QIAO, G. G. 2011. Folic Acid<br />
Conjugated Amino Acid-Based Star Polymers for Active Targeting of<br />
Cancer Cells. <em>Biomacromolecules,</em> 12<strong>,</strong><br />
3469-3477.</p><br />
<p><br />
TANAKA, N., KUSAKABE, Y., ITO, K., YOSHIMOTO, T. &amp; NAKAMURA, K.<br />
T. 2002. Crystal Structure of Formaldehyde Dehydrogenase from&lt;<br />
i&gt; Pseudomonas putida: the Structural Origin of the Tightly<br />
Bound Cofactor in Nicotinoprotein Dehydrogenases. Journal of molecular<br />
biology, 324, 519-533.</p><br />
<p><br />
WIRADHARMA, N., LIU, S. Q. &amp; YANG, Y. Y. 2012. Branched and<br />
4-Arm Starlike α-Helical Peptide Structures with Enhanced Antimicrobial<br />
Potency and Selectivity. <em>Small,</em> 8<strong>,</strong><br />
362-366.</p><br />
<p><br />
YU, Z., WANG, Q., MA, Q. &amp; ZHANG, R. 2013. Secretory expression<br />
of lacticin Q fused with SUMO in Bacillus subtilis. <em>Protein<br />
Expression and Purification,</em> 89<strong>,</strong><br />
51-55.</p><br />
<p><br />
ZASLOFF, M. 1987. Magainins, a class of antimicrobial peptides from<br />
Xenopus skin: isolation, characterization of two active forms, and<br />
partial cDNA sequence of a precursor. <em>Proceedings of the<br />
National Academy of Sciences,</em> 84<strong>,</strong><br />
5449-5453.</p><br />
<p>&nbsp;</p><br />
<a name="Oxford"></a><br />
<h1>Collaboration with Oxford</h1><br />
<p>To strengthen the sense of iGEM community, we contacted <a<br />
target="_blank" href="https://2014.igem.org/Team:Oxford">University<br />
of Oxford iGEM team</a> to discuss the possibility of<br />
collaboration between our teams. The Oxford project aims to develop a<br />
system that can dispose of the carcinogenic, hazardous solvent<br />
dichloromethane (DCM). To do this, Oxford team has proposed the use of<br />
the DcmA enzyme. This enzyme degrades DCM to form a toxic intermediate<br />
which in turn is converted by another enzyme, FdhA, into a neutral<br />
molecule. Schematically this can be presented in the following way:</p><br />
<p><br />
Reaction 1: DCM +&nbsp;DcmA -&gt; <strong>toxic<br />
intermediate</strong><br><br />
Reaction 2: <strong>toxic intermediate</strong> + FdhA<br />
-&gt; neutral product.</p><br />
<p>The problem for their system lies in bringing the two enzymes<br />
together to ensure efficient reaction kinetics. An idea that we<br />
discussed with Oxford was to use the star peptide platform to link the<br />
two enzymes together, in a structure similar to the following: </p><br />
<img<br />
src="https://static.igem.org/mediawiki/2014/2/2e/Melbourne_Collab_1.png"<br />
alt="" height="325" width="500"><br />
<p>We worked with Oxford to study this system from a theoretical<br />
standpoint. Our team studied the 3D structure of both enzymes involved<br />
to confirm that the enzyme could in theory be attached to a linker in<br />
this manner, while Oxford team did stochastic modelling to determine<br />
how reaction rate changes when the linker length, labeled D on the<br />
diagram, changes. </p><br />
<p><br />
As a first check, it was necessary to determine whether anchoring these<br />
enzymes to our star would be sterically possible. We studied the<br />
structures of FdhA and DcmA determined by crystallography using data<br />
from the protein data bank. As no crystallographic data was available<br />
in the databank for DcmA, GST was examined as a proxy, as GST is<br />
structurally homologous to DcmA. </p><br />
<p><br />
The crystalographically resolved structure of FdhA enzyme (Tanaka et<br />
al., 2002) from the protein data bank revealed the following image:</p><br />
<img<br />
src="https://static.igem.org/mediawiki/2014/d/dc/Melbourne_Collab_2.png"<br />
alt="" height="355" width="700"><br />
<p>The crystalographically resolved structure of GST was as<br />
follows (Chang et al., 2014):</p><br />
<img<br />
src="https://static.igem.org/mediawiki/2014/e/e9/Melbourne_Collab_3.png"<br />
alt="" height="361" width="800"><br />
<p>It can be seen that the amino- and carboxy-terminals are<br />
located away from the active site. This suggests that both enzymes,<br />
DcmA and FdhA, might be linked together via linkers that are used in<br />
our star peptide. That is, the active site will not be sterically<br />
hindered by attachment to the star peptide. That said, it is difficult<br />
to predict how anchoring the protein to the star will affect its<br />
tertiary structure. Further, it is difficult to predict how limiting<br />
the rotational degrees of freedom will affect enzyme activity.</p><br />
<p>The Oxford team modeled this system and found that, indeed,<br />
the star peptide as an enzyme linker was favourable to enzyme kinetics.<br />
Their work can be found <a<br />
href="https://2014.igem.org/Team:Oxford/alternatives_to_microcompartments#show2"><br />
here</a>.<br />
</p><br />
</body><br />
</html></div>Slowehttp://2014.igem.org/Team:Melbourne/AttributionsTeam:Melbourne/Attributions2014-12-11T13:46:57Z<p>Slowe: </p>
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<img src="https://static.igem.org/mediawiki/2014/9/9b/Melbourne_Banner.png" alt="Banner" height="225" width="1000"><br />
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Home</a> </td><br />
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<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Team"style="color:#000000"><br />
Team</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Project"style="color:#000000"><br />
Project</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
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Human Practices</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Achievements"style="color:#000000"><br />
Achievements</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Notebook"style="color:#000000"><br />
Notebook</a></td><br />
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<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Protocols"style=" color:#000000"> <br />
Protocols</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Safety"style="color:#000000"><br />
Safety</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Sponsors"style="color:#000000"><br />
Sponsors</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Attributions"style="color:#000000"><br />
Attributions</a></td><br />
<br />
<br />
<td align="right"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="46px"></a> </td><br />
</tr><br />
</table><br />
<br />
<br />
<h1 >Attributions</h1><br />
<p>The University of Melbourne iGEM team was responsible for almost all aspects of running the team, from purchasing supplies, coming up with the project concept, writing protocols, and executing the lab work. Help received from advisors and others is greatly appreciated and acknowledged below.</p><br />
<p>&nbsp;</p><br />
<p>Special thanks and attributions go to:</p><br />
<p><h3>Associate Professor Heung-Chin Cheng</h3><br><br />
Allowed us to use his lab space and arranged for training on practical procedures, helping with troubleshooting and theory</p><p>&nbsp;</p><br />
<p><h3>Associate Professor Paul Gooley, Dr Angus Johnston, and Associate Professor Neil O&rsquo; Brien-Simpson</h3><br><br />
Provided advice on our gene construct and helped us with brainstorming</p><p>&nbsp;</p><br />
<p><h3>Daisy Cheng</h3><br><br />
Assisted the team with laboratory techniques and reviewed our protocols </p><p>&nbsp;</p><br />
<p><h3>Students of the Cheng Lab, including </h3><h3>Ashfaqul Hoque, Sze-ting Bong, Gahana Advani, Ken Ang, George Cao, David Zula, and Ya Chee</h3><br><br />
Provided incredible amount of help in the lab</p><p>&nbsp;</p><br />
<p><h3>Pat Shilling and the Gooley Lab</h3><br><br />
Were a great help in designing experiments and protocols</p><p>&nbsp;</p><br />
<p><h3>George Grigorian</h3><br><br />
Designed the layout of the website</p><p>&nbsp;</p><br />
<p><h3>Luke Thorburn and Nic Roumeliotis</h3><br><br />
Did the amazing videos visible at our website</p><p>&nbsp;</p><br />
<p><h3>Members of the Bio21 Proteomics Facility </h3><br><br />
Ran our samples through mass spectrometers and helped us to interpret the data</p><br />
<p>&nbsp;</p><br />
<br />
<p><h3>Staff of Bio21 administration, including Peter Coles and Helen Varnavas </h3><br><br />
Were extremely helpful in helping us order supplies and administering the team </p><br><br />
<br />
<p><h3>Bharath Kumar from the 2013 TU Delft iGEM team </h3><br><br />
Provided valuable advice about the Delft SUMO expression system.</p><br><br />
<br />
<p><h3>2014 TU Delft iGEM team </h3><br><br />
Supplied us with their UlpI gene.</p><br><br />
<br />
<br />
<p><h3> Bianna Makogon</h3><br><br />
For work on the book "The Adventures of E. Coli"</p><br><br />
<br />
<br />
<p><h3> Melissa Warner</h3><br><br />
For the design of the Melbourne iGEM logo </p><br><br />
<!-- END EDIT HERE HERE --><br />
</html></div>Slowehttp://2014.igem.org/Team:Melbourne/ProjectTeam:Melbourne/Project2014-12-11T13:45:37Z<p>Slowe: </p>
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style="text-transform: uppercase; font-family: 'Lato',sans-serif;"<br />
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<a href="https://2014.igem.org/Team:Melbourne"<br />
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<a href="https://2014.igem.org/Team:Melbourne/Project"<br />
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<a href="https://2014.igem.org/Team:Melbourne/Protocols"<br />
style="color: rgb(0, 0, 0);"> Protocols</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Safety"<br />
style="color: rgb(0, 0, 0);">Safety</a></td><br />
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onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
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<a href="https://2014.igem.org/Team:Melbourne/Attributions"<br />
style="color: rgb(0, 0, 0);">Attributions</a></td><br />
<td align="right"> <a<br />
href="https://2014.igem.org/Main_Page"> <img<br />
src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png"<br />
width="46"></a> </td><br />
</tr><br />
</tbody><br />
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<h1>Project and results</h1><br />
<p>Read about our experimental work below, or jump to our<br />
theoretical collaboration with the <a href="#Oxford">University<br />
of Oxford</a><br />
</p><br />
<h2>Introduction and theory</h2><br />
<p>Synthetic peptide chemists have long produced peptide-based<br />
materials <em>in vitro</em>. Star-shaped peptides are a<br />
promising type of biomaterial being explored in the field of<br />
nanomedicine (Sulistio et al., 2012). Star peptides can have several<br />
biomedical uses, such as acting as drug delivery vehicles (Sulistio et<br />
al., 2011) or linkers for other biomacromolecules. Star peptides<br />
generally take the form of several linear peptide arms linked together<br />
in a central core. One way of linking these linear peptide arms<br />
together is to used covalent bonds such as those in disulfides.<br />
Typically, disulfide bonds are formed synthetically by taking several<br />
linear arms and treating them with an oxidant <em>in vitro</em>.<br />
Here, we introduce a new approach to forming star peptides using <em>E.<br />
coli</em> and synthetic biology. Thus, we aimed to show how the<br />
peptides synthesis and disulfide bond forming machinery of <em>E.<br />
coli</em> can be used to form disulfide linked star peptides and<br />
key star peptide precursors.</p><br />
<p>&nbsp;</p><br />
<h2>Synthesis approach</h2><br />
<p><em>E. coli</em> naturally possesses the capacity<br />
to form disulfide bonds. In native strains, disulfide bonds are<br />
naturally formed by an array of enzymes which are part of the Dsb<br />
family (e.g. DsbA and DsbC) (Kadokura et al., 2003, Kadokura and<br />
Beckwith, 2009). Normally, these enzymes are found in the oxidizing<br />
periplasm of the cell. However, several new strains of <em>E.<br />
coli</em> have recently been engineered which contain an<br />
oxidizing cytoplasm conducive to disulfide bond formation. One example<br />
of this is the SHuffle cell line (Lobstein et al., 2012). The cell line<br />
contains mutations to key enzymes responsible for the reducing nature<br />
of the cytoplasm, namely thioredoxin reductase (<em>trxB</em>)<br />
and glutathione reductase (<em>gor</em>). Furthermore, the<br />
Shuffle cell line over expresses the disulfide bond isomerase DsbC to<br />
the cytoplasm. Together, these mutations allow SHuffle to fold<br />
disulfide-bonded proteins in the cytoplasm at a higher success rate<br />
compared to non-mutants. We aimed to take advantage of the disulfide<br />
bond forming capabilities of this strain of <em>E. coli</em><br />
to synthesize star peptides in cells. As shown in the figure below, the<br />
synthesis steps may proceed as follows:</p><br />
<p>&nbsp;</p><br />
<ol><br />
<li> Express a short peptide containing two cysteine residues<br />
either to the <em>E. coli</em> periplasm or the cytoplasm<br />
of a <em>trxB gor </em>mutant.</li><br />
<li><em>E. coli</em> disulfide bond forming enzymes<br />
fold the peptide into a hairpin loop structure.<br />
</li><br />
<li>Cut the loop at a protease recognition site engineered into<br />
the peptide. This may be done by extracting the folded peptide from the<br />
cell and treating it with the protease in vitro. Alternatively, the<br />
protease may be co-expressed in the cell to allow for in vivo cleavage.<br />
</li><br />
</ol><br />
<p>&nbsp;</p><br />
<table align="center"><br />
<tbody><br />
<tr><br />
<td align="center"><br />
<img<br />
src="https://static.igem.org/mediawiki/2014/9/9d/Project_concept-v2.png"<br />
alt="" height="600" width="480"></td><br />
</tr><br />
</tbody><br />
</table><br />
<p>&nbsp;</p><br />
<p>This synthesis approach has several benefits over purely <em>in<br />
vitro</em> approaches. Firstly, the exact peptide sequence can be<br />
precisely programmed into <em>E. coli</em> using<br />
recombinant DNA synthesis. Secondly, by performing the disulfide bond<br />
formation in cells and optionally the proteolytic cleavage, several<br />
synthesis steps which would need to be performed <em>in vitro</em><br />
are eliminated. From a scale up perspective, this would eliminate<br />
entire unit operations which would otherwise be required to produce<br />
this product. Given these benefits, in the current study, we aimed to<br />
express a star peptide precursor to the cytoplasm of SHuffle cells,<br />
which would later be extracted and externally digested with the<br />
star-forming protease. In order to achieve this, we first designed<br />
several star peptides which might be amenable to this synthetic<br />
strategy. These peptides are described below.</p><br />
<p>&nbsp;</p><br />
<h2>Rationally designed peptides</h2><br />
<p>There are two approaches to functionalising star peptides. In<br />
the first, the identity of the arms can be chosen to be bioactive<br />
peptide molecules. This is the simplest approach to producing a<br />
biologically-relevant star. In the second, the arms can be<br />
functionalised by ligating molecules to them at any point. We used both<br />
of these approaches to design two separate peptides.</p><br />
<h3>Magainin 1 star and linear peptides</h3><br />
<p>Our first strategy was to make a star peptide using<br />
antimicrobial peptides(AMPs) as building blocks. AMPs are small,<br />
approximately 50 residue peptides secreted by some bacterial and<br />
eukaryotic cells which selectively kill microbial cells. It is thought<br />
that AMPs work by forming pores in the membrane of prokaryotic cells<br />
(Brogden, 2005). AMPs have been recombinantly expressed in a number of<br />
organisms, including <em>E. coli</em> (for a review, see<br />
Li, 2011) and <em>B. Subtilis</em> (Chen et al., 2009, Yu<br />
et al., 2013).</p><br />
<p>Our concept was to design a star peptide with antimicrobial<br />
peptide arms. Wiradharma et al. (2012) first showed that placing linear<br />
antimicrobial peptides in a star configuration could lead to enhanced<br />
antimicrobial activity and decreased hemolytic activity. Although it is<br />
unclear why this is the case, it may be due to the ability of<br />
neighbouring antimicrobial peptide arms to interact with each other to<br />
synergistically rupture the membrane.<br><br />
While Wiradharma used a synthetic peptide sequence, we designed a<br />
peptide using the naturally occurring AMP, Magainin 1 (Zasloff, 1987).<br />
Magainin 1 peptides will be placed to the ends of each star arm.</p><br />
<p>Magainin 1 was chosen because the Magainins are one of the<br />
major classes of antimicrobial peptides, being well studied and<br />
characterised. In addition, we were concerned that tethering the<br />
antimicrobial peptide to the star peptide might interfere with its<br />
antimicrobial activity. Magainin 1, however, has previously been<br />
tethered to surfaces, where it has imparted the surfaces with<br />
microbicidal properties (Glinel et al., 2008; Humblot et al., 2009). We<br />
surmised that if Magainin 1 maintained its activity while anchored to<br />
surfaces, it may also maintain its activity while anchored to a star<br />
peptide.</p><br />
<p>The sequence for Magainin 1 is: GIGKFLHSAGKFGKAFVGEIMKS.</p><br />
<p>When attached to a star peptide it will have the following<br />
structure:</p><br />
<img<br />
src="https://static.igem.org/mediawiki/2014/2/27/MAGAININ_1_peptide.png"<br />
alt="" height="300" width="720"><br />
<p>There are several design elements to note:</p><br />
<ul><br />
<li><br />
The antimicrobial peptide star will be expressed with a SUMO fusion<br />
protein. This is because without the fusion, it is likely that the<br />
peptide would be toxic to the host cell.</li><br />
<li>The peptide includes a Factor X cutting site between the<br />
two cysteines for eventual proteolytic cleavage and formation of the<br />
star peptide.</li><br />
</ul><br />
<p>In addition to the star peptide Magainin 1, we synthesised a<br />
gene for a linear Magainin 1 peptide as well. This is identical to the<br />
construct above, except that there is only one Magainin 1 peptide<br />
attached to the SUMO fusion.</p><br />
<h3>Unstructured Peptide (USP) Construct</h3><br />
<p>In the second approach, we designed a peptide which can be<br />
functionalised using chemical approaches. This peptide was designed to<br />
have flexible, unstructured arms and was termed the USP construct.<br />
Unlike the Magainin 1 star, the arms of this peptide serve not as<br />
active peptides themselves, but as inert structural linkers.</p><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/USPI_Peptide.png"<br />
alt="" height="216" width="720"><br />
<p>The arms were designed with the following elements in mind:</p><br />
<ul><br />
<li><br />
<b>Lack of structure.</b> The arms were designed with a<br />
bioinspired approach, using the FxFG motif of nucleoporins (where x is<br />
a variable amino acid residue). Such segments naturally repeat in<br />
nucleoporins and are thought to lead to disorder/lack of stable<br />
secondary structure. Nucleoporins are found in mammalian cells, serving<br />
as flexible brushes around nuclear pores (Ader et al., 2010).<br />
</li><br />
<li><b>Water-soluble.</b> The arms were designed<br />
with several charged amino acids to improve solubility.<br />
</li><br />
<li><b>Designed to form a disulfide bond.</b><br />
Although it is difficult to rationally ensure that the disulfide bond<br />
will form between two cysteines in our peptide, we incorporated a beta<br />
turn between the two cysteines which may encourage the peptide to fold<br />
at the apex of the hairpin loop. This may bring the cysteines into<br />
closer proximity, providing more probable bond formation.</li><br />
</ul><br />
<p>The ultimate utility of this peptide lies in its ability to be<br />
functionalised with other biomacromolecules. For example, the technique<br />
of Native Chemical Ligation(NCL) can be used to join peptides,<br />
proteins, and other ligands to the arms (Dawson et al., 1994). The idea<br />
of attaching enzymes to the star peptide was explored by the University<br />
of Oxford iGEM 2014 team in a collaborative effort between our two<br />
teams (See Supplementary Project Work at the end of this page). </p><br />
<p>&nbsp;</p><br />
<h2>Expression system</h2><br />
<p>In order to successfully express our constructs, we designed<br />
our protein expression vectors to include a fusion protein. The fusion<br />
protein was necessary for two reasons. First, some of our constructs<br />
are very small (e.g. the non-star Magainin 1), and expression levels of<br />
very small peptides can be difficult without a fusion partner. Second,<br />
two of our constructs code for antimicrobial peptides. Without a fusion<br />
partner, it is likely that these genes would be toxic to their hosts<br />
upon induction.</p><br />
<p>To find a suitable expression system, we looked towards the<br />
Registry of Standard Parts. We used the SUMO protein expression system<br />
designed by TU Delft 2014 (for example, see <a target="_blank"<br />
href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1022101">http://parts.igem.org/wiki/index.php?title=Part:BBa_K1022101</a>).<br />
This system essentially consists of a N-terminal HIS-tag followed by<br />
the SUMO protein (also known as UlpI).</p><br />
<p><br />
The strategy of expressing toxic AMPs using SUMO has been successfully<br />
reported in the literature (Bommarius et al., 2010). We surmise that<br />
the SUMO protein could inhibit the antimicrobial activity of single,<br />
linear peptides, and that it may also inhibit the activity of our star<br />
antimicrobial peptide.</p><br />
<p><br />
SUMO as a fusion protein also has the benefit of leaving no residues at<br />
the C-terminal end of the cleavage site. This means that upon cleavage<br />
with the SUMO protease, the native protein can be recovered. In our<br />
case, this means that one of the arms of the star can be designed<br />
without the need to take into account the addition of any amino acid<br />
residues left behind by the protease.<br><br />
We used the SUMO peptide sequence reported by TU Delft. However, our<br />
construct contained the following unique features:</p><br />
<ol><br />
<li> Standardisation of the biobrick by substituting the T7<br />
promoter and RBS (the origins of which are both not specified in the<br />
Delft documentation) with the standard T7 promoter and RBS BioBrick <a<br />
target="_blank" href="http://parts.igem.org/Part:BBa_K525998">BBa_K525998</a>.<br />
In addition to supporting the principle of standardization, using the<br />
well-characterized promoter BioBrick should help assure expression<br />
levels.<br><br />
</li><br />
<li>Biobrick BBa_K1022101 lacks a terminator sequence (this was<br />
presumably because the part was meant to be integrated into a larger<br />
genetic construct with a terminator). A terminator from the registry of<br />
standard parts was added (specifically, the wild type terminator from<br />
T7 bacteriophage,&nbsp;<a target="_blank"<br />
href="http://parts.igem.org/Part:BBa_K731721">BBa_K731721</a>).<br />
</li><br />
<li>The original biobrick BBa_K1022101 codes for three amino<br />
acid residues before the HIS-tag (ASM), which appeared to be redundant.<br />
Correspondence with the 2013 TU Delft team suggested that these<br />
residues were unnecessary and appear to be cleaved within the cell as<br />
part of the cells post-translational modifications. However, their<br />
presence complicates the addition of additional tags at the N-terminus<br />
of the protein (e.g. periplasmic export tags), and therefore were not<br />
included.<br><br />
</li><br />
<li>The SUMO sequence was codon optimised for E. coli during<br />
synthesis. As the Delft documentation did not specify whether the gene<br />
was codon optimal, codon optomisation was undertaken to potentially<br />
improve expression levels. The linear Magainin 1 construct, however,<br />
was not codon optimised in the SUMO region in order to provide a<br />
control condition.</li><br />
</ol><br />
<p>To summarise, the protein expression devices used in our<br />
project to the following form:</p><br />
<img<br />
src="https://static.igem.org/mediawiki/2014/d/d0/Gene_circuit_export.png"<br />
alt="" height="91" width="720"><br />
<p>The protein coding region consisted of a 6x-HIS tag followed<br />
by the SUMO protein and the relevant star or linear peptide.</p><br />
<p>&nbsp;</p><br />
<h2>Plasmid preparation: Cloning and acquisition of the genetic<br />
constructs</h2><br />
<h3>Magainin 1 Star Peptide</h3><br />
<p>This construct was synthesised in the standard shipping<br />
plasmid from Life Technologies- plasmid pMK. We had originally planned<br />
to express our protein to the periplasm of E. coli, and therefore had<br />
included in the synthesis a periplasmic export tag, the TorTss signal<br />
sequence (Steiner et al., 2006).</p><br />
<p><br />
Cytoplasmic and periplasmic expression may both allow for disulfide<br />
bond formation in the cell. We decided, however, to focus on<br />
cytoplasmic expression. There are distinct advantages to cytoplasmic<br />
expression (e.g. the absence of several periplasmic proteases and<br />
potentially higher expression levels (Baneyx, 1999)).</p><br />
<p><br />
Another reason the cytoplasm was chosen was to allow us to test the<br />
effects of an oxidising versus reducing intracellular environment on<br />
disulfide bond formation. We planned to express the construct in both<br />
SHuffle T7 cells (oxidising cytoplasm) and BL21(DE3) (reducing<br />
cytoplasm) to probe whether there was a difference in disulfide bond<br />
formation. Therefore, we needed to remove the periplasmic export tag<br />
from the gene.</p><br />
<p><br />
To do this, we use the following cloning strategy (noting that the top<br />
row represents the gene initially synthesised in plasmid pMK):</p><br />
<img<br />
src="https://static.igem.org/mediawiki/2014/2/2c/Melbourne_cloning_strategy_diagram.jpg"<br />
alt="" height="437" width="900"><br />
<p>The gene was inserted into plasmid pSB1C3 containing the T7<br />
promoter and ribosome binding site, BBa_K525998. It was inserted after<br />
the promoter and before the BioBrick suffix. This was accomplished by<br />
digesting the destination vector with SpeI and PstI. At the same time,<br />
PCR was used to amplify the segment of the gene containing the SUMO<br />
fusion and the Magainin 1 Star peptide, adding XbaI and keeping the<br />
PstI site existing in the gene.</p><br />
<p><br />
Digestion of the PCR product then allowed for ligation of the insert<br />
and destination vector using the PstI sites and XbaI and SpeI (XbaI and<br />
SpeI have compatible sticky ends). Note that after the ligation, there<br />
will be a scar in the gene where the XbaI and the SpeI sites were<br />
ligated.</p><br />
<p><br />
The ligation appeared to be successful. The ligation mixture was<br />
transformed to DH5α competent cells and plated onto<br />
chloramphenicol-containing agar plates.</p><br />
<p><br />
Plasmid from 5 colonies (C1, C2, C3, C4, and C5) was cultured,<br />
extracted, and then digested with EcoRI and PstI.</p><br />
<p><br />
As evident in the figure below, at least 4 of the colonies appeared to<br />
contain the insert. The empty, linearised pSB1C3 backbone ran at<br />
approximately the correct size (2070 bps), as did the insert (740 bps).</p><br />
<center><img<br />
src="https://static.igem.org/mediawiki/2014/9/95/Magainin_1_subcloning.png"<br />
alt="" height="400" width="353"></center><br />
<p>N.B. MW marker is the 100 bp Ladder from Axygen. * indicates<br />
the colony picked for sequencing and eventual transformation to the<br />
expression cell lines.</p><br />
<p>The DNA from Colony C2 was confirmed using Sanger sequencing<br />
at the Australian Genome Research Facility. This DNA appears in the<br />
registry of standard parts as BioBrick <a target="_blank"<br />
href="http://parts.igem.org/Part:BBa_K1394000">BBa_K1394000</a>.</p><br />
<p><br />
For the USP peptide and the linear Magainin 1 peptide, the genes were<br />
synthesised by GenScript and delivered in pSB1C3. The expression<br />
vectors had identical gene regulatory elements to that used for the<br />
Magainin 1 Star peptide. They only differed in the codon optimisation<br />
used, and they also lacked the assembly scar described above.</p><br />
<h2><br />
Protein expression and characterisation<br />
</h2><br />
<p><br />
After acquiring our genes, the project moved to protein expression and<br />
purification. We expressed both star peptides (Magainin 1 Star Peptide<br />
and the USP peptide) in both the SHuffle T7 and BL21(DE3) cell lines,<br />
both sourced from New England Biolabs. As the Linear Magainin 1 peptide<br />
does not have any special disulfide bonding<br />
requirements, we expressed it in BL21(DE3).<br />
</p><br />
<p><br />
The plasmids were transformed to the expression cells, and a single<br />
colony was cultured and induced overnight at 17 °C. A whole cell sample<br />
both before<br />
IPTG induction (-IPTG) and after the induction period (+IPTG) were<br />
boiled in SDS-PAGE sample buffer and loaded on a 15% tris-glycine gel.<br />
The whole cell<br />
Coomassie stained gel is shown below alongside the NEB P7712S molecular<br />
weight marker.<br />
</p><br />
<br><br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/2/24/Whole_cell_CS.png"<br />
height="330" width="439"></p><br />
<br><br />
<p><br />
From theoretical prediction of the peptide masses, we would expect the<br />
following distribution of molecular weights:<br />
</p><br />
<br><br />
<table align="center" border="1" cellpadding="0"<br />
cellspacing="0"><br />
<tbody><br />
<tr><br />
<td width="156"><br />
<p align="center">Magainin 1 Star Peptide (Mag1<br />
Star)<br />
</p><br />
</td><br />
<td width="145"><br />
<p align="center">USP<br />
</p><br />
</td><br />
<td width="156"><br />
<p align="center">Linear Magainin 1 (Linear Mag1)<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="156"><br />
<p align="center">22.15 kDa<br />
</p><br />
</td><br />
<td width="145"><br />
<p align="center">29.3 kDa<br />
</p><br />
</td><br />
<td width="156"><br />
<p align="center">14.32 kDa<br />
</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<br><br />
<p><br />
We looked for bands corresponding to overexpression of the proteins. We<br />
observed a thick band, post IPTG around 32 kDa in the BL21(DE3) cells<br />
carrying the<br />
USP plasmid. However, we saw this same band appearing in the Linear<br />
Magainin 1 lane but the Linear Magainin 1 protein should have a much<br />
lower molecular<br />
weight than what was observed. Therefore, we did not assume that we had<br />
overexpression overexpression of the USP 1 protein.<br />
</p><br />
<p><br />
We also noted bands in all post-IPTG lanes around 17 kDa. This would be<br />
roughly consistent with both the Linear Magainin 1 and Magainin 1 Star<br />
Peptide,<br />
noting that small proteins may not run at their expected molecular<br />
weight. Again, the analysis is complicated by the fact that the band<br />
also appears in the<br />
lane corresponding to the larger molecular weight protein, USP I. Given<br />
this ambiguity, we decided to purify all the protein in the sample<br />
using Ni-NTA<br />
purification.<br />
</p><br />
<h3><br />
Purification and further characterisation<br />
</h3><br />
<p><br />
A small-scale purification was carried out according to our <a<br />
href="https://static.igem.org/mediawiki/2014/e/e1/MELBOURNE_D1V1_Column_Purification_of_His-Tagged_Proteins.pdf">protocol</a>.<br />
The cells were lysed with iodoacetamide, an alkylating agent, added to<br />
the lysis buffer. Iodoacetamide blocks all free cysteines on proteins<br />
with a short alkyl group. This was added<br />
because we wanted to determine if a disulphide bond had formed inside<br />
the cell. If we did not block free cysteines, then any bond that had<br />
formed could be<br />
attributed to spontaneous/air oxidation of disulfides outside the cell.<br />
</p><br />
<p><br />
After purification, we ran a concurrent Western Blot and Coomassie<br />
stain on both the pre-induction samples from above and the purified<br />
protein (namely, the<br />
first elution from the batch purification).<br />
</p><br />
<p><br />
The Western Blot used mouse monoclonal antibodies against the<br />
N-terminal HIS-tag as primary antibodies and anti-mouse secondary<br />
antibodies:<br />
</p><br />
<p align="center"><br />
<img<br />
src="https://static.igem.org/mediawiki/2014/5/54/Western_Blot_Melb.png"<br />
border="0" height="400" width="460"></p><br />
<p><br />
The corresponding Coomassie stain is show below:<br />
</p><br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f0/Purified_CS.png"<br />
border="0" height="365" width="486"></p><br />
<p><br />
There are several interesting aspects of the Western Blot. Firstly,<br />
there was a strong band between 25 and 32 kDa in most lanes. However,<br />
it<br />
appears in all of the pre-induction controls, suggesting non-specific<br />
binding of the anti-HIS antibodies.<br />
</p><br />
<p><br />
Secondly, we observed a band in most of the lanes around 17 kDa (with<br />
the exception being the USP protein in SHuffle cells). As these bands<br />
were not<br />
present in the pre-induction controls, we suspected they were a result<br />
of the induction.<br />
</p><br />
<h3><br />
Mass spectroscopy<br />
</h3><br />
<p><br />
In order to assess the identity of the bands, we performed an in-gel<br />
tryptic digestion on select bands. We digested bands in the Coomassie<br />
stain which<br />
corresponded to the HIS-tagged bands in the Western Blot. This was<br />
followed by mass spec analysis (LC MS/MS). We focused our analysis on<br />
the Magainin 1<br />
Star Peptide bands and the Linear Magainin 1 band. Our USP peptide was,<br />
by design, highly rich in basic residues, greatly reducing the<br />
likelihood that<br />
tryptic fragments would be detected by the mass spec.<br />
</p><br />
<p><br />
We found that the Linear Magainin 1 peptide appeared to be present in<br />
the approximately 17 kDa band, with the following detected tryptic<br />
fragments in the<br />
sequence below (bold; basic residues underlined):<br />
</p><br />
<p><br />
<img<br />
src="https://static.igem.org/mediawiki/2014/1/1b/Tryptic_digest_linear_mag1.PNG"<br />
border="0" height="63" width="601"></p><br />
<p><br />
Note that spaces have been added in the sequence to emphasise distinct<br />
domains in the protein (in this case, the fusion protein versus the<br />
Magainin 1<br />
peptide). Together with the fact that the protein runs close to the<br />
expected molecular weight, this seems to provide good evidence that the<br />
protein is<br />
being expressed.<br />
</p><br />
<p><br />
We then examined the bands corresponding to the Magainin 1 Star<br />
Peptide.<br />
</p><br />
<p><br />
Again, we digested bands in the Coomassie stained gel corresponding to<br />
the prominent HIS-tagged bands on the <em>Western Blot</em><br />
(near 17 kDa).<br />
</p><br />
<p><br />
The mass spec coverage for the Magainin 1 Star Peptide expressed in<br />
SHuffle cells was as follows:<br />
</p><br />
<p><br />
<img<br />
src="https://static.igem.org/mediawiki/2014/6/63/Tryptic_digest_Sample_A_Star_Mag_Shuffle.PNG"<br />
border="0" height="90" width="601"></p><br />
<p><br />
The coverage for the same peptide expressed in BL21(DE3) was found to<br />
be:<br />
</p><br />
<p><br />
<img<br />
src="https://static.igem.org/mediawiki/2014/d/db/Tryptic_digest_Sample_A_Star_Mag_BL21%28DE3%29.PNG"<br />
border="0" height="85" width="602"></p><br />
<p><br />
Again, we can see that tryptic peptides from the expressed protein<br />
appear to present in the gel band under analysis.<br />
</p><br />
<p><br />
There is a relatively lower sequence coverage. This could be a<br />
limitation of our procedure: for example, improper destaining during<br />
the in gel digestion<br />
procedure can inhibit tryptic digestion.<br />
</p><br />
<p><br />
However, even if the digestion was complete, the mass spec will only<br />
detect fragments which ionize well. The Magainin 1 Star peptide<br />
consists of four<br />
repeats of the Magainin 1 sequence (corresponding to the arms of the<br />
star). If the two tryptic fragments within Magainin 1 do not ionize<br />
well, then indeed<br />
the entire peptide would not be read.<br />
</p><br />
<p><br />
Finally, it is possible that the protein has been cleaved or degraded.<br />
This would account for the slightly lower-than-expected mass on the SDS<br />
page gel.<br />
</p><br />
<p><br />
Nonetheless, it is a possibility that the peptide fragments are present<br />
but not detected. Replication of the in-gel mass spec would be useful<br />
in clarifying this point, or provided the purity of the sample is<br />
acceptable, it may be possible to run an intact mass spec. Digestion<br />
with alternative enzymes may also provide a more robust sequence<br />
coverage, particularly for the USP peptide.<br />
</p><br />
<h2><br />
Conclusions<br />
</h2><br />
<p><br />
We designed several novel star peptides based on several rational<br />
design criteria. Further, we began the optimisation process required to<br />
express these peptides in <em>E. coli</em>.<br />
</p><br />
<p><br />
To this end, we constructed an expression system based on the SUMO<br />
protein and standard BioBrick parts. The function of this system was<br />
confirmed, as it<br />
appears it does produce HIS-tagged SUMO fusion protein in <em>E.<br />
coli</em>, as expected. Nonetheless, we hope to continue to<br />
characterise our parts, likely through further purification and<br />
measurement of the mass. </p><br />
<p>Given the success of SUMO in the protein expression community,<br />
we hope our BioBrick<br />
will encourage further use of the fusion domain within iGEM. In<br />
addition, we hope that our ideas and concepts will inspire future<br />
iGEM efforts to explore the applications of synthetic biology for<br />
material science. We continue to believe that bacteria have great<br />
promise for the <em>in vivo</em><br />
construction of unique biomaterials, and we look forward to seeing how<br />
the synthetic biology community will develop in this area.<br />
</p><br />
<h2>References</h2><br />
<p>ADER, C., FREY, S., MAAS, W., SCHMIDT, H. B., GÖRLICH, D.<br />
&amp; BALDUS, M. 2010. Amyloid-like interactions within nucleoporin<br />
FG hydrogels. <em>Proceedings of the National Academy of<br />
Sciences,</em> 107<strong>,</strong> 6281-6285.</p><br />
<p><br />
BANEYX, F. 1999. Recombinant protein expression in Escherichia coli. <em>Current<br />
Opinion in Biotechnology,</em> 10<strong>,</strong><br />
411-421.</p><br />
<p><br />
BOMMARIUS, B., JENSSEN, H., ELLIOTT, M., KINDRACHUK, J., PASUPULETI,<br />
M., GIEREN, H., JAEGER, K. E., HANCOCK, R. E. W. &amp; KALMAN, D.<br />
2010. Cost-effective expression and purification of antimicrobial and<br />
host defense peptides in Escherichia coli. <em>Peptides,</em><br />
31<strong>,</strong> 1957-1965.</p><br />
<p><br />
BROGDEN, K. A. 2005. Antimicrobial peptides: Pore formers or metabolic<br />
inhibitors in bacteria? <em>Nature Reviews Microbiology,</em><br />
3<strong>,</strong> 238-250.</p><br />
<p><br />
CHANG, C., CHHOR, G., CLANCY, S. &amp; JOACHIMIAK, A. 2014. Crystal<br />
Structure of Glutathione S-transferase Domain Protein From Haliangium<br />
Ochraceum DSM 14365 [Online]. ''NCBI. Available:<br />
http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=4w66'<br />
[Accessed September 9 2014].</p><br />
<p><br />
CHEN, X., ZHU, F., CAO, Y. &amp; QIAO, S. 2009. Novel expression<br />
vector for secretion of cecropin AD in Bacillus subtilis with enhanced<br />
antimicrobial activity. <em>Antimicrobial agents and<br />
chemotherapy,</em> 53<strong>,</strong> 3683-3689.</p><br />
<p><br />
GLINEL, K., JONAS, A. M., JOUENNE, T., LEPRINCE, J., GALAS, L.<br />
&amp; HUCK, W. T. 2008. Antibacterial and antifouling polymer<br />
brushes incorporating antimicrobial peptide. <em>Bioconjug Chem,</em><br />
20<strong>,</strong> 71-77.</p><br />
<p><br />
HUMBLOT, V., YALA, J.-F., THEBAULT, P., BOUKERMA, K., HÉQUET, A.,<br />
BERJEAUD, J.-M. &amp; PRADIER, C.-M. 2009. The antibacterial<br />
activity of Magainin I immobilized onto mixed thiols self-assembled<br />
monolayers. <em>Biomaterials,</em> 30<strong>,</strong><br />
3503-3512.</p><br />
<p><br />
DAWSON, P. E., MUIR, T. W., CLARK-LEWIS, I. &amp; KENT, S. 1994.<br />
Synthesis of proteins by native chemical ligation. <em>Science,</em><br />
266<strong>,</strong> 776-779.</p><br />
<p><br />
KADOKURA, H. &amp; BECKWITH, J. 2009. Detecting folding<br />
intermediates of a protein as it passes through the bacterial<br />
translocation channel. <em>Cell,</em> 138<strong>,</strong><br />
1164-1173.</p><br />
<p><br />
KADOKURA, H., KATZEN, F. &amp; BECKWITH, J. 2003. Protein disulfide<br />
bond formation in prokaryotes. <em>Annual review of biochemistry,</em><br />
72<strong>,</strong> 111-135.</p><br />
<p><br />
LI, Y. 2011. Recombinant production of antimicrobial peptides<br />
in&lt; i&gt; Escherichia coli&lt;/i&gt;: A review. <em>Protein<br />
Expression and Purification,</em> 80<strong>,</strong><br />
260-267.</p><br />
<p><br />
LOBSTEIN, J., EMRICH, C. A., JEANS, C., FAULKNER, M., RIGGS, P.<br />
&amp; BERKMEN, M. 2012. SHuffle, a novel Escherichia coli protein<br />
expression strain capable of correctly folding disulfide bonded<br />
proteins in its cytoplasm. <em>Microb Cell Fact,</em> 11<strong>,</strong><br />
56-56.</p><br />
<p><br />
PROTEIN MODEL PORTAL. Query result, GST and DcmA [Online]. Available:<br />
http://www.proteinmodelportal.org/query/uniprot/P21161 [Accessed<br />
September 9 2014].</p><br />
<p><br />
STEINER, D., FORRER, P., STUMPP, M. T. &amp; PLUCKTHUN, A. 2006.<br />
Signal sequences directing cotranslational translocation expand the<br />
range of proteins amenable to phage display. <em>Nat Biotech,</em><br />
24<strong>,</strong> 823-831.</p><br />
<p><br />
SULISTIO, A., GURR, P. A., BLENCOWE, A. &amp; QIAO, G. G. 2012.<br />
Peptide-Based Star Polymers: The Rising Star in Functional Polymers. <em>Australian<br />
Journal of Chemistry,</em> 65<strong>,</strong><br />
978-984.</p><br />
<p><br />
SULISTIO, A., LOWENTHAL, J., BLENCOWE, A., BONGIOVANNI, M. N., ONG, L.,<br />
GRAS, S. L., ZHANG, X. &amp; QIAO, G. G. 2011. Folic Acid<br />
Conjugated Amino Acid-Based Star Polymers for Active Targeting of<br />
Cancer Cells. <em>Biomacromolecules,</em> 12<strong>,</strong><br />
3469-3477.</p><br />
<p><br />
TANAKA, N., KUSAKABE, Y., ITO, K., YOSHIMOTO, T. &amp; NAKAMURA, K.<br />
T. 2002. Crystal Structure of Formaldehyde Dehydrogenase from&lt;<br />
i&gt; Pseudomonas putida: the Structural Origin of the Tightly<br />
Bound Cofactor in Nicotinoprotein Dehydrogenases. Journal of molecular<br />
biology, 324, 519-533.</p><br />
<p><br />
WIRADHARMA, N., LIU, S. Q. &amp; YANG, Y. Y. 2012. Branched and<br />
4-Arm Starlike α-Helical Peptide Structures with Enhanced Antimicrobial<br />
Potency and Selectivity. <em>Small,</em> 8<strong>,</strong><br />
362-366.</p><br />
<p><br />
YU, Z., WANG, Q., MA, Q. &amp; ZHANG, R. 2013. Secretory expression<br />
of lacticin Q fused with SUMO in Bacillus subtilis. <em>Protein<br />
Expression and Purification,</em> 89<strong>,</strong><br />
51-55.</p><br />
<p><br />
ZASLOFF, M. 1987. Magainins, a class of antimicrobial peptides from<br />
Xenopus skin: isolation, characterization of two active forms, and<br />
partial cDNA sequence of a precursor. <em>Proceedings of the<br />
National Academy of Sciences,</em> 84<strong>,</strong><br />
5449-5453.</p><br />
<p>&nbsp;</p><br />
<a name="Oxford"></a><br />
<h1>Collaboration with Oxford</h1><br />
<p>To strengthen the sense of iGEM community, we contacted <a<br />
target="_blank" href="https://2014.igem.org/Team:Oxford">University<br />
of Oxford iGEM team</a> to discuss the possibility of<br />
collaboration between our teams. The Oxford project aims to develop a<br />
system that can dispose of the carcinogenic, hazardous solvent<br />
dichloromethane (DCM). To do this, Oxford team has proposed the use of<br />
the DcmA enzyme. This enzyme degrades DCM to form a toxic intermediate<br />
which in turn is converted by another enzyme, FdhA, into a neutral<br />
molecule. Schematically this can be presented in the following way:</p><br />
<p><br />
Reaction 1: DCM +&nbsp;DcmA -&gt; <strong>toxic<br />
intermediate</strong><br><br />
Reaction 2: <strong>toxic intermediate</strong> + FdhA<br />
-&gt; neutral product.</p><br />
<p>The problem for their system lies in bringing the two enzymes<br />
together to ensure efficient reaction kinetics. An idea that we<br />
discussed with Oxford was to use the star peptide platform to link the<br />
two enzymes together, in a structure similar to the following: </p><br />
<img<br />
src="https://static.igem.org/mediawiki/2014/2/2e/Melbourne_Collab_1.png"<br />
alt="" height="325" width="500"><br />
<p>We worked with Oxford to study this system from a theoretical<br />
standpoint. Our team studied the 3D structure of both enzymes involved<br />
to confirm that the enzyme could in theory be attached to a linker in<br />
this manner, while Oxford team did stochastic modelling to determine<br />
how reaction rate changes when the linker length, labeled D on the<br />
diagram, changes. </p><br />
<p><br />
As a first check, it was necessary to determine whether anchoring these<br />
enzymes to our star would be sterically possible. We studied the<br />
structures of FdhA and DcmA determined by crystallography using data<br />
from the protein data bank. As no crystallographic data was available<br />
in the databank for DcmA, GST was examined as a proxy, as GST is<br />
structurally homologous to DcmA. </p><br />
<p><br />
The crystalographically resolved structure of FdhA enzyme (Tanaka et<br />
al., 2002) from the protein data bank revealed the following image:</p><br />
<img<br />
src="https://static.igem.org/mediawiki/2014/d/dc/Melbourne_Collab_2.png"<br />
alt="" height="355" width="700"><br />
<p>The crystalographically resolved structure of GST was as<br />
follows (Chang et al., 2014):</p><br />
<img<br />
src="https://static.igem.org/mediawiki/2014/e/e9/Melbourne_Collab_3.png"<br />
alt="" height="361" width="800"><br />
<p>It can be seen that the amino- and carboxy-terminals are<br />
located away from the active site. This suggests that both enzymes,<br />
DcmA and FdhA, might be linked together via linkers that are used in<br />
our star peptide. That is, the active site will not be sterically<br />
hindered by attachment to the star peptide. That said, it is difficult<br />
to predict how anchoring the protein to the star will affect its<br />
tertiary structure. Further, it is difficult to predict how limiting<br />
the rotational degrees of freedom will affect enzyme activity.</p><br />
<p>The Oxford team modeled this system and found that, indeed,<br />
the star peptide as an enzyme linker was favourable to enzyme kinetics.<br />
Their work can be found <a<br />
href="https://2014.igem.org/Team:Oxford/alternatives_to_microcompartments#show2"><br />
here</a>.<br />
</p><br />
</body><br />
</html></div>Slowehttp://2014.igem.org/Team:Melbourne/RecruitmentTeam:Melbourne/Recruitment2014-12-07T02:32:09Z<p>Slowe: </p>
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<a href="https://2014.igem.org/Team:Melbourne/Project"<br />
style="color: rgb(0, 0, 0);">Project</a> </td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Human_Practices"<br />
style="color: rgb(0, 0, 0);"> Human Practices</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Achievements"<br />
style="color: rgb(0, 0, 0);">Achievements</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Notebook"<br />
style="color: rgb(0, 0, 0);">Notebook</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Protocols"<br />
style="color: rgb(0, 0, 0);"> Protocols</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Safety"<br />
style="color: rgb(0, 0, 0);">Safety</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Sponsors"<br />
style="color: rgb(0, 0, 0);">Sponsors</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Attributions"<br />
style="color: rgb(0, 0, 0);">Attributions</a></td><br />
<td align="right"> <a<br />
href="https://2014.igem.org/Main_Page"> <img<br />
src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png"<br />
width="46"></a> </td><br />
</tr><br />
</tbody><br />
</table><br />
<p class="MsoNormal" style="margin-top: 12pt;">Thank<br />
you for your interest in the<br />
2015 Melbourne University iGEM Team! Please read the following<br />
information<br />
about iGEM, the team, and what you need to do to get involved.</p><br />
<h1>What is iGEM?<o:p></o:p></h1><br />
<p class="MsoNormal"><span class="GramE"><span<br />
class="usercontent">iGEM</span></span><span<br />
class="usercontent"> is a unique opportunity to get involved<br />
in an enterprising<br />
student research group and make a scientific impact.</span><br><br />
<br><br />
<span class="GramE"><span class="textexposedshow">iGEM</span></span><span<br />
class="textexposedshow"> is short for the International<br />
Genetically Engineering<br />
Machine competition. <span class="GramE">iGEM</span><br />
is an undergraduate science<br />
competition held each November in Boston. In the months leading up to<br />
iGEM,<br />
university teams use the latest tools from synthetic<br />
biology/biotechnology to<br />
create a novel single celled organism or “biological machine”.</span></p><br />
<p class="MsoNormal"><span data-ft="{&quot;tn&quot;:&quot;K&quot;}"<br />
class="userContent"><span class="text_exposed_show">Each<br />
year, students from around the globe form<br />
teams at their respective universities with the goal of building a<br />
biological system or tool, which they present at the iGEM global<br />
conference. Students in the past have designed bacteria that<br />
produce new types of drugs and biofuels, act as biosensors of toxic<br />
pollutants, and even serve&nbsp; as biological computation<br />
platforms&nbsp; (for examples of past<br />
projects, see <a href="https://igem.org/About"<br />
rel="nofollow nofollow" target="_blank"<br />
onmouseover='LinkshimAsyncLink.swap(this, "http:\/\/igem.org\/About");'<br />
onclick='LinkshimAsyncLink.swap(this, "http:\/\/l.facebook.com\/l.php?u=http\u00253A\u00252F\u00252Figem.org\u00252FAbout&h=zAQFmUzcK&enc=AZNGkoGC-HF5q40u8IHnH2vOwvgHdyy0ClVop-mb6cjZGHojVOVS9IRhqf-Bi3W3tjr6Zt8No6t5_Rcx8yzlpP7oLfAxZyO28QciO52K7yqag82-VE7nwRVRmBKK-HgbxqVsq9pAa5Lv-OJPoe-BAhfl&s=1");'>https://igem.org/About</a>)</span></span>.<span<br />
class="textexposedshow"> </span><o:p></o:p></p><br />
<p class="MsoNormal"><span class="GramE"><span<br />
class="textexposedshow">iGEM</span></span><span<br />
class="textexposedshow"> teams manage everything from the<br />
conception to the<br />
execution of the iGEM project, with the aid of faculty supervisors. </span><br><br />
<br><br />
<span class="textexposedshow">The benefits to<br />
participating in iGEM include:</span><br><br />
<span class="textexposedshow">-Get valuable scientific and<br />
leadership experience</span><br><br />
<span class="textexposedshow">-Develop a useful, novel<br />
biotechnology and gain experience<br />
in the latest in interdisciplinary biological research<o:p></o:p></span></p><br />
<p class="MsoNormal"><span class="textexposedshow">-Make<br />
an impact</span><br><br />
<span class="textexposedshow">-Have fun!</span><br><br />
<br><br />
</p><br />
<h1><span class="GramE"></span>What is<br />
involved and what is needed</h1><br />
<p class="MsoNormal">We are searching for enthusiastic<br />
students with<br />
an interest in science.<o:p></o:p></p><br />
<p class="MsoNormal"><o:p>&nbsp;</o:p></p><br />
<p class="MsoNormal" style="page-break-after: avoid;">As<br />
part of the iGEM team, you would help with the<br />
following tasks:<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpFirst"<br />
style="text-indent: -18pt; page-break-after: avoid;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->Research<br />
on the iGEM project ideas. The ideas<br />
for the iGEM project are student driven, and team members often need to<br />
answer<br />
specific research question to design new experiments or come up with<br />
new ideas.<br />
This will typically involve doing&nbsp; searches of the literature<br />
using Google<br />
Scholar or other tools and reading scientific articles.<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpMiddle"<br />
style="text-indent: -18pt; page-break-after: avoid;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->Help<br />
with developing experimental methods. The<br />
2014 team has built up a library of protocols and experimental methods.<br />
However, the project for 2015 will likely require new methods. You will<br />
need to<br />
look up protocols in the literature and adapt them to the project<br />
requirements.<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpMiddle"<br />
style="text-indent: -18pt;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->Wet<br />
lab work OR modelling/computation OR engineering<br />
design. By joining the iGEM team, you will have an opportunity to<br />
participate in<br />
the lab and learn many standard techniques in molecular biology.<br />
Alternatively, many iGEM teams make use of the skills of<br />
engineers,<br />
computer scientists, and other non-biological science disciplines.<br />
&nbsp;For example, this<br />
may<br />
take the form of <span class="SpellE">modeling</span><br />
a biological<br />
system using software like <span class="SpellE">Matlab</span>,<br />
designing a microfluidic device with biological applications or<br />
designing an electrical device which interfaces with a biological<br />
system. If<br />
you have an interest in interdisciplinary research between your field<br />
and biology,<br />
it is likely iGEM will be able to accommodate it.<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpLast"<br />
style="text-indent: -18pt;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->The<br />
scope of iGEM also extends into<br />
non-traditional science areas, including biotechnology<br />
entrepreneurship,<br />
biotechnology ethics and the law, and science outreach, and so we are<br />
actively<br />
seeking students with business, law, design, and arts backgrounds. You<br />
could,<br />
for example, create a business plan for <span class="GramE">a</span><br />
iGEM-created<br />
company, examine bioethics within synthetic biology, or even explore<br />
the realm&nbsp; of biologically-inspired art.<o:p></o:p></p><br />
<p class="MsoNormal"><o:p>&nbsp;</o:p></p><br />
<p class="MsoNormal">There are several traits needed on<br />
the iGEM team:<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpFirst"<br />
style="text-indent: -18pt;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]--><span<br />
class="GramE">Research skills. IGEM</span> is an<br />
exciting<br />
opportunity to undertake self-directed research in synthetic biology.<br />
We will<br />
therefore need students who are keenly interested and adept in<br />
research. To<br />
participate, you will need to have the capacity to quickly get up to<br />
speed in<br />
the field of biotechnology and to eventually excel in a lab with<br />
limited<br />
instruction.<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpMiddle"<br />
style="text-indent: -18pt;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->We’re<br />
looking for team members from a range of<br />
backgrounds. iGEM is about cross-disciplinary research, so in addition<br />
to<br />
biological and biomedical science students, we welcome students from<br />
engineering (electrical, mechanical, chemical, software etc.), computer<br />
science, maths, physics, chemistry, and other physical sciences. Also,<br />
students<br />
from non-science backgrounds are very welcome to get in touch to<br />
explore how they<br />
can contribute to the team.<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpLast"<br />
style="text-indent: -18pt;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->Participation<br />
would be most suited to students<br />
with a high level of academic maturity. Typically, this includes third<br />
year,<br />
honours, or Masters <span class="GramE">students</span>,<br />
<span class="SpellE">but students</span><br />
from all levels who can demonstrate an aptitude for research or<br />
leadership are welcome<br />
to apply.<o:p></o:p></p><br />
<p class="MsoNormal">A biological science background is<br />
helpful, but not<br />
required. Team members will need to use knowledge from second-level<br />
biology subjects.<br />
However, we have had team members without a biological science<br />
background who<br />
have excelled. <o:p></o:p></p><br />
<h1>Recruitment</h1><br />
<p class="MsoNormal">If you are interested in being a part<br />
of iGEM, please email<br />
Sean Lowe at MelbourneUniIGem@gmail.com for more information. <o:p></o:p></p><br />
<p class="MsoNormal">Further information about iGEM and<br />
updates about the<br />
recruitment process will also be made available at <a<br />
href="https://www.facebook.com/MelbourneUniIGem">https://www.facebook.com/MelbourneUniIGem</a>.<o:p></o:p></p><br />
<p class="MsoNormal">Over the summer, we are looking for<br />
students to form part of a founding team for 2015. As part of this<br />
team, you would work to set the framework and goals for next year. You<br />
would work with the current team to liaise with<br />
faculty and sponsors to set up support systems for next year. You would<br />
need to be able to generate a vision for what<br />
a student-based science team can and should be, it’s potential and<br />
opportunities.</p><br />
<p class="MsoNormal"><br />
Recruitment is now open. Roles will filled in December and throughout<br />
the summer. If you wish to be involved over the summer, please get in<br />
touch with us as soon as possible and before 5 January 2015.<br />
Any positions which are not filled over the summer will be<br />
filled in weeks 1-2 of semester 1, 2015. Note that applications at the<br />
start of the semester can be competitive,&nbsp;so if<br />
interested, please get in touch over the summer.</p><br />
<p class="MsoNormal">If you decide you would like to<br />
apply, you will need to<br />
send a CV and answer a short questionnaire about iGEM (available by<br />
email). </p><br />
<p class="MsoNormal"></p><br />
<h1>Further information</h1><br />
<p class="MsoNormal">Learn more about iGEM in<br />
general at: <a<br />
href="http://www.facebook.com/l.php?u=http%3A%2F%2Figem.org%2FAbout&amp;h=HAQEjYmHL&amp;s=1"<br />
target="_blank">https://igem.org/About</a><br<br />
style=""><br />
</p><br />
<!--[if !supportLineBreakNewLine]--><br style=""><br />
<!--[endif]--><o:p></o:p><br />
<p class="MsoNormal">Learn about this year’s team by<br />
clicking on the links above.<o:p></o:p></p><br />
<p class="MsoNormal"><br><br />
<span class="textexposedshow">Also browse the previous<br />
Melbourne <span class="SpellE">Uni</span> iGEM team<br />
at<span class="GramE">:</span></span><br><br />
<a href="http://openwetware.org/wiki/IGEM:Melbourne/2008"<br />
target="_blank">http://openwetware.org/wiki/IGEM:Melbourne/2008</a><br><br />
<a<br />
href="http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-melbourne-university-te"<br />
target="_blank">http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-<span<br />
class="SpellE">melbourne</span>-university-<span<br />
class="SpellE">te</span></a><span<br />
class="textexposedshow"><o:p></o:p></span></p><br />
<!-- END EDIT HERE HERE --><br />
</body><br />
</html></div>Slowehttp://2014.igem.org/Team:Melbourne/RecruitmentTeam:Melbourne/Recruitment2014-12-06T10:22:17Z<p>Slowe: </p>
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<img src="https://static.igem.org/mediawiki/2014/9/9b/Melbourne_Banner.png"<br />
alt="Banner" height="225" width="1000"><br />
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style="text-transform: uppercase; font-family: 'Lato',sans-serif;"<br />
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bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne"<br />
style="color: rgb(0, 0, 0);">Home</a> </td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Team"<br />
style="color: rgb(0, 0, 0);">Team</a> </td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Project"<br />
style="color: rgb(0, 0, 0);">Project</a> </td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Human_Practices"<br />
style="color: rgb(0, 0, 0);"> Human Practices</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Achievements"<br />
style="color: rgb(0, 0, 0);">Achievements</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Notebook"<br />
style="color: rgb(0, 0, 0);">Notebook</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Protocols"<br />
style="color: rgb(0, 0, 0);"> Protocols</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Safety"<br />
style="color: rgb(0, 0, 0);">Safety</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Sponsors"<br />
style="color: rgb(0, 0, 0);">Sponsors</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Attributions"<br />
style="color: rgb(0, 0, 0);">Attributions</a></td><br />
<td align="right"> <a<br />
href="https://2014.igem.org/Main_Page"> <img<br />
src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png"<br />
width="46"></a> </td><br />
</tr><br />
</tbody><br />
</table><br />
<p class="MsoNormal" style="margin-top: 12pt;">Thank<br />
you for your interest in the<br />
2015 Melbourne University iGEM Team! Please read the following<br />
information<br />
about iGEM, the team, and what you need to do to get involved.</p><br />
<h1>What is iGEM?<o:p></o:p></h1><br />
<p class="MsoNormal"><span class="GramE"><span<br />
class="usercontent">iGEM</span></span><span<br />
class="usercontent"> is a unique opportunity to get involved<br />
in an enterprising<br />
student research group and make a scientific impact.</span><br><br />
<br><br />
<span class="GramE"><span class="textexposedshow">iGEM</span></span><span<br />
class="textexposedshow"> is short for the International<br />
Genetically Engineering<br />
Machine competition. <span class="GramE">iGEM</span><br />
is an undergraduate science<br />
competition held each November in Boston. In the months leading up to<br />
iGEM,<br />
university teams use the latest tools from synthetic<br />
biology/biotechnology to<br />
create a novel single celled organism or “biological machine”.</span></p><br />
<p class="MsoNormal"><span data-ft="{&quot;tn&quot;:&quot;K&quot;}"<br />
class="userContent"> iGEM is an international competition<br />
based on the new scienc<span class="text_exposed_show">e<br />
of synthetic biology. Each year, students from around the globe form<br />
teams at their respective universities with the goal of building a<br />
biological system or tool, which they present at the iGEM global<br />
conference in Boston. Students in the past have designed bacteria that<br />
produce new types of drugs and biofuels, act as biosensors of toxic<br />
pollutants, and even perform simple computation (for examples of past<br />
projects, see <a href="https://igem.org/About"<br />
rel="nofollow nofollow" target="_blank"<br />
onmouseover='LinkshimAsyncLink.swap(this, "http:\/\/igem.org\/About");'<br />
onclick='LinkshimAsyncLink.swap(this, "http:\/\/l.facebook.com\/l.php?u=http\u00253A\u00252F\u00252Figem.org\u00252FAbout&h=zAQFmUzcK&enc=AZNGkoGC-HF5q40u8IHnH2vOwvgHdyy0ClVop-mb6cjZGHojVOVS9IRhqf-Bi3W3tjr6Zt8No6t5_Rcx8yzlpP7oLfAxZyO28QciO52K7yqag82-VE7nwRVRmBKK-HgbxqVsq9pAa5Lv-OJPoe-BAhfl&s=1");'>https://igem.org/About</a>)</span></span>.<span<br />
class="textexposedshow"> </span><o:p></o:p></p><br />
<p class="MsoNormal"><span class="GramE"><span<br />
class="textexposedshow">iGEM</span></span><span<br />
class="textexposedshow"> teams manage everything from the<br />
conception to the<br />
execution of the iGEM project, with the aid of faculty supervisors. </span><br><br />
<br><br />
<span class="textexposedshow">The benefits to<br />
participating in iGEM include:</span><br><br />
<span class="textexposedshow">-Get valuable scientific and<br />
leadership experience</span><br><br />
<span class="textexposedshow">-Develop a useful, novel<br />
biotechnology and gain experience<br />
in the latest in interdisciplinary biological research<o:p></o:p></span></p><br />
<p class="MsoNormal"><span class="textexposedshow">-Make<br />
an impact</span><br><br />
<span class="textexposedshow">-Have an incredible<br />
experience!</span><br><br />
<br><br />
</p><br />
<h1><span class="GramE"></span>What is<br />
involved and what is needed</h1><br />
<p class="MsoNormal">We are searching for enthusiastic<br />
students with<br />
an interest in science.<o:p></o:p></p><br />
<p class="MsoNormal"><o:p>&nbsp;</o:p></p><br />
<p class="MsoNormal" style="page-break-after: avoid;">As<br />
part of the iGEM team, you would help with the<br />
following tasks:<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpFirst"<br />
style="text-indent: -18pt; page-break-after: avoid;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->Research<br />
on the iGEM project ideas. The ideas<br />
for the iGEM project are student driven, and team members often need to<br />
answer<br />
specific research question to design new experiments or come up with<br />
new ideas.<br />
This will typically involve doing quick searches of the literature<br />
using Google<br />
Scholar and reading scientific articles.<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpMiddle"<br />
style="text-indent: -18pt; page-break-after: avoid;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->Help<br />
with developing experimental methods. The<br />
2014 team has built up a library of protocols and experimental methods.<br />
However, the project for 2015 will likely require new methods. You will<br />
need to<br />
look up protocols in the literature and adapt them to the project<br />
requirements.<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpMiddle"<br />
style="text-indent: -18pt;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->Wet<br />
lab work OR modelling/computation OR engineering<br />
design. By joining the iGEM team, you will have an opportunity to<br />
participate in<br />
the lab and learn many standard techniques in molecular biology from<br />
fellow lab<br />
members. Alternatively, many iGEM teams make use of the skills of<br />
engineers,<br />
computer scientists, and other non-biological science disciplines. This<br />
may<br />
take the form of, for example, <span class="SpellE">modeling</span><br />
a biological<br />
system using software like <span class="SpellE">Matlab</span>,<br />
designing microfluidic devices with biological applications or<br />
designing an electrical device which interfaces with a biological<br />
system. If<br />
you have an interest in interdisciplinary research between your field<br />
and biology,<br />
it is likely iGEM will be able to accommodate it.<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpLast"<br />
style="text-indent: -18pt;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->The<br />
scope of iGEM also extends into<br />
non-traditional science areas, including biotechnology<br />
entrepreneurship,<br />
biotechnology ethics and the law, and science outreach, and so we are<br />
actively<br />
seeking students with business, law, design, and arts backgrounds. You<br />
could,<br />
for example, create a business plan for <span class="GramE">a</span><br />
iGEM-created<br />
company, examine bioethics within synthetic biology, or even explore<br />
the<br />
potential of biologically-inspired art.<o:p></o:p></p><br />
<p class="MsoNormal"><o:p>&nbsp;</o:p></p><br />
<p class="MsoNormal">There are several traits needed on<br />
the iGEM team:<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpFirst"<br />
style="text-indent: -18pt;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]--><span<br />
class="GramE">Research skills. IGEM</span> is an<br />
exciting<br />
opportunity to undertake self-directed research in synthetic biology.<br />
We will<br />
therefore need students who are keenly interested and adept in<br />
research. To<br />
participate, you will need to have the capacity to quickly get up to<br />
speed in<br />
the field of biotechnology and to eventually excel in a lab with<br />
limited<br />
instruction.<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpMiddle"<br />
style="text-indent: -18pt;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->We’re<br />
looking for team members from a range of<br />
backgrounds. iGEM is about cross-disciplinary research, so in addition<br />
to<br />
biological and biomedical science students, we welcome students from<br />
engineering (electrical, mechanical, chemical, software etc.), computer<br />
science, maths, physics, chemistry, and other physical sciences. Also,<br />
students<br />
from non-science backgrounds are very welcome to get in touch to<br />
explore how they<br />
can contribute to the team.<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpLast"<br />
style="text-indent: -18pt;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->Participation<br />
would be most suited to students<br />
with a high level of academic maturity. Typically, this includes third<br />
year,<br />
honours, or Masters <span class="GramE">students</span>,<br />
<span class="SpellE">but students</span><br />
from all levels who can demonstrate an aptitude for research or<br />
leadership are welcome<br />
to apply.<o:p></o:p></p><br />
<p class="MsoNormal">A biological science background is<br />
helpful, but not<br />
required. Team members will need to use knowledge from second-level<br />
biology subjects.<br />
However, we have had team members without a biological science<br />
background who<br />
have excelled. <o:p></o:p></p><br />
<h1>Recruitment</h1><br />
<p class="MsoNormal">If you are interested in being a part<br />
of iGEM, please email<br />
Sean Lowe at MelbourneUniIGem@gmail.com and indicate the role(s) you<br />
are<br />
interested in. <o:p></o:p></p><br />
<p class="MsoNormal">Further information about iGEM and<br />
updates about the<br />
recruitment process will be made available at <a<br />
href="https://www.facebook.com/MelbourneUniIGem">https://www.facebook.com/MelbourneUniIGem</a>.<o:p></o:p></p><br />
<br />
<p class="MsoNormal">We are currently seeking expressions of interest from keen students who<br />
wish to get involved with iGEM. Over the summer, we are looking for<br />
students to form part of the founding team for 2015. As part of this<br />
team, you would work to set the framework and goals for next year. With<br />
the guidance and assistance of the current team, you would liaise with<br />
faculty and sponsors to set up support systems for the team. Most<br />
importantly, you will be need to be able to generate a vision for what<br />
a student-based science team can and should be, it’s potential and<br />
opportunities.</p><br />
<p class="MsoNormal"><br />
Recruitment is now open. Roles will filled in December and throughout<br />
the summer. If you wish to be involved over the summer, please get in<br />
touch with us as soon as possible and before 5 January 2015.<br />
Any positions on the team which are not filled over the summer will be<br />
filled in weeks 1-2 of semester 1, 2015. Note that applications at the<br />
start of the semester can be very competitive,&nbsp;so if<br />
interested, please get in touch over the summer.</p><br />
<br />
<p class="MsoNormal"><br />
If your skills and interests match up with the team, you will need to<br />
send a CV and answer a short questionnaire about iGEM (available by<br />
email). <p class="MsoNormal"><br />
<h1>Further information</h1><br />
<span class="textexposedshow">Learn more about iGEM in<br />
general at: </span><a<br />
href="http://www.facebook.com/l.php?u=http%3A%2F%2Figem.org%2FAbout&amp;h=HAQEjYmHL&amp;s=1"<br />
target="_blank">https://igem.org/About</a><br<br />
style=""><br />
<!--[if !supportLineBreakNewLine]--><br style=""><br />
<!--[endif]--><o:p></o:p><br />
<p class="MsoNormal">Learn about this year’s team by<br />
clicking on the links above.<o:p></o:p></p><br />
<p class="MsoNormal"><br><br />
<span class="textexposedshow">Also browse the previous<br />
Melbourne <span class="SpellE">Uni</span> iGEM team<br />
at<span class="GramE">:</span></span><br><br />
<a href="http://openwetware.org/wiki/IGEM:Melbourne/2008"<br />
target="_blank">http://openwetware.org/wiki/IGEM:Melbourne/2008</a><br><br />
<a<br />
href="http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-melbourne-university-te"<br />
target="_blank">http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-<span<br />
class="SpellE">melbourne</span>-university-<span<br />
class="SpellE">te</span></a><span<br />
class="textexposedshow"><o:p></o:p></span></p><br />
<!-- END EDIT HERE HERE --><br />
</body><br />
</html></div>Slowehttp://2014.igem.org/Team:Melbourne/RecruitmentTeam:Melbourne/Recruitment2014-12-06T10:11:56Z<p>Slowe: </p>
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table {<br />
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html, body {<br />
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<img src="https://static.igem.org/mediawiki/2014/9/9b/Melbourne_Banner.png"<br />
alt="Banner" height="225" width="1000"><br />
<table<br />
style="text-transform: uppercase; font-family: 'Lato',sans-serif;"<br />
width="100%"><br />
<tbody><br />
<tr height="10"><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne"<br />
style="color: rgb(0, 0, 0);">Home</a> </td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Team"<br />
style="color: rgb(0, 0, 0);">Team</a> </td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Project"<br />
style="color: rgb(0, 0, 0);">Project</a> </td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Human_Practices"<br />
style="color: rgb(0, 0, 0);"> Human Practices</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Achievements"<br />
style="color: rgb(0, 0, 0);">Achievements</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Notebook"<br />
style="color: rgb(0, 0, 0);">Notebook</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Protocols"<br />
style="color: rgb(0, 0, 0);"> Protocols</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Safety"<br />
style="color: rgb(0, 0, 0);">Safety</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Sponsors"<br />
style="color: rgb(0, 0, 0);">Sponsors</a></td><br />
<td onmouseover="this.bgColor='#d3d3d3'"<br />
onmouseout="this.bgColor='#e7e7e7'" align="center"<br />
bgcolor="#e7e7e7" height="30"><br />
<a href="https://2014.igem.org/Team:Melbourne/Attributions"<br />
style="color: rgb(0, 0, 0);">Attributions</a></td><br />
<td align="right"> <a<br />
href="https://2014.igem.org/Main_Page"> <img<br />
src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png"<br />
width="46"></a> </td><br />
</tr><br />
</tbody><br />
</table><br />
<p class="MsoNormal" style="margin-top: 12pt;">Thank<br />
you for your interest in the<br />
2015 Melbourne University iGEM Team! Please read the following<br />
information<br />
about iGEM, the team, and what you need to do to get involved.</p><br />
<h1>What is iGEM?<o:p></o:p></h1><br />
<p class="MsoNormal"><span class="GramE"><span<br />
class="usercontent">iGEM</span></span><span<br />
class="usercontent"> is a unique opportunity to get involved<br />
in an enterprising<br />
student research group and make a scientific impact.</span><br><br />
<br><br />
<span class="GramE"><span class="textexposedshow">iGEM</span></span><span<br />
class="textexposedshow"> is short for the International<br />
Genetically Engineering<br />
Machine competition. <span class="GramE">iGEM</span><br />
is an undergraduate science<br />
competition held each November in Boston. In the months leading up to<br />
iGEM,<br />
university teams use the latest tools from synthetic<br />
biology/biotechnology to<br />
create a novel single celled organism or “biological machine”.</span></p><br />
<p class="MsoNormal"><span data-ft="{&quot;tn&quot;:&quot;K&quot;}"<br />
class="userContent"> iGEM is an international competition<br />
based on the new scienc<span class="text_exposed_show">e<br />
of synthetic biology. Each year, students from around the globe form<br />
teams at their respective universities with the goal of building a<br />
biological system or tool, which they present at the iGEM global<br />
conference in Boston. Students in the past have designed bacteria that<br />
produce new types of drugs and biofuels, act as biosensors of toxic<br />
pollutants, and even perform simple computation (for examples of past<br />
projects, see <a href="https://igem.org/About"<br />
rel="nofollow nofollow" target="_blank"<br />
onmouseover='LinkshimAsyncLink.swap(this, "http:\/\/igem.org\/About");'<br />
onclick='LinkshimAsyncLink.swap(this, "http:\/\/l.facebook.com\/l.php?u=http\u00253A\u00252F\u00252Figem.org\u00252FAbout&h=zAQFmUzcK&enc=AZNGkoGC-HF5q40u8IHnH2vOwvgHdyy0ClVop-mb6cjZGHojVOVS9IRhqf-Bi3W3tjr6Zt8No6t5_Rcx8yzlpP7oLfAxZyO28QciO52K7yqag82-VE7nwRVRmBKK-HgbxqVsq9pAa5Lv-OJPoe-BAhfl&s=1");'>https://igem.org/About</a>)</span></span>.<span<br />
class="textexposedshow"> </span><o:p></o:p></p><br />
<p class="MsoNormal"><span class="GramE"><span<br />
class="textexposedshow">iGEM</span></span><span<br />
class="textexposedshow"> teams manage everything from the<br />
conception to the<br />
execution of the iGEM project, with the aid of faculty supervisors. </span><br><br />
<br><br />
<span class="textexposedshow">The benefits to<br />
participating in iGEM include:</span><br><br />
<span class="textexposedshow">-Get valuable scientific and<br />
leadership experience</span><br><br />
<span class="textexposedshow">-Develop a useful, novel<br />
biotechnology and gain experience<br />
in the latest in interdisciplinary biological research<o:p></o:p></span></p><br />
<p class="MsoNormal"><span class="textexposedshow">-Make<br />
an impact</span><br><br />
<span class="textexposedshow">-Have an incredible<br />
experience!</span><br><br />
<br><br />
</p><br />
<h1><span class="GramE"></span>What is<br />
involved and what is needed</h1><br />
<p class="MsoNormal">We are searching for enthusiastic<br />
students with<br />
an interest in science.<o:p></o:p></p><br />
<p class="MsoNormal"><o:p>&nbsp;</o:p></p><br />
<p class="MsoNormal" style="page-break-after: avoid;">As<br />
part of the iGEM team, you would help with the<br />
following tasks:<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpFirst"<br />
style="text-indent: -18pt; page-break-after: avoid;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->Research<br />
on the iGEM project ideas. The ideas<br />
for the iGEM project are student driven, and team members often need to<br />
answer<br />
specific research question to design new experiments or come up with<br />
new ideas.<br />
This will typically involve doing quick searches of the literature<br />
using Google<br />
Scholar and reading scientific articles.<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpMiddle"<br />
style="text-indent: -18pt; page-break-after: avoid;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->Help<br />
with developing experimental methods. The<br />
2014 team has built up a library of protocols and experimental methods.<br />
However, the project for 2015 will likely require new methods. You will<br />
need to<br />
look up protocols in the literature and adapt them to the project<br />
requirements.<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpMiddle"<br />
style="text-indent: -18pt;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->Wet<br />
lab work OR modelling/computation OR engineering<br />
design. By joining the iGEM team, you will have an opportunity to<br />
participate in<br />
the lab and learn many standard techniques in molecular biology from<br />
fellow lab<br />
members. Alternatively, many iGEM teams make use of the skills of<br />
engineers,<br />
computer scientists, and other non-biological science disciplines. This<br />
may<br />
take the form of, for example, <span class="SpellE">modeling</span><br />
a biological<br />
system using software like <span class="SpellE">Matlab</span>,<br />
designing microfluidic devices with biological applications or<br />
designing an electrical device which interfaces with a biological<br />
system. If<br />
you have an interest in interdisciplinary research between your field<br />
and biology,<br />
it is likely iGEM will be able to accommodate it.<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpLast"<br />
style="text-indent: -18pt;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->The<br />
scope of iGEM also extends into<br />
non-traditional science areas, including biotechnology<br />
entrepreneurship,<br />
biotechnology ethics and the law, and science outreach, and so we are<br />
actively<br />
seeking students with business, law, design, and arts backgrounds. You<br />
could,<br />
for example, create a business plan for <span class="GramE">a</span><br />
iGEM-created<br />
company, examine bioethics within synthetic biology, or even explore<br />
the<br />
potential of biologically-inspired art.<o:p></o:p></p><br />
<p class="MsoNormal"><o:p>&nbsp;</o:p></p><br />
<p class="MsoNormal">There are several traits needed on<br />
the iGEM team:<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpFirst"<br />
style="text-indent: -18pt;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]--><span<br />
class="GramE">Research skills. IGEM</span> is an<br />
exciting<br />
opportunity to undertake self-directed research in synthetic biology.<br />
We will<br />
therefore need students who are keenly interested and adept in<br />
research. To<br />
participate, you will need to have the capacity to quickly get up to<br />
speed in<br />
the field of biotechnology and to eventually excel in a lab with<br />
limited<br />
instruction.<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpMiddle"<br />
style="text-indent: -18pt;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->We’re<br />
looking for team members from a range of<br />
backgrounds. iGEM is about cross-disciplinary research, so in addition<br />
to<br />
biological and biomedical science students, we welcome students from<br />
engineering (electrical, mechanical, chemical, software etc.), computer<br />
science, maths, physics, chemistry, and other physical sciences. Also,<br />
students<br />
from non-science backgrounds are very welcome to get in touch to<br />
explore how they<br />
can contribute to the team.<o:p></o:p></p><br />
<p class="MsoListParagraphCxSpLast"<br />
style="text-indent: -18pt;"><!--[if !supportLists]--><span<br />
style="font-family: Symbol;"><span style="">·<span<br />
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><!--[endif]-->Participation<br />
would be most suited to students<br />
with a high level of academic maturity. Typically, this includes third<br />
year,<br />
honours, or Masters <span class="GramE">students</span>,<br />
<span class="SpellE">but students</span><br />
from all levels who can demonstrate an aptitude for research or<br />
leadership are welcome<br />
to apply.<o:p></o:p></p><br />
<p class="MsoNormal">A biological science background is<br />
helpful, but not<br />
required. Team members will need to use knowledge from second-level<br />
biology subjects.<br />
However, we have had team members without a biological science<br />
background who<br />
have excelled. <o:p></o:p></p><br />
<h1>Recruitment</h1><br />
<p class="MsoNormal">If you are interested in being a part<br />
of iGEM, please email<br />
Sean Lowe at MelbourneUniIGem@gmail.com and indicate the role(s) you<br />
are<br />
interested in. <o:p></o:p></p><br />
<p class="MsoNormal">Further information about iGEM and<br />
updates about the<br />
recruitment process will be made available at <a<br />
href="https://www.facebook.com/MelbourneUniIGem">https://www.facebook.com/MelbourneUniIGem</a>.<o:p></o:p></p><br />
<br><br />
We are currently seeking expressions of interest from keen students who<br />
wish to get involved with iGEM. Over the summer, we are looking for<br />
students to form part of the founding team for 2015. As part of this<br />
team, you would work to set the framework and goals for next year. With<br />
the guidance and assistance of the current team, you would liaise with<br />
faculty and sponsors to set up support systems for the team. Most<br />
importantly, you will be need to be able to generate a vision for what<br />
a student-based science team can and should be, it’s potential and<br />
opportunities.<br><br />
<br><br />
Recruitment is now open. Roles will filled in December and throughout<br />
the summer. If you wish to be involved over the summer, please get in<br />
touch with us as soon as possible and before 5 January 2015.<br><br />
Any positions on the team which are not filled over the summer will be<br />
filled in weeks 1-2 of semester 1, 2015. Note that applications at the<br />
start of the semester can be very competitive,&nbsp;so if<br />
interested, please get in touch now. <br><br />
<br><br />
If your skills and interests match up with the team, you will need to<br />
send a CV and answer a short questionnaire about iGEM (available by<br />
email). <br><br />
<h1>Further information</h1><br />
<span class="textexposedshow">Learn more about iGEM in<br />
general at: </span><a<br />
href="http://www.facebook.com/l.php?u=http%3A%2F%2Figem.org%2FAbout&amp;h=HAQEjYmHL&amp;s=1"<br />
target="_blank">https://igem.org/About</a><br<br />
style=""><br />
<!--[if !supportLineBreakNewLine]--><br style=""><br />
<!--[endif]--><o:p></o:p><br />
<p class="MsoNormal">Learn about this year’s team by<br />
clicking on the links above.<o:p></o:p></p><br />
<p class="MsoNormal"><br><br />
<span class="textexposedshow">Also browse the previous<br />
Melbourne <span class="SpellE">Uni</span> iGEM team<br />
at<span class="GramE">:</span></span><br><br />
<a href="http://openwetware.org/wiki/IGEM:Melbourne/2008"<br />
target="_blank">http://openwetware.org/wiki/IGEM:Melbourne/2008</a><br><br />
<a<br />
href="http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-melbourne-university-te"<br />
target="_blank">http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-<span<br />
class="SpellE">melbourne</span>-university-<span<br />
class="SpellE">te</span></a><span<br />
class="textexposedshow"><o:p></o:p></span></p><br />
<!-- END EDIT HERE HERE --><br />
</body><br />
</html></div>Slowehttp://2014.igem.org/File:Melbourne_homepage_bg_low_qual.jpgFile:Melbourne homepage bg low qual.jpg2014-12-04T10:03:50Z<p>Slowe: uploaded a new version of &quot;File:Melbourne homepage bg low qual.jpg&quot;</p>
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<div></div>Slowehttp://2014.igem.org/Team:MelbourneTeam:Melbourne2014-12-04T09:47:26Z<p>Slowe: </p>
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/**<br />
* 1. Remove default vertical scrollbar in IE 8/9.<br />
* 2. Improve readability and alignment in all browsers.<br />
*/<br />
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textarea {<br />
overflow: auto; /* 1 */<br />
vertical-align: top; /* 2 */<br />
}<br />
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/* ==========================================================================<br />
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<br />
/**<br />
* Remove most spacing between table cells.<br />
*/<br />
<br />
table {<br />
border-collapse: collapse;<br />
border-spacing: 0;<br />
}<br />
<br />
html, body {<br />
color: #111;<br />
background-color: transparent;<br />
width: 100%;<br />
height: 100%;<br />
margin: 0;<br />
padding: 0;<br />
display:block;<br />
line-height:normal;<br />
<br />
}<br />
<br />
h1 {<br />
font-weight:bold;<br />
font-size:52px; <br />
margin:0;<br />
text-transform:uppercase;<br />
font-family: 'Lato', sans-serif;<br />
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h2 {<br />
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<div id="intro"><br />
<img src="https://static.igem.org/mediawiki/2014/7/73/Site_logo_invert.png" alt="Mel Uni iGEM Logo" width="706" height="175"><br />
<br />
<p>Welcome to 2014 University of Melbourne iGEM Wiki. At the University of Melbourne, we’ve been developing systems for expressing star peptides in E. coli. We welcome you to step into our scientific stratosphere to learn more about star peptides and the Melbourne Uni iGEM team.</p><br />
<br />
</div><br />
<br />
<br />
<br />
<table width="100%" style="text-transform: uppercase; font-family: 'Lato', sans-serif;"><br />
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<a href="https://2014.igem.org/Team:Melbourne"style="color:#000000"><br />
Home</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Team"style="color:#000000"><br />
Team</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Project"style="color:#000000"><br />
Project</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Human_Practices"style="color:#000000"> <br />
Human Practices</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Achievements"style="color:#000000"><br />
Achievements</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Notebook"style="color:#000000"><br />
Notebook</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Protocols"style=" color:#000000"> <br />
Protocols</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Safety"style="color:#000000"><br />
Safety</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Sponsors"style="color:#000000"><br />
Sponsors</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Attributions"style="color:#000000"><br />
Attributions</a></td><br />
<br />
<br />
<td align="right"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="46px"></a> </td><br />
</tr><br />
</table><br />
<br />
<h1 >University of Melbourne<br>iGEM team </h1><br />
<h3>Star peptides are an exciting type of peptide architecture, with the many different types of star peptides forming a constellation of useful molecules. Watch the video below to find out more.</h3><br />
<br />
<table align="center"><br />
<tr><br />
<td align="center"><br />
<iframe width="828" height="465" src="//www.youtube.com/embed/3g2MlA2py8c" frameborder="0" allowfullscreen></iframe></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<p>Direct download of the video is available on the iGEM server <a href="https://2014.igem.org/File:University_of_Melbourne_intro_video.mov">here</a>.</p><br />
<br />
<h3>Browse through our website to learn more details of our star peptide design concept here at the University of Melbourne.</h3><br />
<p>&nbsp;</p><br />
<h4>Also visit us on Facebook at <a href="https://www.facebook.com/MelbourneUniIGem"style="color:#000000"><br />
https://www.facebook.com/MelbourneUniIGem</a> or get in touch at MelbourneUniIgem@gmail.com.</h4><br />
<br />
<br><br />
<br />
<table width="100%" height="1" border="1"><br />
<tr><td></td></tr></table><br />
<p>&nbsp;</p><br />
<h3>Sponsors:</h3><br />
<p>&nbsp;</p><br />
<table width="889"><br />
<tr><br />
<td width="364" ><p><img src="https://static.igem.org/mediawiki/2014/4/4c/Melbourne_DSI.jpg" width="363" height="128" alt=""/></p></td><br />
<td width="167"><p><img src="https://static.igem.org/mediawiki/2014/e/e8/Melbourne_Bio21_Institute.jpg" width="115" height="196" alt=""/></p></td><br />
<td width="342" ><p><img src="https://static.igem.org/mediawiki/2014/6/61/Melbourne_Department_of_biochemistry.png" width="252" height="185" alt=""/></p></td><br />
</tr><br />
</table><br />
<table width="800"><br />
<tr><br />
<td width="365"><table width="800"><br />
<tr><br />
<td width="80"><img src="https://static.igem.org/mediawiki/2014/5/58/Melbourne_Genesearch.png" width="170" height="53" alt=""/></td><br />
<td width="78"><img src="https://static.igem.org/mediawiki/2014/d/df/Melbourne_GenScript.png" width="183" height="56" alt=""/></td><br />
<td width="109"><img src="https://static.igem.org/mediawiki/2014/7/71/Melbourne_New_England_bio_labs.png" width="171" height="67" alt=""/></td><br />
<td width="365"><img src="https://static.igem.org/mediawiki/2014/a/af/Melbourne_Mettler_Toledo.png" width="188" height="111" alt=""/></td><br />
<td width="365"><img src="https://static.igem.org/mediawiki/2014/9/9c/Melbounre_IndieMosh.png" width="149" height="40" alt=""/></td><br />
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</html></div>Slowehttp://2014.igem.org/File:Melbourne_homepage_bg_low_qual.jpgFile:Melbourne homepage bg low qual.jpg2014-12-04T09:45:57Z<p>Slowe: </p>
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<div></div>Slowehttp://2014.igem.org/Team:Melbourne/RecruitmentTeam:Melbourne/Recruitment2014-12-03T09:26:02Z<p>Slowe: </p>
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<img src="https://static.igem.org/mediawiki/2014/9/9b/Melbourne_Banner.png" alt="Banner" height="225" width="1000"><br />
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<br />
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<a href="https://2014.igem.org/Team:Melbourne"style="color:#000000"><br />
Home</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Team"style="color:#000000"><br />
Team</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Project"style="color:#000000"><br />
Project</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Human_Practices"style="color:#000000"> <br />
Human Practices</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Achievements"style="color:#000000"><br />
Achievements</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Notebook"style="color:#000000"><br />
Notebook</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Protocols"style=" color:#000000"> <br />
Protocols</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Safety"style="color:#000000"><br />
Safety</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Sponsors"style="color:#000000"><br />
Sponsors</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Attributions"style="color:#000000"><br />
Attributions</a></td><br />
<br />
<br />
<td align="right"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="46px"></a> </td><br />
</tr><br />
</table><br />
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<br />
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<br />
<br />
<br />
<p class=MsoNormal style='margin-top:12.0pt'>Thank you for your interest in the<br />
2015 Melbourne University iGEM Team! Please read the following information<br />
about iGEM, the team, and what you need to do to get involved.</p><br />
<br />
<h1>What is iGEM?<o:p></o:p></h1><br />
<br />
<p class=MsoNormal><span class=GramE><span class=usercontent>iGEM</span></span><span<br />
class=usercontent> is a unique opportunity to get involved in an enterprising<br />
student research group and make a scientific impact.</span><br><br />
<br><br />
<span class=GramE><span class=textexposedshow>iGEM</span></span><span<br />
class=textexposedshow> is short for the International Genetically Engineering<br />
Machine competition. <span class=GramE>iGEM</span> is an undergraduate science<br />
competition held each November in Boston. In the months leading up to iGEM,<br />
university teams use the latest tools from synthetic biology/biotechnology to<br />
create a novel single celled organism or “biological machine”. </span><o:p></o:p></p><br />
<br />
<br />
<br />
<p class=MsoNormal><span class=GramE><span class=textexposedshow>iGEM</span></span><span<br />
class=textexposedshow> teams manage everything from the conception to the<br />
execution of the iGEM project, with the aid of faculty supervisors. </span><br><br />
<br><br />
<span class=textexposedshow>The benefits to participating in iGEM include:</span><br><br />
<span class=textexposedshow>-Get valuable scientific and leadership experience</span><br><br />
<span class=textexposedshow>-Develop a useful, novel biotechnology and gain experience<br />
in the latest in interdisciplinary biological research<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal><span class=textexposedshow>-Make an impact</span><br><br />
<span class=textexposedshow>-Have an amazing experience!</span><br><br />
<br><br />
<br />
<br />
<h1><span class=GramE></span>What is involved and what is needed</h1><br />
<br />
<p class=MsoNormal>We are searching for enthusiastic students with<br />
an interest in science.<o:p></o:p></p><br />
<br />
<br />
<br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal style='mso-pagination:widow-orphan lines-together;<br />
page-break-after:avoid'>As part of the iGEM team, you would help with the<br />
following tasks:<o:p></o:p></p><br />
<br />
<p class=MsoListParagraphCxSpFirst style='text-indent:-18.0pt;mso-pagination:<br />
widow-orphan lines-together;page-break-after:avoid;mso-list:l1 level1 lfo1'><![if !supportLists]><span<br />
style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family:<br />
Symbol'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]>Research on the iGEM project ideas. The ideas<br />
for the iGEM project are student driven, and team members often need to answer<br />
specific research question to design new experiments or come up with new ideas.<br />
This will typically involve doing quick searches of the literature using Google<br />
Scholar and reading scientific articles.<o:p></o:p></p><br />
<br />
<p class=MsoListParagraphCxSpMiddle style='text-indent:-18.0pt;mso-pagination:<br />
widow-orphan lines-together;page-break-after:avoid;mso-list:l1 level1 lfo1'><![if !supportLists]><span<br />
style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family:<br />
Symbol'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]>Help with developing experimental methods. The<br />
2014 team has built up a library of protocols and experimental methods.<br />
However, the project for 2015 will likely require new methods. You will need to<br />
look up protocols in the literature and adapt them to the project requirements.<o:p></o:p></p><br />
<br />
<p class=MsoListParagraphCxSpMiddle style='text-indent:-18.0pt;mso-list:l1 level1 lfo1'><![if !supportLists]><span<br />
style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family:<br />
Symbol'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]>Wet lab work OR modelling/computation OR engineering<br />
design. By joining the iGEM team, you will have an opportunity to participate in<br />
the lab and learn many standard techniques in molecular biology from fellow lab<br />
members. Alternatively, many iGEM teams make use of the skills of engineers,<br />
computer scientists, and other non-biological science disciplines. This may<br />
take the form of, for example, <span class=SpellE>modeling</span> a biological<br />
system using software like <span class=SpellE>Matlab</span>, designing microfluidic devices with biological applications or<br />
designing an electrical device which interfaces with a biological system. If<br />
you have an interest in interdisciplinary research between your field and biology,<br />
it is likely iGEM will be able to accommodate it.<o:p></o:p></p><br />
<br />
<p class=MsoListParagraphCxSpLast style='text-indent:-18.0pt;mso-list:l1 level1 lfo1'><![if !supportLists]><span<br />
style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family:<br />
Symbol'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]>The scope of iGEM also extends into<br />
non-traditional science areas, including biotechnology entrepreneurship,<br />
biotechnology ethics and the law, and science outreach, and so we are actively<br />
seeking students with business, law, design, and arts backgrounds. You could,<br />
for example, create a business plan for <span class=GramE>a</span> iGEM-created<br />
company, examine bioethics within synthetic biology, or even explore the<br />
potential of biologically-inspired art.<o:p></o:p></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal>There are several traits needed on the iGEM team:<o:p></o:p></p><br />
<br />
<p class=MsoListParagraphCxSpFirst style='text-indent:-18.0pt;mso-list:l0 level1 lfo2'><![if !supportLists]><span<br />
style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family:<br />
Symbol'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span class=GramE>Research skills. IGEM</span> is an exciting<br />
opportunity to undertake self-directed research in synthetic biology. We will<br />
therefore need students who are keenly interested and adept in research. To<br />
participate, you will need to have the capacity to quickly get up to speed in<br />
the field of biotechnology and to eventually excel in a lab with limited<br />
instruction.<o:p></o:p></p><br />
<br />
<p class=MsoListParagraphCxSpMiddle style='text-indent:-18.0pt;mso-list:l0 level1 lfo2'><![if !supportLists]><span<br />
style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family:<br />
Symbol'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]>We’re looking for team members from a range of<br />
backgrounds. iGEM is about cross-disciplinary research, so in addition to<br />
biological and biomedical science students, we welcome students from<br />
engineering (electrical, mechanical, chemical, software etc.), computer<br />
science, maths, physics, chemistry, and other physical sciences. Also, students<br />
from non-science backgrounds are very welcome to get in touch to explore how they<br />
can contribute to the team.<o:p></o:p></p><br />
<br />
<p class=MsoListParagraphCxSpLast style='text-indent:-18.0pt;mso-list:l0 level1 lfo2'><![if !supportLists]><span<br />
style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family:<br />
Symbol'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]>Participation would be most suited to students<br />
with a high level of academic maturity. Typically, this includes third year,<br />
honours, or Masters <span class=GramE>students</span>, <span class=SpellE>but students</span><br />
from all levels who can demonstrate an aptitude for research or leadership are welcome<br />
to apply.<o:p></o:p></p><br />
<br />
<p class=MsoNormal>A biological science background is helpful, but not<br />
required. Team members will need to use knowledge from second-level biology subjects.<br />
However, we have had team members without a biological science background who<br />
have excelled. <o:p></o:p></p><br />
<br />
<br />
<br><br />
<p class=MsoNormal>If you are interested in being a part of iGEM, please email<br />
Sean Lowe at MelbourneUniIGem@gmail.com and indicate the role(s) you are<br />
interested in. <o:p></o:p></p><br />
<br />
<br />
<br />
<p class=MsoNormal>Further information about iGEM and updates about the<br />
recruitment process will be made available at <a<br />
href="https://www.facebook.com/MelbourneUniIGem">https://www.facebook.com/MelbourneUniIGem</a>.<o:p></o:p></p><br />
<br />
<br />
<br />
<p class=MsoNormal>Thank you again for your interest and we look forward to<br />
hearing from you.<o:p></o:p></p><br />
<br />
<br><br />
<br />
<span class=textexposedshow>Learn more about iGEM in general at: </span><a<br />
href="http://www.facebook.com/l.php?u=http%3A%2F%2Figem.org%2FAbout&amp;h=HAQEjYmHL&amp;s=1"<br />
target="_blank">https://igem.org/About</a><br style='mso-special-character:line-break'><br />
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'><br />
<![endif]><o:p></o:p></p><br />
<br />
<p class=MsoNormal>Learn about this year’s team by clicking on the links above.<o:p></o:p></p><br />
<br />
<p class=MsoNormal><br><br />
<span class=textexposedshow>Also browse the previous Melbourne <span<br />
class=SpellE>Uni</span> iGEM team at<span class=GramE>:</span></span><br><br />
<a href="http://openwetware.org/wiki/IGEM:Melbourne/2008" target="_blank">http://openwetware.org/wiki/IGEM:Melbourne/2008</a><br><br />
<a<br />
href="http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-melbourne-university-te"<br />
target="_blank">http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-<span<br />
class=SpellE>melbourne</span>-university-<span class=SpellE>te</span></a><span<br />
class=textexposedshow><o:p></o:p></span></p><br />
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<img src="https://static.igem.org/mediawiki/2014/9/9b/Melbourne_Banner.png" alt="Banner" height="225" width="1000"><br />
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<table width="100%" style="text-transform: uppercase; font-family: 'Lato', sans-serif;"><br />
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<a href="https://2014.igem.org/Team:Melbourne"style="color:#000000"><br />
Home</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Team"style="color:#000000"><br />
Team</a> </td><br />
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<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Project"style="color:#000000"><br />
Project</a> </td><br />
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<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Human_Practices"style="color:#000000"> <br />
Human Practices</a></td><br />
<br />
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<a href="https://2014.igem.org/Team:Melbourne/Achievements"style="color:#000000"><br />
Achievements</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Notebook"style="color:#000000"><br />
Notebook</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Protocols"style=" color:#000000"> <br />
Protocols</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Safety"style="color:#000000"><br />
Safety</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Sponsors"style="color:#000000"><br />
Sponsors</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Attributions"style="color:#000000"><br />
Attributions</a></td><br />
<br />
<br />
<td align="right"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="46px"></a> </td><br />
</tr><br />
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<br />
<p class=MsoNormal style='margin-top:12.0pt'>Thank you for your interest in the<br />
2015 Melbourne University iGEM Team! Please read the following information<br />
about iGEM, the team, and what you need to do to get involved.<o:p></o:p></p><br />
<br />
<h1>What is iGEM?<o:p></o:p></h1><br />
<br />
<p class=MsoNormal><span class=GramE><span class=usercontent>iGEM</span></span><span<br />
class=usercontent> is a unique opportunity to get involved in enterprising<br />
student research and make a scientific impact.</span><br><br />
<br><br />
<span class=GramE><span class=textexposedshow>iGEM</span></span><span<br />
class=textexposedshow> is short for the International Genetically Engineering<br />
Machine competition. <span class=GramE>iGEM</span> is an undergraduate science<br />
competition held each November in Boston. In the months leading up to iGEM,<br />
university teams use the latest tools from synthetic biology/biotechnology to<br />
create a novel single celled organism or “biological machine”. </span><o:p></o:p></p><br />
<br />
<br />
<br />
<p class=MsoNormal><span class=GramE><span class=textexposedshow>iGEM</span></span><span<br />
class=textexposedshow> teams manage everything from the conception to the<br />
execution of the iGEM project, with the aid of faculty supervisors. </span><br><br />
<br><br />
<span class=textexposedshow>The benefits to participating in iGEM include:</span><br><br />
<span class=textexposedshow>-Get valuable scientific and leadership experience</span><br><br />
<span class=textexposedshow>-Develop a useful, novel biotechnology and gain experience<br />
in the latest in interdisciplinary biological research<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal><span class=textexposedshow>-Make an impact</span><br><br />
<span class=textexposedshow>-Have an amazing experience!</span><br><br />
<br><br />
<span class=textexposedshow>Learn more about iGEM in general at: </span><a<br />
href="http://www.facebook.com/l.php?u=http%3A%2F%2Figem.org%2FAbout&amp;h=HAQEjYmHL&amp;s=1"<br />
target="_blank">https://igem.org/About</a><br style='mso-special-character:line-break'><br />
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'><br />
<![endif]><o:p></o:p></p><br />
<br />
<p class=MsoNormal>Learn about this year’s team by clicking on the links above.<o:p></o:p></p><br />
<br />
<p class=MsoNormal><br><br />
<span class=textexposedshow>Also browse the previous Melbourne <span<br />
class=SpellE>Uni</span> iGEM team at<span class=GramE>:</span></span><br><br />
<a href="http://openwetware.org/wiki/IGEM:Melbourne/2008" target="_blank">http://openwetware.org/wiki/IGEM:Melbourne/2008</a><br><br />
<a<br />
href="http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-melbourne-university-te"<br />
target="_blank">http://www.bio21.unimelb.edu.au/news/the-bioclock---bacteria-to-mark-time-for-<span<br />
class=SpellE>melbourne</span>-university-<span class=SpellE>te</span></a><span<br />
class=textexposedshow><o:p></o:p></span></p><br />
<br />
<h1><span class=GramE></span>What is involved and what is needed</h1><br />
<br />
<p class=MsoNormal>We are always on the lookout for enthusiastic students with<br />
an interest in science.<o:p></o:p></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal>A biological science background is helpful, but not<br />
required. Team members will need to use knowledge from second-level biology subjects.<br />
However, we have had team members without a biological science background who<br />
have excelled. <o:p></o:p></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal style='mso-pagination:widow-orphan lines-together;<br />
page-break-after:avoid'>As part of the iGEM team, you would help with the<br />
following tasks:<o:p></o:p></p><br />
<br />
<p class=MsoListParagraphCxSpFirst style='text-indent:-18.0pt;mso-pagination:<br />
widow-orphan lines-together;page-break-after:avoid;mso-list:l1 level1 lfo1'><![if !supportLists]><span<br />
style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family:<br />
Symbol'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]>Research on the iGEM project ideas. The ideas<br />
for the iGEM project are student driven, and team members often need to answer<br />
specific research question to design new experiments or come up with new ideas.<br />
This will typically involve doing quick searches of the literature using Google<br />
Scholar and reading scientific articles.<o:p></o:p></p><br />
<br />
<p class=MsoListParagraphCxSpMiddle style='text-indent:-18.0pt;mso-pagination:<br />
widow-orphan lines-together;page-break-after:avoid;mso-list:l1 level1 lfo1'><![if !supportLists]><span<br />
style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family:<br />
Symbol'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]>Help with developing experimental methods. The<br />
2014 team has built up a library of protocols and experimental methods.<br />
However, the project for 2015 will likely require new methods. You will need to<br />
look up protocols in the literature and adapt them to the project requirements.<o:p></o:p></p><br />
<br />
<p class=MsoListParagraphCxSpMiddle style='text-indent:-18.0pt;mso-list:l1 level1 lfo1'><![if !supportLists]><span<br />
style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family:<br />
Symbol'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]>Wet lab work OR modelling/computation OR engineering<br />
design. By joining the iGEM team, you will have an opportunity to participate in<br />
the lab and learn many standard techniques in molecular biology from fellow lab<br />
members. Alternatively, many iGEM teams make use of the skills of engineers,<br />
computer scientists, and other non-biological science disciplines. This may<br />
take the form of, for example, <span class=SpellE>modeling</span> a biological<br />
system using software like <span class=SpellE>Matlab</span>. It might also<br />
involve designing microfluidic devices with biological applications or<br />
designing an electrical device which interfaces with a biological system. If<br />
you have an interest in interdisciplinary research between your field and biology,<br />
it is likely iGEM will be able to accommodate it.<o:p></o:p></p><br />
<br />
<p class=MsoListParagraphCxSpLast style='text-indent:-18.0pt;mso-list:l1 level1 lfo1'><![if !supportLists]><span<br />
style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family:<br />
Symbol'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]>The scope of iGEM also extends into<br />
non-traditional science areas, including biotechnology entrepreneurship,<br />
biotechnology ethics and the law, and science outreach, and so we are actively<br />
seeking students with business, law, design, and arts backgrounds. You could,<br />
for example, create a business plan for <span class=GramE>a</span> iGEM-created<br />
company, examine bioethics within synthetic biology, or even explore the<br />
potential of biologically-inspired art.<o:p></o:p></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal>There are several traits needed on the iGEM team:<o:p></o:p></p><br />
<br />
<p class=MsoListParagraphCxSpFirst style='text-indent:-18.0pt;mso-list:l0 level1 lfo2'><![if !supportLists]><span<br />
style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family:<br />
Symbol'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span class=GramE>Research skills. IGEM</span> is an exciting<br />
opportunity to undertake self-directed research in synthetic biology. We will<br />
therefore need students who are keenly interested and adept in research. To<br />
participate, you will need to have the capacity to quickly get up to speed in<br />
the field of biotechnology and to eventually excel in a lab with limited<br />
instruction.<o:p></o:p></p><br />
<br />
<p class=MsoListParagraphCxSpMiddle style='text-indent:-18.0pt;mso-list:l0 level1 lfo2'><![if !supportLists]><span<br />
style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family:<br />
Symbol'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]>We’re looking for team members from a range of<br />
backgrounds. iGEM is about cross-disciplinary research, so in addition to<br />
biological and biomedical science students, we welcome students from<br />
engineering (electrical, mechanical, chemical, software etc.), computer<br />
science, maths, physics, chemistry, and other physical sciences. Also, students<br />
from non-science backgrounds are very welcome to get in touch to explore how they<br />
can contribute to the team.<o:p></o:p></p><br />
<br />
<p class=MsoListParagraphCxSpLast style='text-indent:-18.0pt;mso-list:l0 level1 lfo2'><![if !supportLists]><span<br />
style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family:<br />
Symbol'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]>Participation would be most suited to students<br />
with a high level of academic maturity. Typically, this includes third year,<br />
honours, or Masters <span class=GramE>students</span>, <span class=SpellE>butstudents</span><br />
from all levels who can demonstrate an aptitude for research or leadership are welcome<br />
to apply.<o:p></o:p></p><br />
<br />
<br><br />
<p class=MsoNormal>If you are interested in being a part of iGEM, please email<br />
Sean Lowe at MelbourneUniIGem@gmail.com and indicate the role(s) you are<br />
interested in. <o:p></o:p></p><br />
<br />
<br />
<br />
<p class=MsoNormal>Further information about iGEM and updates about the<br />
recruitment process will be made available at <a<br />
href="https://www.facebook.com/MelbourneUniIGem">https://www.facebook.com/MelbourneUniIGem</a>.<o:p></o:p></p><br />
<br />
<br />
<br />
<p class=MsoNormal>Thank you again for your interest and we look forward to<br />
hearing from you.<o:p></o:p></p><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
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Achievements</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Notebook"style="color:#000000"><br />
Notebook</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Protocols"style=" color:#000000"> <br />
Protocols</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Safety"style="color:#000000"><br />
Safety</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Sponsors"style="color:#000000"><br />
Sponsors</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Attributions"style="color:#000000"><br />
Attributions</a></td><br />
<br />
<br />
<td align="right"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="46px"></a> </td><br />
</tr><br />
</table><br />
<br />
<h1 >Team</h1><br />
<br />
<h3>About The University of Melbourne</h3><br />
<p>Located in Melbourne, Victoria, The University of Melbourne was founded in 1853 and is Australia’s second oldest university. The university is consistently ranked among the leading universities in the world, with international rankings of world universities placing it at number 1 in Australia and number 34 in the world (Times Higher Education World University Ranking 2013-2014).</p><br />
<br />
<h3>About The 2014 Melbourne iGEM Team</h3><br />
<p>The 2014 Melbourne iGEM team consists of 19 highly-motivated undergraduate students and 1 postgraduate student from a range of disciplines, including biochemistry and molecular biology, chemistry, chemical engineering and bioengineering. The team’s academic and research credentials are well-tested. This year, the team comprises three of The University of Melbourne’s Chancellor’s Scholars (top 0.1% of all Australian high school students) and several team members with research experience at Melbourne’s high-profile research institutes, such as the prestigious Walter and Eliza Hall Institute, The Peter MacCallum Cancer Centre, and The Baker IDI Heart and Diabetes Institute. The team is supervised by Associate Professor Heung-Chin Cheng, Associate Professor Paul Gooley, Associate Professor Neil O’Brien-Simpson, and Dr. Angus Johnston.<br />
The Melbourne iGEM team was founded by a group of enthusiastic and motivated students in 2013. The 2013 team played a key role in establishing the foundations of this year’s team and generating this year’s project. Unfortunately many of the 2013 team members could not continue their work for iGEM in 2014, so credit must also be given to Pedro Avellar Costa, Michelle Tie, Georgina Panshem, Morgana Cerqueira, Jeong Yoon Esther Kim, Winnie Tan, Hannah Nguyen, Mary Teo, Cathy Pitt and Joyce Kant.</p><br />
<br />
<h3>Faculty Supervisors</h3><br />
<table width="100%"><br />
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<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/d/d4/Melbourne_Cheng.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Heung-Chin Cheng (primary supervisor)</strong></p><br />
An Associate Professor with the Department of Biochemistry and Molecular Biology, Heung-Chin has been studying the biochemical basis of the regulation of protein kinases and phosphatases since he was a graduate student at the University of California in 1982. His PhD project unravelled the active site structure of c-AMP-dependent protein kinase, how the kinase recognises its protein substrates and how the kinase is selectively inhibited by its endogenous inhibitor. Heung-Chin was the Melbourne iGEM team&rsquo;s supervisor and he kindly offered us space to work in his lab and assisted us when we had questions about practical procedures, troubleshooting or theory.</td><br />
</tr><br />
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<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/7/74/Melbourne_Gooley.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Paul Gooley</strong></p><br />
Associate Professor Paul Gooley directs a structural biology research group that focuses on the application of NMR spectroscopy to elucidate structure and protein interactions. He obtained his degrees at The University of New South Wales and spent 10 years in the USA, including 5 years at the pharmaceutical company Merk and Co. Over the last 10 years his group has conducted and published NMR structural and dynamical analyses on a number of protein domains and systems that have biological functions in stress and infection, in lipid transport, in protein and membrane trafficking, and in receptor signalling. Paul assisted the 2014 Melbourne iGEM team by providing advice, answering questions and brainstorming.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/9/97/Melbourne_Angus.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Angus Johnston</strong></p><br />
Dr Angus Johnston is a researcher at the Monash Institute of Pharmaceutical Science. Angus is an ARC Future Fellow who’s work focuses on developing better ways to deliver drugs, making them more therapeutically active and limiting side effects. He has extensive knowledge and expertise in nanomaterials assembly, material characterisation, cellular interactions and advanced imaging techniques. Angus’ research interests include targeted vaccine therapy, sensors for cellular imaging, understanding cellular processing of nanoparticles, self assembling peptides as drug carrier and the toxicology of nanomaterials. Angus assisted the 2014 Melbourne iGEM team by providing advice, answering questions and brainstorming.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/f/fd/Melbourne_Neil_O%E2%80%99Brien-Simpson.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Neil O’Brien-Simpson</strong></p><br />
Associate Professor Neil O’Brien-Simpson is a researcher at the Royal Dental Hospital in Melbourne and has an interdisciplinary background, combining organic and peptide chemistry with immunology to develop novel vaccines and therapeutics and investigating the immune response to pathogens. His research into vaccine design and periodontitis has resulted in Neil being awarded the Colgate Prize for Dental Research (1999), The IADR Hatton Award (2000) and the Oral Biology Award (2003). In 2004 he became program co-leader for the Novel Diagnostics, Vaccines and Pharmaceuticals Research Program in the Oral Health CRC. Neil assisted the 2014 Melbourne iGEM team by providing advice, answering questions and brainstorming.</td><br />
</tr><br />
</table><br />
<br />
<h3>Team Members</h3><br />
<table width="100%"><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/2/25/Melbourne_Sean.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Sean Lowe (Team Leader)</strong></p><br />
Sean started the 2014 Melbourne iGEM team while completing his Master of Engineering in chemical engineering. As the team leader, Sean was responsible for communicating with supervisors and sponsors, setting up the lab, and making sure the team ran smoothly. Sean is passionate about science and saw iGEM as an amazing opportunity to combine his interests in chemical engineering and biotechnology. He found that the best part of iGEM was the creative process, and thoroughly enjoyed discussing many great ideas with his bright and capable teammates. He looks forward to passing on the torch to the next generation of Melbourne Uni iGEMers.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/a/a6/Melbourne_Lizzy.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Elizabeth Brookes</strong></p><br />
Elizabeth is in her second year of the Bachelor of Biomedicine degree and is majoring in Immunology. In the future Elizabeth hopes to continue to develop her passion for research while studying postgraduate medicine. Elizabeth was new to iGEM in 2014 and contributed to Melbourne’s iGEM project by working in the laboratory, developing protocols and performing various administrative duties. For Elizabeth, the most enjoyable aspect of iGEM was getting hands-on experience while meeting like-minded people. In her free time Elizabeth enjoys playing netball and discovering new restaurants with friends.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/f/f6/Melbourne_Peter.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Peter Collins</strong></p><br />
Peter is in his final year of the Bachelor of Science at The Australian National University in Canberra and is majoring in Science Communications, as well as completing his first year of the Bachelor of Environments at The University of Melbourne. In the future, Peter aims to work on The Human Variome Project, develop the iGEM competition in Australia and work for UNESCO. Peter joined the Melbourne iGEM team in 2014 and was in charge of human practices and outreach, working to establish relations with high schools in Melbourne for on-going public outreach efforts in the future. As such, Peter has enjoyed getting an insight into managing science innovation teams and the tremendous array of challenges that accompany this. In his spare time, Peter enjoys learning about esoteric topics that may have unrealised commercial applications.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/1/1a/Melbourne_Sheryl.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Sheryl Ding</strong></p><br />
Sheryl is in the first year of a Bachelor of Biomedicine degree and is hoping to major in Bioengineering Systems. In the future, Sheryl would like to work in biomedical research, preferably specialising in engineering or molecular and cellular biology. After joining the iGEM team in early 2014, Sheryl contributed to the laboratory work and some administrative tasks for the project. Sheryl enjoyed all aspects of this experience, but particularly the opportunity to gain practical insight into laboratory research and meet the people on the iGEM team. Outside of science, Sheryl enjoys visual art and watching TV shows.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/6/66/Melbourne_Robyn.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Robyn Esterbauer</strong></p><br />
As a second year student of the Bachelor of Science with a major in Microbiology and Immunology, Robyn hopes to undertake a masters and PhD, specialising in infectious disease research. After joining the team in March 2014, Robyn contributed to the team with her work in the laboratory, research into cysteine bond formation and computational protein design analysis, and public outreach activities. Robyn has enjoyed experiencing a group research environment and learning some very useful practical laboratory skills. In her free time, Robyn loves to read cutting edge infectious disease research and playing soccer.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/d/d4/Melbourne_Tobias.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Tobias Gustavsson</strong></p><br />
Tobias is in his second year of the Bachelor of Biomedicine degree and is majoring in Immunology. In the future Tobias hopes to study medicine and continue to pursue research in immunology. Tobias was new to iGEM in 2014 and contributed to the theoretical design and research of the team’s project. iGEM presented Tobias with the opportunity to finally partake in the laboratory work that he had heard so much about in lectures.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/e/e8/Melbourne_Henry.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Henry Howard</strong></p><br />
Henry has just completed his Honours year for the Bachelor of Biomedicine and in the future he hopes to undertake a PhD and work in Melbourne’s bustling medical research sector. After joining the iGEM team in February 2013, Henry predominantly contributed to the theoretical design of the project. He most enoyed having access to resources that have allowed us to undertake serious scientific research of our own design. Outside of iGEM, Henry is a black belt in Tang Soo Tao and enjoys planting trees and completing jigsaw puzzles.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/8/8f/Melbourne_Aishwarya.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Aishwarya Kulkarni</strong></p><br />
Aishwarya is in her second year of the Bachelor of Science and is majoring in Biochemistry and Molecular Biology. Her future plans include completing a PhD and research in the area of translational bioinformatics and personalised medicine. As part of the Melbourne iGEM 2014 team, Aishwarya contributed to laboratory and budgeting tasks. She enjoyed both the atmosphere of the team and the challenges with which we were confronted when trying to work independently to solve problems. Outside of iGEM, Aishwarya also enjoys playing tennis.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/2/2a/Melbourne_Ranit.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Ranit Lahmy</strong></p><br />
Ranit is in her final year of the Bachelor of Science degree and is completing her major in Chemistry. Ranit joined the Melbourne iGEM team in early 2013 and has contributed to many facets of the project, including laboratory work, administration and human outreach. As a consequence, Ranit has really enjoyed the chance to meet new people while learning research and laboratory skills. When not working on iGEM, Ranit enjoys reading books, playing tennis and hanging out with friends.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/b/bc/Melbourne_Jeffrey.JPG" width="150" height="196" alt=""/></td><br />
<td><p><strong>Jeffrey Lai</strong></p><br />
Jeffrey is in his first year of the Bachelor of Biomedicine at The University of Melbourne and in the future he would like to become a medical practitioner or researcher. After joining the Melbourne team in July 2014, Jeffrey helped out in the laboratory work and the iGEM website. Jeffrey found it incredibly rewarding when we finally saw some promising results after putting so much time and effort into the project. In his spare time, Jeffrey likes to play the piano and compose symphonic music.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/1/10/Melbourne_Melissa.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Melissa Leckie</strong></p><br />
Melissa is in her final year of the Bachelor of Science and is completing a major in biochemistry and molecular biology. Competing in her first iGEM jamboree in 2008, Melissa joined the current team in 2013 and has contributed to project design, administration and human outreach. Through this experience Melissa has enjoyed making life long friends and facing the challenges of being part of a student run team. Melissa wishes to pursue a career in Medical Technology and in her spare time she enjoys reading and travelling.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/3/3c/Melbourne_Keit.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Keit Loi</strong></p><br />
Keit is in his final year of the Bachelor of Biomedicine and is majoring in infection and immunity. Since joining the Melbourne iGEM team in early 2014, Keit has mainly contributed to the laboratory work for the project and he has enjoyed that this has given him a good understanding of what working in research would be like. In the future, Keit would like to pursue this passion and work towards a PhD and university lecturing. Outside of iGEM, Keit works on projects with high school students and enjoys chilling with friends and kicking back with some video games.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/0/07/Melbourne_Darcy.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Darcy Marum</strong></p><br />
Darcy is in his second year of the Bachelor of Science degree and is majoring in Microbiology and Immunology. Darcy hopes to pursue research in these exciting fields after completing an honours year. After joining the team in early 2014, Darcy predominantly worked on laboratory work for the project and enjoyed the practical experience that he gained.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/2/2e/Melbourne_Gayle.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Gayle Pereira</strong></p><br />
Gayle is in her final year of the Bachelor of Science and is majoring in Pathology. In the future Gayle hopes to pursue a career either as a clinical pathologist or secondary school teacher. Gayle joined the iGEM team in 2013 and was the senior laboratory leader for the group, tirelessly working and teaching others in the laboratory. Working on the project, Gayle appreciated gaining the laboratory experience that she requires for her future ambitions. In her free time Gayle enjoys supporting her team in the Australian Football League and doing handicraft-based hobbies.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/b/b1/Melbourne_Lumi.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Lumi Tomren</strong></p><br />
Lumi is in her first year of the Bachelor of Biomedicine at The University of Melbourne and is planning on majoring in Microbiology and Immunology. With a desire to learn more about synthetic biology and the microscopic world around us, Lumi joined the team in Spring 2014. Lumi has contributed to the team by performing lab work and she has really enjoyed learning a lot of new practical techniques. She has also enjoyed discovering many of the possibilities of synthetic biology by researching past projects undertaken by iGEM teams. Outside of iGEM and science, Lumi enjoys visual art.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/d/d6/Melbourne_Michael.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Michael Wei</strong></p><br />
Michael is in his first year of the Bachelor of Biomedicine and plans on majoring in neuroscience. Michael was new to iGEM in 2014 and worked primarily in the laboratory and designing primers. Michael is interested in working towards a career in neurosurgery or cancer tumour research, looking specifically at the epigenetic mechanisms underlying cancer regulation. As part of the iGEM team, Michael has enjoyed meeting like-minded individuals who share similar interests. Michael is fascinated by marine life and enjoys reading in his leisure time.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/0/07/Melbourne_Stan.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Stanislav Yatskevich</strong></p><br />
Although Stanislav started studying his first year of a Bachelor of Biomedicine at The University of Melbourne, he has now commenced study at The University of Oxford reading Biochemistry. Stanislav joined the Melbourne iGEM team in March 2014 and contributed greatly to the theoretical project design and laboratory work. After moving to Oxford, Stanislav also facilitated communication between our team and the Oxford iGEM team. In the future, Stanislav would like to pursue research in Biochemistry and he has appreciated the amount of experience he was able to gain in such a short time while taking part in iGEM. Outside of Biochemistry, Stanislav enjoys playing football and procrastinating his study.</td><br />
</tr><br />
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<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/d/dc/Melbourne_Nicholas.JPG" width="150" height="196" alt=""/></td><br />
<td><p><strong>Nicholas Yee</strong></p><br />
Nicholas has just completed the Honours year of his Bachelor of Biomedicine degree, studying novel pathway inhibitors to elucidate mechanisms of brain tumours. He plans to continue his research into brain tumours by undertaking a PhD and potentially studying medicine. After joining the team in 2014, Nicholas enjoyed helping the younger students in the team by teaching them new laboratory techniques and troubleshooting their experiments. When not in the laboratory, Nicholas enjoys hanging out with friends and playing badminton.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/1/1b/Melbourne_Michelle.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Michelle Zheng</strong></p><br />
Michelle is in her second year of the Bachelor of Science and is undertaking the Biochemistry major. At the completion of her degree, Michelle seeks to pursue further research through the honours or masters programmes. Michelle joined the Melbourne iGEM team in 2014 and completed many hours of laboratory work contributing to the project. Being part of a team dedicated to science, the laboratory work and the challenge of overcoming troubleshooting problems were the aspects of iGEM that excited Michelle most. In her free time she enjoys playing the violin.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/0/0f/Melbourne_Wayne.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Wayne Zheng</strong></p><br />
Wayne is in his first year of the Bachelor of Biomedicine and hopes to eventually major in Human Structure and Function. In the future, Wayne would like to continue his involvement in biomedical research while also studying and practising medicine. In contributing to the 2014 Melbourne iGEM team Wayne performed laboratory work as well as some theoretical research. Wayne loved learning new techniques in the laboratory and reading into a wide range of research topics.</td><br />
</tr><br />
<br />
</table><br />
<br />
<br />
<h3>Advisors</h3><br />
<p>A special mention must also be made to our Honours, Masters and PhD student advisors in the Cheng Lab who tirelessly and patiently assisted with our troubleshooting and planning of experimental procedures. These students were: <br />
<br />
Ashfaqul Hoque (PhD student), Gahana Advani (PhD student), George Cao (Masters student), Ken Ang (Honours student) and David Zula (Honours student).</p><br />
<br />
<table><br />
<tr><br />
<td width="152" ><img src="https://static.igem.org/mediawiki/2014/5/58/Melbourne_Ashfaqul.jpg" width="150" height="196" alt=""/></td><br />
<td width="10" ></td><br />
<td width="152" ><img src="https://static.igem.org/mediawiki/2014/d/da/Melbourne_Gahana.jpg" width="150" height="196" alt=""/></td><br />
<td width="10" ></td><br />
<td width="152" ><img src="https://static.igem.org/mediawiki/2014/f/f8/Melbourne_George.jpg" width="150" height="196" alt=""/></td><br />
<td width="10" ></td><br />
<td width="152" ><img src="https://static.igem.org/mediawiki/2014/a/aa/Melbourne_Ken.jpg" width="150" height="196" alt=""/></td><br />
<td width="10" ></td><br />
<td width="152" ><img src="https://static.igem.org/mediawiki/2014/9/9c/Melbourne_Dave.jpg" width="150" height="196" alt=""/></td><br />
</tr><br />
<br />
<tr><br />
<td align="center"><strong>Ashfaqul Hoque</strong></td><br />
<td></td><br />
<td align="center"> <strong>Gahana Advani</strong></td><br />
<td></td><br />
<td align="center"> <strong>George Cao</strong></td><br />
<td></td><br />
<td align="center"><strong>Ken Ang</strong></td><br />
<td></td><br />
<td align="center"><strong>David Zula</strong></td><br />
</tr><br />
<br />
</table><br />
<br />
<p>&nbsp;</p><br />
<p>Many thanks also go to <strong>Sze-Ting Bong</strong> and <strong>Anna Gakusurudoi</strong>, Masters students in the Cheng lab whose assistance with practical methods was extremely helpful.<br />
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/**<br />
* Address style set to `bolder` in Firefox 4+, Safari 5, and Chrome.<br />
*/<br />
<br />
b,<br />
strong {<br />
font-weight: bold;<br />
}<br />
<br />
/**<br />
* Address styling not present in Safari 5 and Chrome.<br />
*/<br />
<br />
dfn {<br />
font-style: italic;<br />
}<br />
<br />
/**<br />
* Address differences between Firefox and other browsers.<br />
*/<br />
<br />
hr {<br />
-moz-box-sizing: content-box;<br />
box-sizing: content-box;<br />
height: 0;<br />
}<br />
<br />
/**<br />
* Address styling not present in IE 8/9.<br />
*/<br />
<br />
mark {<br />
background: #ff0;<br />
color: #000;<br />
}<br />
<br />
/**<br />
* Correct font family set oddly in Safari 5 and Chrome.<br />
*/<br />
<br />
code,<br />
kbd,<br />
pre,<br />
samp {<br />
font-family: monospace, serif;<br />
font-size: 1em;<br />
}<br />
<br />
/**<br />
* Improve readability of pre-formatted text in all browsers.<br />
*/<br />
<br />
pre {<br />
white-space: pre-wrap;<br />
}<br />
<br />
/**<br />
* Set consistent quote types.<br />
*/<br />
<br />
q {<br />
quotes: "\201C" "\201D" "\2018" "\2019";<br />
}<br />
<br />
/**<br />
* Address inconsistent and variable font size in all browsers.<br />
*/<br />
<br />
small {<br />
font-size: 80%;<br />
}<br />
<br />
/**<br />
* Prevent `sub` and `sup` affecting `line-height` in all browsers.<br />
*/<br />
<br />
sub,<br />
sup {<br />
font-size: 75%;<br />
line-height: 0;<br />
position: relative;<br />
vertical-align: baseline;<br />
}<br />
<br />
sup {<br />
top: -0.5em;<br />
}<br />
<br />
sub {<br />
bottom: -0.25em;<br />
}<br />
<br />
/* ==========================================================================<br />
Embedded content<br />
========================================================================== */<br />
<br />
/**<br />
* Remove border when inside `a` element in IE 8/9.<br />
*/<br />
<br />
img {<br />
border: 0;<br />
}<br />
<br />
/**<br />
* Correct overflow displayed oddly in IE 9.<br />
*/<br />
<br />
svg:not(:root) {<br />
overflow: hidden;<br />
}<br />
<br />
/* ==========================================================================<br />
Figures<br />
========================================================================== */<br />
<br />
/**<br />
* Address margin not present in IE 8/9 and Safari 5.<br />
*/<br />
<br />
figure {<br />
margin: 0;<br />
}<br />
<br />
/* ==========================================================================<br />
Forms<br />
========================================================================== */<br />
<br />
/**<br />
* Define consistent border, margin, and padding.<br />
*/<br />
<br />
fieldset {<br />
border: 1px solid #c0c0c0;<br />
margin: 0 2px;<br />
padding: 0.35em 0.625em 0.75em;<br />
}<br />
<br />
/**<br />
* 1. Correct `color` not being inherited in IE 8/9.<br />
* 2. Remove padding so people aren't caught out if they zero out fieldsets.<br />
*/<br />
<br />
legend {<br />
border: 0; /* 1 */<br />
padding: 0; /* 2 */<br />
}<br />
<br />
/**<br />
* 1. Correct font family not being inherited in all browsers.<br />
* 2. Correct font size not being inherited in all browsers.<br />
* 3. Address margins set differently in Firefox 4+, Safari 5, and Chrome.<br />
*/<br />
<br />
button,<br />
input,<br />
select,<br />
textarea {<br />
font-family: inherit; /* 1 */<br />
font-size: 100%; /* 2 */<br />
margin: 0; /* 3 */<br />
}<br />
<br />
/**<br />
* Address Firefox 4+ setting `line-height` on `input` using `!important` in<br />
* the UA stylesheet.<br />
*/<br />
<br />
button,<br />
input {<br />
line-height: normal;<br />
}<br />
<br />
/**<br />
* Address inconsistent `text-transform` inheritance for `button` and `select`.<br />
* All other form control elements do not inherit `text-transform` values.<br />
* Correct `button` style inheritance in Chrome, Safari 5+, and IE 8+.<br />
* Correct `select` style inheritance in Firefox 4+ and Opera.<br />
*/<br />
<br />
button,<br />
select {<br />
text-transform: none;<br />
}<br />
<br />
/**<br />
* 1. Avoid the WebKit bug in Android 4.0.* where (2) destroys native `audio`<br />
* and `video` controls.<br />
* 2. Correct inability to style clickable `input` types in iOS.<br />
* 3. Improve usability and consistency of cursor style between image-type<br />
* `input` and others.<br />
*/<br />
<br />
button,<br />
html input[type="button"], /* 1 */<br />
input[type="reset"],<br />
input[type="submit"] {<br />
-webkit-appearance: button; /* 2 */<br />
cursor: pointer; /* 3 */<br />
}<br />
<br />
/**<br />
* Re-set default cursor for disabled elements.<br />
*/<br />
<br />
button[disabled],<br />
html input[disabled] {<br />
cursor: default;<br />
}<br />
<br />
/**<br />
* 1. Address box sizing set to `content-box` in IE 8/9.<br />
* 2. Remove excess padding in IE 8/9.<br />
*/<br />
<br />
input[type="checkbox"],<br />
input[type="radio"] {<br />
box-sizing: border-box; /* 1 */<br />
padding: 0; /* 2 */<br />
}<br />
<br />
/**<br />
* 1. Address `appearance` set to `searchfield` in Safari 5 and Chrome.<br />
* 2. Address `box-sizing` set to `border-box` in Safari 5 and Chrome<br />
* (include `-moz` to future-proof).<br />
*/<br />
<br />
input[type="search"] {<br />
-webkit-appearance: textfield; /* 1 */<br />
-moz-box-sizing: content-box;<br />
-webkit-box-sizing: content-box; /* 2 */<br />
box-sizing: content-box;<br />
}<br />
<br />
/**<br />
* Remove inner padding and search cancel button in Safari 5 and Chrome<br />
* on OS X.<br />
*/<br />
<br />
input[type="search"]::-webkit-search-cancel-button,<br />
input[type="search"]::-webkit-search-decoration {<br />
-webkit-appearance: none;<br />
}<br />
<br />
/**<br />
* Remove inner padding and border in Firefox 4+.<br />
*/<br />
<br />
button::-moz-focus-inner,<br />
input::-moz-focus-inner {<br />
border: 0;<br />
padding: 0;<br />
}<br />
<br />
/**<br />
* 1. Remove default vertical scrollbar in IE 8/9.<br />
* 2. Improve readability and alignment in all browsers.<br />
*/<br />
<br />
textarea {<br />
overflow: auto; /* 1 */<br />
vertical-align: top; /* 2 */<br />
}<br />
<br />
/* ==========================================================================<br />
Tables<br />
========================================================================== */<br />
<br />
/**<br />
* Remove most spacing between table cells.<br />
*/<br />
<br />
table {<br />
border-collapse: collapse;<br />
border-spacing: 0;<br />
}<br />
<br />
html, body {<br />
color: #111;<br />
background-color: transparent;<br />
width: 100%;<br />
height: 100%;<br />
margin: 0;<br />
padding: 0;<br />
display:block;<br />
line-height:normal;<br />
<br />
}<br />
<br />
h1 {<br />
font-weight:bold;<br />
font-size:52px;<br />
margin:0;<br />
text-transform:uppercase;<br />
font-family: 'Lato', sans-serif;<br />
font-weight:900;<br />
text-align:center;<br />
/*font-family: "Courier New", Courier, "Lucida Sans Typewriter", "Lucida Typewriter", monospace;*/<br />
}<br />
h2 {<br />
font-weight:bold;<br />
font-size:24px;<br />
text-transform:uppercase;<br />
font-family: 'Lato', sans-serif;<br />
font-weight:900;<br />
/*font-family: "Courier New", Courier, "Lucida Sans Typewriter", "Lucida Typewriter", monospace;*/<br />
}<br />
p {<br />
font-family: 'Lato', sans-serif;<br />
/*font-family: "Courier New", Courier, "Lucida Sans Typewriter", "Lucida Typewriter", monospace;*/<br />
}<br />
img {margin:10px;}<br />
<br />
<br />
<br />
.firstHeading { display:none;}<br />
<br />
#content {border:none !important; line-height:normal !important;}<br />
<br />
h1, h2, h3, h4 {border:none !important;}<br />
<br />
#content, #gloabalWrapper {position:static !important;}<br />
<br />
<br />
<br />
/*#menubar, .left-menu, .left-menu a {<br />
color: white !important;<br />
background-color: #555555;<br />
<br />
}*/<br />
<br />
#top-section {<br />
border:none !important;<br />
}<br />
#p-logo {display:none;}<br />
<br />
#top-section {height:0px;}<br />
<br />
#search-controls {<br />
display:none;<br />
}<br />
#footer-box {<br />
display:none;<br />
}<br />
<br />
#catlinks {<br />
display:none;<br />
}<br />
<br />
</style><br />
<img src="https://static.igem.org/mediawiki/2014/9/9b/Melbourne_Banner.png" alt="Banner" height="225" width="1000"><br />
<br />
<br />
<table width="100%" style="text-transform: uppercase; font-family: 'Lato', sans-serif;"><br />
<br />
<tr height="10px"><br />
<br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne"style="color:#000000"><br />
Home</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Team"style="color:#000000"><br />
Team</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Project"style="color:#000000"><br />
Project</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Human_Practices"style="color:#000000"> <br />
Human Practices</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Achievements"style="color:#000000"><br />
Achievements</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Notebook"style="color:#000000"><br />
Notebook</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Protocols"style=" color:#000000"> <br />
Protocols</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Safety"style="color:#000000"><br />
Safety</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Sponsors"style="color:#000000"><br />
Sponsors</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Attributions"style="color:#000000"><br />
Attributions</a></td><br />
<br />
<br />
<td align="right"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="46px"></a> </td><br />
</tr><br />
</table><br />
<br />
<br />
<h1>Achievements</h1><br />
<h2>Bronze medal requirements</h2><br />
<ul><br />
<li>Register the team, have a great summer, and plan to have<br />
fun at the Giant Jamboree.</li><br />
<ul><br />
<li>We have met this criterion</li><br />
</ul><br />
<li>Successfully complete and submit this iGEM 2014 Judging<br />
form.</li><br />
<ul><br />
<li>We have met this criterion</li><br />
</ul><br />
<li>Create and share a Description of the team's project using<br />
the iGEM wiki and the team's parts using the Registry of Standard<br />
Biological Parts.</li><br />
<ul><br />
<li>We have met this criterion</li><br />
</ul><br />
<li>Plan to present a Poster and Talk at the iGEM Jamboree.</li><br />
<ul><br />
<li>We meet this criterion</li><br />
</ul><br />
<li>The description of each project must clearly attribute work<br />
done by the students and distinguish it from work done by others,<br />
including host labs, advisors, instructors, sponsors, professional<br />
website designers, artists, and commercial services.</li><br />
<ul><br />
<li>This is documented on the <a<br />
href="https://2014.igem.org/Team:Melbourne/Attributions">Attributions<br />
Page</a>.</li><br />
</ul><br />
<li>Document at least one new standard BioBrick Part or Device<br />
used in your project/central to your project and submit this part to<br />
the iGEM Registry</li><br />
<ul><br />
<li>Submitted &nbsp;<a<br />
href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1394000">BBa_K1394000</a>,<br />
<a<br />
href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1394001">BBa_K1394001</a>,<br />
and <a<br />
href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1394002">BBa_K1394002</a>,<br />
the parts central to our project.</li><br />
</ul><br />
</ul><br />
<h2>Silver medal requirements</h2><br />
<ul><br />
<li>Experimentally validate that at least one new BioBrick Part<br />
or Device of your own design and construction works as expected.</li><br />
<ul><br />
<li>As described on our <a<br />
href="https://2014.igem.org/Team:Melbourne/Project">project<br />
page</a>, we validated that our protein expression BioBricks<br />
worked and led to protein expression (e.g. <a<br />
href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1394001">BBa_K1394001</a>).</li><br />
</ul><br />
<li>Document the characterization of this part in the Main Page<br />
section of that Part's/Device's Registry entry.</li><br />
<ul><br />
<li>These characterization efforts have been documented.</li><br />
</ul><br />
<li>Submit this new part to the iGEM Parts Registry<br />
(submissions must adhere to the iGEM Registry guidelines)</li><br />
<ul><br />
<li>These parts have been submitted.</li><br />
</ul><br />
<li>iGEM projects involve important questions beyond the bench,<br />
for example relating to (but not limited to) ethics, sustainability,<br />
social justice, safety, security, or intellectual property rights.<br />
Articulate at least one question encountered by your team, and describe<br />
how your team considered the(se) question(s) within your project.<br />
Include attributions to all experts and stakeholders consulted.</li><br />
<ul><br />
<li>We asked the general question "how can we improve access<br />
to science education across a broad age range, including early<br />
childhood education?" With this effort came additional questions,<br />
including "How can we make biology more accessible to young children?"<br />
and (for high school aged students) "How can we improve access to<br />
science education for disadvantaged youth in Australia?" We consulted<br />
with a variety of stakeholders within the science education community<br />
in order to answer these questions, as described in our <a<br />
href="https://2014.igem.org/Team:Melbourne/Public_Outreach">Human<br />
Practices</a> page.</li><br />
<li>Our project concept could potentially have commercial<br />
value and therefore we had to address intellectual property issues. We<br />
consulted with a number of experts and stakeholders in the intellectual<br />
property space, as described in our <a<br />
href="https://2014.igem.org/Team:Melbourne/Public_Outreach">Human<br />
Practices</a> page.</li><br />
</ul><br />
</ul><br />
<h2>Gold medal requirements</h2><br />
<ul><br />
<li>Improve the function OR characterization of an existing<br />
BioBrick Part or Device (created by another team or your own<br />
institution in a previous year), enter this information in the<br />
Registry. Please see the Registry help page on how to document a<br />
contribution to an existing part.</li><br />
<ul><br />
<li>Our BioBrick protein expression device improves the 2013<br />
TU Delft SUMO fusion <a<br />
href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1022101">BioBrick</a>.<br />
This discussed in detail on our <a<br />
href="https://2014.igem.org/Team:Melbourne/Project">project<br />
page</a>.</li><br />
</ul><br />
<li>Help any registered iGEM team from another school or<br />
institution by, for example, characterizing a part, debugging a<br />
construct, or modeling or simulating their system.</li><br />
<ul><br />
<li>Our team formed a collaborative relationship with the<br />
University of Oxford. We assisted Oxford by providing them with a<br />
design concept for attaching enzymes to star peptides, discussing how<br />
we could improve their enzymatic system with star peptides, and<br />
providing research on linking their particular enzymes to a peptide<br />
linker. See our <a<br />
href="https://2014.igem.org/Team:Melbourne/Project#Oxford">project<br />
page</a> for details.</li><br />
<li>Our team helped significantly with the promotion of the<br />
University of Sydney's outreach program, the Strange Nature writing<br />
competition. We created a promotional video for the competition which<br />
was distributed to high school teachers and students across Australia.<br />
Further, we contacted high school teachers directly to help distribute<br />
video and word of the competition. See our <a<br />
href="https://2014.igem.org/Team:Melbourne/Public_Outreach">Human<br />
Practices</a><br />
page.</li><br />
</ul><br />
</ul><br />
<br />
<br />
<br />
</html></div>Slowehttp://2014.igem.org/Team:Melbourne/Human_PracticesTeam:Melbourne/Human Practices2014-11-26T14:36:51Z<p>Slowe: </p>
<hr />
<div><html><br />
<link href='http://fonts.googleapis.com/css?family=Lato:400,900' rel='stylesheet' type='text/css'><br />
<style><br />
/* CSS Document */<br />
/*! normalize.css v2.1.2 | MIT License | git.io/normalize */<br />
<br />
/* ==========================================================================<br />
HTML5 display definitions<br />
========================================================================== */<br />
<br />
/**<br />
* Correct `block` display not defined in IE 8/9.<br />
*/<br />
<br />
article,<br />
aside,<br />
details,<br />
figcaption,<br />
figure,<br />
footer,<br />
header,<br />
hgroup,<br />
main,<br />
nav,<br />
section,<br />
summary {<br />
display: block;<br />
}<br />
<br />
/**<br />
* Correct `inline-block` display not defined in IE 8/9.<br />
*/<br />
<br />
audio,<br />
canvas,<br />
video {<br />
display: inline-block;<br />
}<br />
<br />
/**<br />
* Prevent modern browsers from displaying `audio` without controls.<br />
* Remove excess height in iOS 5 devices.<br />
*/<br />
<br />
audio:not([controls]) {<br />
display: none;<br />
height: 0;<br />
}<br />
<br />
/**<br />
* Address styling not present in IE 8/9.<br />
*/<br />
<br />
[hidden] {<br />
display: none;<br />
}<br />
<br />
/* ==========================================================================<br />
Base<br />
========================================================================== */<br />
<br />
/**<br />
* 1. Set default font family to sans-serif.<br />
* 2. Prevent iOS text size adjust after orientation change, without disabling<br />
* user zoom.<br />
*/<br />
<br />
html {<br />
font-family: sans-serif; /* 1 */<br />
-ms-text-size-adjust: 100%; /* 2 */<br />
-webkit-text-size-adjust: 100%; /* 2 */<br />
}<br />
<br />
/**<br />
* Remove default margin.<br />
*/<br />
<br />
body {<br />
margin: 0;<br />
}<br />
<br />
/* ==========================================================================<br />
Links<br />
========================================================================== */<br />
<br />
/**<br />
* Address `outline` inconsistency between Chrome and other browsers.<br />
*/<br />
<br />
a:focus {<br />
outline: thin dotted;<br />
}<br />
<br />
/**<br />
* Improve readability when focused and also mouse hovered in all browsers.<br />
*/<br />
<br />
a:active,<br />
a:hover {<br />
outline: 0;<br />
}<br />
<br />
/* ==========================================================================<br />
Typography<br />
========================================================================== */<br />
<br />
/**<br />
* Address variable `h1` font-size and margin within `section` and `article`<br />
* contexts in Firefox 4+, Safari 5, and Chrome.<br />
*/<br />
<br />
h1 {<br />
font-size: 2em;<br />
margin: 0.67em 0;<br />
}<br />
<br />
/**<br />
* Address styling not present in IE 8/9, Safari 5, and Chrome.<br />
*/<br />
<br />
abbr[title] {<br />
border-bottom: 1px dotted;<br />
}<br />
<br />
/**<br />
* Address style set to `bolder` in Firefox 4+, Safari 5, and Chrome.<br />
*/<br />
<br />
b,<br />
strong {<br />
font-weight: bold;<br />
}<br />
<br />
/**<br />
* Address styling not present in Safari 5 and Chrome.<br />
*/<br />
<br />
dfn {<br />
font-style: italic;<br />
}<br />
<br />
/**<br />
* Address differences between Firefox and other browsers.<br />
*/<br />
<br />
hr {<br />
-moz-box-sizing: content-box;<br />
box-sizing: content-box;<br />
height: 0;<br />
}<br />
<br />
/**<br />
* Address styling not present in IE 8/9.<br />
*/<br />
<br />
mark {<br />
background: #ff0;<br />
color: #000;<br />
}<br />
<br />
/**<br />
* Correct font family set oddly in Safari 5 and Chrome.<br />
*/<br />
<br />
code,<br />
kbd,<br />
pre,<br />
samp {<br />
font-family: monospace, serif;<br />
font-size: 1em;<br />
}<br />
<br />
/**<br />
* Improve readability of pre-formatted text in all browsers.<br />
*/<br />
<br />
pre {<br />
white-space: pre-wrap;<br />
}<br />
<br />
/**<br />
* Set consistent quote types.<br />
*/<br />
<br />
q {<br />
quotes: "\201C" "\201D" "\2018" "\2019";<br />
}<br />
<br />
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<img src="https://static.igem.org/mediawiki/2014/9/9b/Melbourne_Banner.png" alt="Banner" height="225" width="1000"><br />
<br />
<br />
<table width="100%" style="text-transform: uppercase; font-family: 'Lato', sans-serif;"><br />
<br />
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<br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne"style="color:#000000"><br />
Home</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Team"style="color:#000000"><br />
Team</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Project"style="color:#000000"><br />
Project</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Human_Practices"style="color:#000000"> <br />
Human Practices</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Achievements"style="color:#000000"><br />
Achievements</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Notebook"style="color:#000000"><br />
Notebook</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Protocols"style=" color:#000000"> <br />
Protocols</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Safety"style="color:#000000"><br />
Safety</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Sponsors"style="color:#000000"><br />
Sponsors</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Attributions"style="color:#000000"><br />
Attributions</a></td><br />
<br />
<br />
<td align="right"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="46px"></a> </td><br />
</tr><br />
</table><br />
<br />
<br />
<h1>Public Outreach</h1><br />
<h2>&lsquo;Synthetic biology for kids and adults and everyone in between&rsquo; </h2><br />
<p>The University of Melbourne took up the challenge of teaching synthetic biology to all ages, embracing a &lsquo;science for everyone&rsquo; approach. In so doing, we engaged in education outreach activities with kindergarten students from three childcare centres as well as both university and high school students from across Australia.</p><br />
<p>&nbsp; </p><br />
<h3>For kindergarten students </h3><br />
<table width="100%"><br />
<tr><br />
<td width="195" ><img src="https://static.igem.org/mediawiki/2014/4/4b/Melbourne_Adventure_of_E_coli.jpg" width="176" height="264" alt="Adventure of E coli"/></td><br />
<td width="594"><p>We decided to write the first in a series of children&rsquo;s books for pre-schoolers aged 4-6 themed in the spirit of iGEM entitled &lsquo;The Adventures of E. coli&rsquo; for a number of reasons:</p><br />
<ul><br />
<li> Children aged 4-6 are in a key learning stage, during which their interest in science can significantly influence their long-term science education outcomes (Bowman, Donovan, &amp; Burns, 2001, pp. 8-9). </li><br />
<li>There are lots of resources about science for older kids but not so many for kids at such a young age. </li><br />
<li>Picture books provide a springboard for further classroom activities </li><br />
</ul><br />
<p>With the assistance of IndieMosh (Publishers link is here: <A href="http://www.indiemosh.com.au/author-42-iGEM-Melbourne-childrens-biology-science" rel="nofollow">http://www.indiemosh.com.au/author-42-iGEM-Melbourne-childrens-biology-science</A>) – a self-publishing facilitator – we managed to get our book in print and on Amazon, please see <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/</A></p><br />
<p>After we successfully published our book, it was time to take it to the audience. We approached childcare centres across Melbourne and negotiated for our book to be read to pre-schoolers and for our team members to come along and teach the kids about science.</p></td><br />
<br />
</tr><br />
</table><p>The book reading was well received and was followed by playing some science-related games, and having a chat about science in groups. Please check out the video we made about the day: </p><br />
<br />
<iframe class="centervid" width="560" height="315" src="//www.youtube.com/embed/RNV_lPKcrSc" frameborder="0" allowfullscreen></iframe><br />
<br />
<p> A big thanks to Kids Time Early Learning Centre (Moorabbin), Kids Time Early Learning Centre (Bentleigh East) and Coco's Early Learning Centre for inviting us to present our picture book to the kids. </p><br />
<br />
<p>We believe that through this book, young children can learn about basic concepts and the language of biology, forming a scientific foundation to be built on in future years. Moreover, it is our hope that this book will inspire children to continue to learn about science and to appreciate all the wonders that it holds. </p><br />
<p>The Kindle book is available from here: <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon-ebook/dp/B00OJ691DI/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon-ebook/dp/B00OJ691DI/</A></p><br />
<p>Paperback available on Amazon here: <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/</A></p><br />
<p>Fixed layout epub (for iPad and Android tablets, phones etc) available on Smashwords here: <A href="http://www.smashwords.com/books/view/484305" rel="nofollow">http://www.smashwords.com/books/view/484305</A></p><br />
<br />
<br />
<p> <strong> References: </strong> </p><br />
<p> Bowman, Barbara T.; Donovan, M. Suzanne; & Burns, M. Susan (Eds.). (2001). Eager to learn: Educating our preschoolers. Washington, DC: National Academy Press. </p><br />
<br />
<h3>&nbsp;</h3><br />
<h3>For high school students </h3><br />
<p>In an effort to broaden the accessibility of the iGEM competition to those who appeared to be currently underrepresented in the competition (e.g. disadvantaged students, females and Australian high school students), we approached a private high school in Victoria with the following aim: </p><br />
<p>To find a well-resourced, private high school to partner with an iGEM team (e.g. UoM) to field a team consisting of half private high school students/half underrepresented students – who would have their flights to Boston funded by fundraising done primarily by the private high school. </p><br />
<p>After extensive email &amp; phone correspondence spanning several months, the school decided it was not currently in the position to go ahead with the iGEM team proposal due to upcoming infrastructure developments, though would be interested in fielding a team after these are completed (scheduled for 2016). Nonetheless, the school did encourage us that other APS schools would likely be interested in hearing about iGEM and our team concept. </p><br />
<p>Whilst we didn&rsquo;t achieve our ultimate goal, this activity did raise awareness of the iGEM competition with all the key science staff members at the school which requested not to be named on this wiki (for further details please contact our team leader Sean). </p><br />
<p>Finally, we would encourage all Australian teams to work on the following goals: </p><br />
<ul><br />
<li>improve iGEM's accessibility to those of disadvantaged backgrounds </li><br />
<li>increase female representation in science &amp; iGEM </li><br />
<li>increase the participation of Australian/international high school students in the iGEM competition. </li><br />
</ul><br />
<p><BR><br />
In addition to our negotiations with the private high school, we also aimed at widening the participation of high school students in the iGEM competition by collaborating with the University of Sydney on The Strange Nature Writing competition. </p><br />
<p>We spruiked the competition not only on our project flyers which described the iGEM competition, our project and the Strange Nature Writing Competition, but also through our development of a promotional video designed to be shown in high school assemblies as a way of promoting both iGEM and the Strange Nature Writing competition. Please see the video below: </p><br />
<br />
<iframe class="centervid" width="560" height="315" src="//www.youtube.com/embed/ZuSoO3HVMjo" frameborder="0" allowfullscreen></iframe><br />
<br />
<p>Furthermore, we wrote and published articles on Comet.is which is aimed at high school students, university students and recent university graduates, please see 'For university students' for a list of the articles we wrote. </p><br />
<h3>&nbsp;</h3><br />
<h3>For university students </h3><br />
<p>Our main form of outreach toward university students was in writing and publishing articles on a burgeoning and government sponsored website Comet.is which is aimed at high school students, university students and recent graduates. Our articles covered a range of topics from our iGEM experience to considerations of ethical issues of synthetic biology and the potential of synthetic biology. </p><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/d/d5/Comet.jpg" width="640" height="400" alt="Adventure of E coli"/><br />
<br />
<br />
</p> <strong>Here are some excerpts:</strong> </p><br />
<br />
<blockquote></p> "All this leads me to refer to synthetic biology as a ménage à trois of a discipline, utilizing engineering, biology and computer science to produce a fantastically creative activity." </p></blockquote><br />
<br />
<blockquote></p> "Given assistance in the form of grants or human resources, entrepreneurial forums along with student competitions like InnovationACT and iGEM have the chance to inspire entrepreneurialism in students. It is in these opportunities for students to go on entrepreneurial adventures, replete with late nights, red bulls and a shot at winning a grand prize – that the next generation of entrepreneurs is born." </p></blockquote><br />
<br />
<blockquote></p> "A prime example of this is in the International Genetically Engineered Machine (iGEM) competition, which I am a team member of for the University of Melbourne this year. Our team stretches across many disciplines; from telecommunications and business to chemical engineering and science communication, even information technology - we are practically the UN of fields and backgrounds." </p></blockquote><br />
<br />
<blockquote></p> "Participating in IGEM can definitely be categorized as having an experience and not just joining a team. The competition brings together all the components of completing a project for undergraduate students to be part of, a little like a trial run of a Masters or Doctorate course." </p></blockquote><br />
<br />
<p>Here are the links to our articles: </p><br />
<p><A href="https://comet.is/article/2960" rel="nofollow">https://comet.is/article/2960</A> <A href="https://comet.is/article/3499/" rel="nofollow">https://comet.is/article/3499/</A> <A href="https://comet.is/article/3381/" rel="nofollow">https://comet.is/article/3381/</A> <A href="https://comet.is/article/3561/" rel="nofollow">https://comet.is/article/3561/</A> <A href="https://comet.is/article/3562/" rel="nofollow">https://comet.is/article/3562/</A> <A href="https://comet.is/article/3694/" rel="nofollow">https://comet.is/article/3694/</A> <A href="http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/" rel="nofollow">http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/</A> <A href="http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/" rel="nofollow">http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/</A></p><br />
<p>Furthermore, as part of our outreach directed at university students, we combined our recruitment campaign for an IT whiz to help with our wiki, with a presentation on what iGEM was all about. We lecture bashed over several weeks, presenting over 11 times. </p><br />
<br />
<h3><BR><br />
</h3><br />
<h3>For general public </h3><br />
<p>After promoting the iGEM competition to the Victorian State Chair of National Science Week (NSW) - Nick Besley, our team decided to field a stall in &lsquo;Market of the Mind&rsquo; – a NSW event aimed at the general public located in a high foot traffic area alongside the Yarra River in Southbank across the river from Flinders St Station. Luckily, our stall was located next to the bar, which meant there was a lot of foot traffic directed to our happy team members wearing our iGEM team t-shirts.</p><br />
<img class="centervid" src="https://static.igem.org/mediawiki/2014/d/d7/10641184_10203547262856505_6097351474732015309_n.jpg" width="400" height="300" /><br />
<p> Using a true/false quiz, armed with Ferrero Rochers as rewards for getting all the answers correct, we had over 30 quiz contestants engage with us on the first night and 35+ contestants on the second day. Please see the accompanying video made by the City of Melbourne, which shows our stall/team members from 0:45 </p><br />
<br />
<p><iframe class="centervid" width="560" height="315" src="//www.youtube.com/embed/ahFI3aFBdNc" frameborder="0" allowfullscreen></iframe><p><br />
<br />
<br />
<p> <strong> Our team consulted: </strong> </p><br />
<p>IndieMosh</p><br />
<p>Kids Time Early Learning Centre (Bentleigh East)</p><br />
<p>Kids Time Early Learning Centre (Moorabin)</p><br />
<p>Coco's Early Learning Centre</p><br />
<p>A Victorian private school (one of the 11 Associated Public Schools)</p><br />
<p>USyd-Australia</p><br />
<p>University of Melbourne IT Faculty</p><br />
<p>University of Melbourne Education Faculty</p><br />
<p> University of Melbourne Media and Public Relations Department </p><br />
<p>Victorian State Chair of National Science Week (NSW) - Nick Besley</p><br />
<p> Teachers' Association of Australia (TAA) </p><br />
<br />
<br />
<br />
<h1>Intellectual Property </h1><br />
<br />
<p> <strong> Our team considered the question of ‘how should we proceed if we want to retain IP rights on our project’? </strong> </p><br />
<br />
<p> Before attempting to answer this pertinent IP question, our team leader decided to recruit two more team members who had some intellectual property experience to complement the learning on patent law that a few early team members had acquired in their biotechnology undergraduate degrees. Both the new team members had previously filed provisional patents; one had filed some in Russia and the other in Australia, offering two perspectives on this question of IP. </p><br />
<br />
<p> In answering this question, we approached our universities’ tech transfer office to gain clarity over our team’s discussions and brainstorm sessions on whether our project was patentable. </p><br />
<br />
<p> The tech transfer office advised us that our project may have commercial applications, however it was probably too soon to act on the idea and convert it into a provisional patent, so we decided to hold off and wait till we had a more substantiated grasp on our commercial applications. We were also advised that if at any point we wanted to file for IP protection on the project, it would be really important to not have disclosed the details of our idea to anyone else, as this could result in us losing the right to our IP. </p><br />
<br />
<p> After doing some research on the various law firms who could offer us a provisional patent, we discovered the estimated cost would be in the vicinity of $3500-$6500 depending on the level of detail we wanted to cover e.g. the number and extent of the claims, the full description of the idea, diagrams and experimental protocols as well as the filing & service fees associated. </p><br />
<br />
<p> Due to the relatively high estimates we received, we looked into other options for filing a provisional, including recruiting a law student to join the team and contribute their knowledge to our IP protection. This avenue did not eventuate as our ideas on what we would like to patent continuously evolved over the course of the project and we could not reach a sufficient degree of clarity to bring on a lawyer for the filing of a provisional patent. </p><br />
<br />
<p> Nonetheless, we proceeded with caution, ensuring to follow the advice of the University of Melbourne’s tech transfer office and not disclose our ideas without ensuring our disclosure was protected. An instance when we had to implement this protection was during our scientific collaboration with Oxford, were we ensured Non Disclosure Agreements were signed prior to the disclosure of our project to them. They indicated their interest in using our project as a case study in their ‘iGEM intellectual property guide’, however we felt that this might put our IP rights in jeopardy, so we decided not to proceed as a case study. </p><br />
<br />
<p> At this time, our team is looking to produce at least one provisional patent following the iGEM 2014 competition on at least one of the various techniques we designed during the project. </p><br />
<br />
<br />
<p> <strong> Our team consulted: </strong> </p><br />
<br />
<p> University of Melbourne Technology Transfer Office </p><br />
<p> University of Melbourne Technology Commercialisation Team </p><br />
<p> UoM Commercial </p><br />
<p> Stanislauv Yatskevich (iGEM team member with Russian patent experience) </p><br />
<p> Peter Collins (iGEM team member with Australian patent experience) </p><br />
<br />
<br />
<p> <strong> References: </strong> </p><br />
<br />
<p> http://www.ipaustralia.gov.au/.../factsheet-experimental.../ </p><br />
<br />
<p>http://www.google.com.au/url?sa=t&rct=j&q=&esrc=s...</p></div>Slowehttp://2014.igem.org/Team:Melbourne/TeamTeam:Melbourne/Team2014-11-25T07:55:01Z<p>Slowe: </p>
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Protocols</a></td><br />
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Safety</a></td><br />
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Sponsors</a></td><br />
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Attributions</a></td><br />
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<br />
<h1 >Team</h1><br />
<br />
<h3>About The University of Melbourne</h3><br />
<p>Located in Melbourne, Victoria, The University of Melbourne was founded in 1853 and is Australia’s second oldest university. The university is consistently ranked among the leading universities in the world, with international rankings of world universities placing it at number 1 in Australia and number 34 in the world (Times Higher Education World University Ranking 2013-2014).</p><br />
<br />
<h3>About The 2014 Melbourne iGEM Team</h3><br />
<p>The 2014 Melbourne iGEM team consists of 19 highly-motivated undergraduate students and 1 postgraduate student from a range of disciplines, including biochemistry and molecular biology, chemistry, chemical engineering and bioengineering. The team’s academic and research credentials are well-tested. This year, the team comprises three of The University of Melbourne’s Chancellor’s Scholars (top 0.1% of all Australian high school students) and several team members with research experience at Melbourne’s high-profile research institutes, such as the prestigious Walter and Eliza Hall Institute, The Peter MacCallum Cancer Centre, and The Baker IDI Heart and Diabetes Institute. The team is supervised by Associate Professor Heung-Chin Cheng, Associate Professor Paul Gooley, Associate Professor Neil O’Brien-Simpson, and Dr. Angus Johnston.<br />
The Melbourne iGEM team was founded by a group of enthusiastic and motivated students in 2013. The 2013 team played a key role in establishing the foundations of this year’s team and generating this year’s project. Unfortunately many of the 2013 team members could not continue their work for iGEM in 2014, so credit must also be given to Pedro Avellar Costa, Michelle Tie, Georgina Panshem, Morgana Cerqueira, Jeong Yoon Esther Kim, Winnie Tan, Hannah Nguyen, Mary Teo, Cathy Pitt and Joyce Kant.</p><br />
<br />
<h3>Faculty Supervisors</h3><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/d/d4/Melbourne_Cheng.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Heung-Chin Cheng (Supervisor)</strong></p><br />
An Associate Professor with the Department of Biochemistry and Molecular Biology, Heung-Chin has been studying the biochemical basis of the regulation of protein kinases and phosphatases since he was a graduate student at the University of California in 1982. His PhD project unravelled the active site structure of c-AMP-dependent protein kinase, how the kinase recognises its protein substrates and how the kinase is selectively inhibited by its endogenous inhibitor. Heung-Chin was the Melbourne iGEM team&rsquo;s supervisor and he kindly offered us space to work in his lab and assisted us when we had questions about practical procedures, troubleshooting or theory.</td><br />
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<br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/7/74/Melbourne_Gooley.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Paul Gooley (Advisor)</strong></p><br />
Associate Professor Paul Gooley directs a structural biology research group that focuses on the application of NMR spectroscopy to elucidate structure and protein interactions. He obtained his degrees at The University of New South Wales and spent 10 years in the USA, including 5 years at the pharmaceutical company Merk and Co. Over the last 10 years his group has conducted and published NMR structural and dynamical analyses on a number of protein domains and systems that have biological functions in stress and infection, in lipid transport, in protein and membrane trafficking, and in receptor signalling. Paul assisted the 2014 Melbourne iGEM team by providing advice, answering questions and brainstorming.</td><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/9/97/Melbourne_Angus.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Angus Johnston (Advisor)</strong></p><br />
Dr Angus Johnston is a researcher at the Monash Institute of Pharmaceutical Science. Angus is an ARC Future Fellow who’s work focuses on developing better ways to deliver drugs, making them more therapeutically active and limiting side effects. He has extensive knowledge and expertise in nanomaterials assembly, material characterisation, cellular interactions and advanced imaging techniques. Angus’ research interests include targeted vaccine therapy, sensors for cellular imaging, understanding cellular processing of nanoparticles, self assembling peptides as drug carrier and the toxicology of nanomaterials. Angus assisted the 2014 Melbourne iGEM team by providing advice, answering questions and brainstorming.</td><br />
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<br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/f/fd/Melbourne_Neil_O%E2%80%99Brien-Simpson.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Neil O’Brien-Simpson (Advisor)</strong></p><br />
Associate Professor Neil O’Brien-Simpson is a researcher at the Royal Dental Hospital in Melbourne and has an interdisciplinary background, combining organic and peptide chemistry with immunology to develop novel vaccines and therapeutics and investigating the immune response to pathogens. His research into vaccine design and periodontitis has resulted in Neil being awarded the Colgate Prize for Dental Research (1999), The IADR Hatton Award (2000) and the Oral Biology Award (2003). In 2004 he became program co-leader for the Novel Diagnostics, Vaccines and Pharmaceuticals Research Program in the Oral Health CRC. Neil assisted the 2014 Melbourne iGEM team by providing advice, answering questions and brainstorming.</td><br />
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<br />
<h3>Team Members</h3><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/2/25/Melbourne_Sean.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Sean Lowe (Team Leader)</strong></p><br />
Sean started the 2014 Melbourne iGEM team while completing his Master of Engineering in chemical engineering. As the team leader, Sean was responsible for communicating with supervisors and sponsors, setting up the lab, and making sure the team ran smoothly. Sean is passionate about science and saw iGEM as an amazing opportunity to combine his interests in chemical engineering and biotechnology. He found that the best part of iGEM was the creative process, and thoroughly enjoyed discussing many great ideas with his bright and capable teammates. He looks forward to passing on the torch to the next generation of Melbourne Uni iGEMers.</td><br />
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<br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/a/a6/Melbourne_Lizzy.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Elizabeth Brookes</strong></p><br />
Elizabeth is in her second year of the Bachelor of Biomedicine degree and is majoring in Immunology. In the future Elizabeth hopes to continue to develop her passion for research while studying postgraduate medicine. Elizabeth was new to iGEM in 2014 and contributed to Melbourne’s iGEM project by working in the laboratory, developing protocols and performing various administrative duties. For Elizabeth, the most enjoyable aspect of iGEM was getting hands-on experience while meeting like-minded people. In her free time Elizabeth enjoys playing netball and discovering new restaurants with friends.</td><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/f/f6/Melbourne_Peter.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Peter Collins</strong></p><br />
Peter is in his final year of the Bachelor of Science at The Australian National University in Canberra and is majoring in Science Communications, as well as completing his first year of the Bachelor of Environments at The University of Melbourne. In the future, Peter aims to work on The Human Variome Project, develop the iGEM competition in Australia and work for UNESCO. Peter joined the Melbourne iGEM team in 2014 and was in charge of human practices and outreach, working to establish relations with high schools in Melbourne for on-going public outreach efforts in the future. As such, Peter has enjoyed getting an insight into managing science innovation teams and the tremendous array of challenges that accompany this. In his spare time, Peter enjoys learning about esoteric topics that may have unrealised commercial applications.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/1/1a/Melbourne_Sheryl.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Sheryl Ding</strong></p><br />
Sheryl is in the first year of a Bachelor of Biomedicine degree and is hoping to major in Bioengineering Systems. In the future, Sheryl would like to work in biomedical research, preferably specialising in engineering or molecular and cellular biology. After joining the iGEM team in early 2014, Sheryl contributed to the laboratory work and some administrative tasks for the project. Sheryl enjoyed all aspects of this experience, but particularly the opportunity to gain practical insight into laboratory research and meet the people on the iGEM team. Outside of science, Sheryl enjoys visual art and watching TV shows.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/6/66/Melbourne_Robyn.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Robyn Esterbauer</strong></p><br />
As a second year student of the Bachelor of Science with a major in Microbiology and Immunology, Robyn hopes to undertake a masters and PhD, specialising in infectious disease research. After joining the team in March 2014, Robyn contributed to the team with her work in the laboratory, research into cysteine bond formation and computational protein design analysis, and public outreach activities. Robyn has enjoyed experiencing a group research environment and learning some very useful practical laboratory skills. In her free time, Robyn loves to read cutting edge infectious disease research and playing soccer.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/d/d4/Melbourne_Tobias.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Tobias Gustavsson</strong></p><br />
Tobias is in his second year of the Bachelor of Biomedicine degree and is majoring in Immunology. In the future Tobias hopes to study medicine and continue to pursue research in immunology. Tobias was new to iGEM in 2014 and contributed to the theoretical design and research of the team’s project. iGEM presented Tobias with the opportunity to finally partake in the laboratory work that he had heard so much about in lectures.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/e/e8/Melbourne_Henry.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Henry Howard</strong></p><br />
Henry has just completed his Honours year for the Bachelor of Biomedicine and in the future he hopes to undertake a PhD and work in Melbourne’s bustling medical research sector. After joining the iGEM team in February 2013, Henry predominantly contributed to the theoretical design of the project. He most enoyed having access to resources that have allowed us to undertake serious scientific research of our own design. Outside of iGEM, Henry is a black belt in Tang Soo Tao and enjoys planting trees and completing jigsaw puzzles.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/8/8f/Melbourne_Aishwarya.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Aishwarya Kulkarni</strong></p><br />
Aishwarya is in her second year of the Bachelor of Science and is majoring in Biochemistry and Molecular Biology. Her future plans include completing a PhD and research in the area of translational bioinformatics and personalised medicine. As part of the Melbourne iGEM 2014 team, Aishwarya contributed to laboratory and budgeting tasks. She enjoyed both the atmosphere of the team and the challenges with which we were confronted when trying to work independently to solve problems. Outside of iGEM, Aishwarya also enjoys playing tennis.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/2/2a/Melbourne_Ranit.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Ranit Lahmy</strong></p><br />
Ranit is in her final year of the Bachelor of Science degree and is completing her major in Chemistry. Ranit joined the Melbourne iGEM team in early 2013 and has contributed to many facets of the project, including laboratory work, administration and human outreach. As a consequence, Ranit has really enjoyed the chance to meet new people while learning research and laboratory skills. When not working on iGEM, Ranit enjoys reading books, playing tennis and hanging out with friends.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/b/bc/Melbourne_Jeffrey.JPG" width="150" height="196" alt=""/></td><br />
<td><p><strong>Jeffrey Lai</strong></p><br />
Jeffrey is in his first year of the Bachelor of Biomedicine at The University of Melbourne and in the future he would like to become a medical practitioner or researcher. After joining the Melbourne team in July 2014, Jeffrey helped out in the laboratory work and the iGEM website. Jeffrey found it incredibly rewarding when we finally saw some promising results after putting so much time and effort into the project. In his spare time, Jeffrey likes to play the piano and compose symphonic music.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/1/10/Melbourne_Melissa.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Melissa Leckie</strong></p><br />
Melissa is in her final year of the Bachelor of Science and is completing a major in biochemistry and molecular biology. Competing in her first iGEM jamboree in 2008, Melissa joined the current team in 2013 and has contributed to project design, administration and human outreach. Through this experience Melissa has enjoyed making life long friends and facing the challenges of being part of a student run team. Melissa wishes to pursue a career in Medical Technology and in her spare time she enjoys reading and travelling.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/3/3c/Melbourne_Keit.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Keit Loi</strong></p><br />
Keit is in his final year of the Bachelor of Biomedicine and is majoring in infection and immunity. Since joining the Melbourne iGEM team in early 2014, Keit has mainly contributed to the laboratory work for the project and he has enjoyed that this has given him a good understanding of what working in research would be like. In the future, Keit would like to pursue this passion and work towards a PhD and university lecturing. Outside of iGEM, Keit works on projects with high school students and enjoys chilling with friends and kicking back with some video games.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/0/07/Melbourne_Darcy.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Darcy Marum</strong></p><br />
Darcy is in his second year of the Bachelor of Science degree and is majoring in Microbiology and Immunology. Darcy hopes to pursue research in these exciting fields after completing an honours year. After joining the team in early 2014, Darcy predominantly worked on laboratory work for the project and enjoyed the practical experience that he gained.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/2/2e/Melbourne_Gayle.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Gayle Pereira</strong></p><br />
Gayle is in her final year of the Bachelor of Science and is majoring in Pathology. In the future Gayle hopes to pursue a career either as a clinical pathologist or secondary school teacher. Gayle joined the iGEM team in 2013 and was the senior laboratory leader for the group, tirelessly working and teaching others in the laboratory. Working on the project, Gayle appreciated gaining the laboratory experience that she requires for her future ambitions. In her free time Gayle enjoys supporting her team in the Australian Football League and doing handicraft-based hobbies.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/b/b1/Melbourne_Lumi.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Lumi Tomren</strong></p><br />
Lumi is in her first year of the Bachelor of Biomedicine at The University of Melbourne and is planning on majoring in Microbiology and Immunology. With a desire to learn more about synthetic biology and the microscopic world around us, Lumi joined the team in Spring 2014. Lumi has contributed to the team by performing lab work and she has really enjoyed learning a lot of new practical techniques. She has also enjoyed discovering many of the possibilities of synthetic biology by researching past projects undertaken by iGEM teams. Outside of iGEM and science, Lumi enjoys visual art.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/d/d6/Melbourne_Michael.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Michael Wei</strong></p><br />
Michael is in his first year of the Bachelor of Biomedicine and plans on majoring in neuroscience. Michael was new to iGEM in 2014 and worked primarily in the laboratory and designing primers. Michael is interested in working towards a career in neurosurgery or cancer tumour research, looking specifically at the epigenetic mechanisms underlying cancer regulation. As part of the iGEM team, Michael has enjoyed meeting like-minded individuals who share similar interests. Michael is fascinated by marine life and enjoys reading in his leisure time.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/0/07/Melbourne_Stan.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Stanislav Yatskevich</strong></p><br />
Although Stanislav started studying his first year of a Bachelor of Biomedicine at The University of Melbourne, he has now commenced study at The University of Oxford reading Biochemistry. Stanislav joined the Melbourne iGEM team in March 2014 and contributed greatly to the theoretical project design and laboratory work. After moving to Oxford, Stanislav also facilitated communication between our team and the Oxford iGEM team. In the future, Stanislav would like to pursue research in Biochemistry and he has appreciated the amount of experience he was able to gain in such a short time while taking part in iGEM. Outside of Biochemistry, Stanislav enjoys playing football and procrastinating his study.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/d/dc/Melbourne_Nicholas.JPG" width="150" height="196" alt=""/></td><br />
<td><p><strong>Nicholas Yee</strong></p><br />
Nicholas has just completed the Honours year of his Bachelor of Biomedicine degree, studying novel pathway inhibitors to elucidate mechanisms of brain tumours. He plans to continue his research into brain tumours by undertaking a PhD and potentially studying medicine. After joining the team in 2014, Nicholas enjoyed helping the younger students in the team by teaching them new laboratory techniques and troubleshooting their experiments. When not in the laboratory, Nicholas enjoys hanging out with friends and playing badminton.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/1/1b/Melbourne_Michelle.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Michelle Zheng</strong></p><br />
Michelle is in her second year of the Bachelor of Science and is undertaking the Biochemistry major. At the completion of her degree, Michelle seeks to pursue further research through the honours or masters programmes. Michelle joined the Melbourne iGEM team in 2014 and completed many hours of laboratory work contributing to the project. Being part of a team dedicated to science, the laboratory work and the challenge of overcoming troubleshooting problems were the aspects of iGEM that excited Michelle most. In her free time she enjoys playing the violin.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/0/0f/Melbourne_Wayne.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Wayne Zheng</strong></p><br />
Wayne is in his first year of the Bachelor of Biomedicine and hopes to eventually major in Human Structure and Function. In the future, Wayne would like to continue his involvement in biomedical research while also studying and practising medicine. In contributing to the 2014 Melbourne iGEM team Wayne performed laboratory work as well as some theoretical research. Wayne loved learning new techniques in the laboratory and reading into a wide range of research topics.</td><br />
</tr><br />
<br />
</table><br />
<br />
<br />
<h3>Advisors</h3><br />
<p>A special mention must also be made to our Honours, Masters and PhD student advisors in the Cheng Lab who tirelessly and patiently assisted with our troubleshooting and planning of experimental procedures. These students were: <br />
<br />
Ashfaqul Hoque (PhD student), Gahana Advani (PhD student), George Cao (Masters student), Ken Ang (Honours student) and David Zula (Honours student).</p><br />
<br />
<table><br />
<tr><br />
<td width="152" ><img src="https://static.igem.org/mediawiki/2014/5/58/Melbourne_Ashfaqul.jpg" width="150" height="196" alt=""/></td><br />
<td width="10" ></td><br />
<td width="152" ><img src="https://static.igem.org/mediawiki/2014/d/da/Melbourne_Gahana.jpg" width="150" height="196" alt=""/></td><br />
<td width="10" ></td><br />
<td width="152" ><img src="https://static.igem.org/mediawiki/2014/f/f8/Melbourne_George.jpg" width="150" height="196" alt=""/></td><br />
<td width="10" ></td><br />
<td width="152" ><img src="https://static.igem.org/mediawiki/2014/a/aa/Melbourne_Ken.jpg" width="150" height="196" alt=""/></td><br />
<td width="10" ></td><br />
<td width="152" ><img src="https://static.igem.org/mediawiki/2014/9/9c/Melbourne_Dave.jpg" width="150" height="196" alt=""/></td><br />
</tr><br />
<br />
<tr><br />
<td align="center"><strong>Ashfaqul Hoque</strong></td><br />
<td></td><br />
<td align="center"> <strong>Gahana Advani</strong></td><br />
<td></td><br />
<td align="center"> <strong>George Cao</strong></td><br />
<td></td><br />
<td align="center"><strong>Ken Ang</strong></td><br />
<td></td><br />
<td align="center"><strong>David Zula</strong></td><br />
</tr><br />
<br />
</table><br />
<br />
<p>&nbsp;</p><br />
<p>Many thanks also go to <strong>Sze-Ting Bong</strong> and <strong>Anna Gakusurudoi</strong>, Masters students in the Cheng lab whose assistance with practical methods was extremely helpful.<br />
<!-- END EDIT HERE HERE --><br />
</html></div>Slowehttp://2014.igem.org/Team:Melbourne/ProjectTeam:Melbourne/Project2014-11-25T05:26:58Z<p>Slowe: </p>
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<h1>Project and results</h1><br />
<br />
<p>Read about our experimental work below, or jump to our theoretical collaboration with the <A HREF="#Oxford">University of Oxford</A><br />
<br />
<h2>Introduction and theory</h2><br />
<p>Synthetic peptide chemists have long produced peptide-based materials <em>in vitro</em>. Star-shaped peptides are a promising type of biomaterial being explored in the field of nanomedicine (Sulistio et al., 2012). Star peptides can have several biomedical uses, such as acting as drug delivery vehicles (Sulistio et al., 2011) or linkers for other biomacromolecules. Star peptides generally take the form of several linear peptide arms linked together in a central core. One way of linking these linear peptide arms together is to used covalent bonds such as those in disulfides. Typically, disulfide bonds are formed synthetically by taking several linear arms and treating them with an oxidant <em>in vitro</em>. Here, we introduce a new approach to forming star peptides using <em>E. coli</em> and synthetic biology. Thus, we aimed to show how the peptides synthesis and disulfide bond forming machinery of <em>E. coli</em> can be used to form disulfide linked star peptides and key star peptide precursors.</p><br />
<p>&nbsp;</p><br />
<h2>Synthesis approach</h2><br />
<p><em>E. coli</em> naturally possesses the capacity to form disulfide bonds. In native strains, disulfide bonds are naturally formed by an array of enzymes which are part of the Dsb family (e.g. DsbA and DsbC) (Kadokura et al., 2003, Kadokura and Beckwith, 2009). Normally, these enzymes are found in the oxidizing periplasm of the cell. However, several new strains of <em>E. coli</em> have recently been engineered which contain an oxidizing cytoplasm conducive to disulfide bond formation. One example of this is the SHuffle cell line (Lobstein et al., 2012). The cell line contains mutations to key enzymes responsible for the reducing nature of the cytoplasm, namely thioredoxin reductase (<em>trxB</em>) and glutathione reductase (<em>gor</em>). Furthermore, the Shuffle cell line over expresses the disulfide bond isomerase DsbC to the cytoplasm. Together, these mutations allow SHuffle to fold disulfide-bonded proteins in the cytoplasm at a higher success rate compared to non-mutants. We aimed to take advantage of the disulfide bond forming capabilities of this strain of <em>E. coli</em> to synthesize star peptides in cells. As shown in the figure below, the synthesis steps may proceed as follows:</p><br />
<p>&nbsp;</p><br />
<ol><br />
<li> Express a short peptide containing two cysteine residues either to the <em>E. coli</em> periplasm or the cytoplasm of a <em>trxB gor </em>mutant.</li><br />
<li><em>E. coli</em> disulfide bond forming enzymes fold the peptide into a hairpin loop structure.<br />
</li><br />
<li>Cut the loop at a protease recognition site engineered into the peptide. This may be done by extracting the folded peptide from the cell and treating it with the protease in vitro. Alternatively, the protease may be co-expressed in the cell to allow for in vivo cleavage.<br />
</li><br />
</ol><br />
<p>&nbsp;</p><br />
<table align="center"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9d/Project_concept-v2.png" width="480" height="600" alt=""/></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<p>This synthesis approach has several benefits over purely <em>in vitro</em> approaches. Firstly, the exact peptide sequence can be precisely programmed into <em>E. coli</em> using recombinant DNA synthesis. Secondly, by performing the disulfide bond formation in cells and optionally the proteolytic cleavage, several synthesis steps which would need to be performed <em>in vitro</em> are eliminated. From a scale up perspective, this would eliminate entire unit operations which would otherwise be required to produce this product. Given these benefits, in the current study, we aimed to express a star peptide precursor to the cytoplasm of SHuffle cells, which would later be extracted and externally digested with the star-forming protease. In order to achieve this, we first designed several star peptides which might be amenable to this synthetic strategy. These peptides are described below.</p><br />
<p>&nbsp;</p><br />
<h2>Rationally designed peptides</h2><br />
<p>There are two approaches to functionalising star peptides. In the first, the identity of the arms can be chosen to be bioactive peptide molecules. This is the simplest approach to producing a biologically-relevant star. In the second, the arms can be functionalised by ligating molecules to them at any point. We used both of these approaches to design two separate peptides.</p><br />
<h3>Magainin 1 star and linear peptides</h3><br />
<p>Our first strategy was to make a star peptide using antimicrobial peptides(AMPs) as building blocks. AMPs are small, approximately 50 residue peptides secreted by some bacterial and eukaryotic cells which selectively kill microbial cells. It is thought that AMPs work by forming pores in the membrane of prokaryotic cells (Brogden, 2005). AMPs have been recombinantly expressed in a number of organisms, including <em>E. coli</em> (for a review, see Li, 2011) and <em>B. Subtilis</em> (Chen et al., 2009, Yu et al., 2013).</p><br />
<p>Our concept was to design a star peptide with antimicrobial peptide arms. Wiradharma et al. (2012) first showed that placing linear antimicrobial peptides in a star configuration could lead to enhanced antimicrobial activity and decreased hemolytic activity. Although it is unclear why this is the case, it may be due to the ability of neighbouring antimicrobial peptide arms to interact with each other to synergistically rupture the membrane.<br><br />
While Wiradharma used a synthetic peptide sequence, we designed a peptide using the naturally occurring AMP, Magainin 1 (Zasloff, 1987). Magainin 1 peptides will be placed to the ends of each star arm.</p><br />
<br />
<p>Magainin 1 was chosen because the Magainins are one of the major classes of antimicrobial peptides, being well studied and characterised. In addition, we were concerned that tethering the antimicrobial peptide to the star peptide might interfere with its antimicrobial activity. Magainin 1, however, has previously been tethered to surfaces, where it has imparted the surfaces with microbicidal properties (Glinel et al., 2008; Humblot et al., 2009). We surmised that if Magainin 1 maintained its activity while anchored to surfaces, it may also maintain its activity while anchored to a star peptide.</p><br />
<p>The sequence for Magainin 1 is: GIGKFLHSAGKFGKAFVGEIMKS.</p><br />
<p>When attached to a star peptide it will have the following structure:</p><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/MAGAININ_1_peptide.png" width="720" height="300" alt=""/><br />
<p>There are several design elements to note:</p><br />
<ul><br />
<li><br />
The antimicrobial peptide star will be expressed with a SUMO fusion protein. This is because without the fusion, it is likely that the peptide would be toxic to the host cell.</li><br />
<li>The peptide includes a Factor X cutting site between the two cysteines for eventual proteolytic cleavage and formation of the star peptide.</li><br />
</ul><br />
<p>In addition to the star peptide Magainin 1, we synthesised a gene for a linear Magainin 1 peptide as well. This is identical to the construct above, except that there is only one Magainin 1 peptide attached to the SUMO fusion.</p><br />
<br />
<h3>Unstructured Peptide (USP) Construct</h3><br />
<p>In the second approach, we designed a peptide which can be functionalised using chemical approaches. This peptide was designed to have flexible, unstructured arms and was termed the USP construct. Unlike the Magainin 1 star, the arms of this peptide serve not as active peptides themselves, but as inert structural linkers.</p><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/USPI_Peptide.png" width="720" height="216" alt=""/><br />
<p>The arms were designed with the following elements in mind:</p><br />
<ul><br />
<li><br />
<b>Lack of structure.</b> The arms were designed with a bioinspired approach, using the FxFG motif of nucleoporins (where x is a variable amino acid residue). Such segments naturally repeat in nucleoporins and are thought to lead to disorder/lack of stable secondary structure. Nucleoporins are found in mammalian cells, serving as flexible brushes around nuclear pores (Ader et al., 2010).<br />
</li><br />
<li><b>Water-soluble.</b> The arms were designed with several charged amino acids to improve solubility.<br />
</li><br />
<li><b>Designed to form a disulfide bond.</b> Although it is difficult to rationally ensure that the disulfide bond will form between two cysteines in our peptide, we incorporated a beta turn between the two cysteines which may encourage the peptide to fold at the apex of the hairpin loop. This may bring the cysteines into closer proximity, providing more probable bond formation.</li></ul><br />
<br />
<p>The ultimate utility of this peptide lies in its ability to be functionalised with other biomacromolecules. For example, the technique of Native Chemical Ligation(NCL) can be used to join peptides, proteins, and other ligands to the arms (Dawson et al., 1994). The idea of attaching enzymes to the star peptide was explored by the University of Oxford iGEM 2014 team in a collaborative effort between our two teams (See Supplementary Project Work at the end of this page). </p><br />
<br />
<p>&nbsp;</p><br />
<h2>Expression system</h2><br />
<p>In order to successfully express our constructs, we designed our protein expression vectors to include a fusion protein. The fusion protein was necessary for two reasons. First, some of our constructs are very small (e.g. the non-star Magainin 1), and expression levels of very small peptides can be difficult without a fusion partner. Second, two of our constructs code for antimicrobial peptides. Without a fusion partner, it is likely that these genes would be toxic to their hosts upon induction.</p><br />
<p>To find a suitable expression system, we looked towards the Registry of Standard Parts. We used the SUMO protein expression system designed by TU Delft 2014 (for example, see<a target="_blank" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1022101">http://parts.igem.org/wiki/index.php?title=Part:BBa_K1022101</a>). This system essentially consists of a N-terminal HIS-tag followed by the SUMO protein (also known as UlpI).</p><br />
<p><br />
The strategy of expressing toxic AMPs using SUMO has been successfully reported in the literature (Bommarius et al., 2010). We surmise that the SUMO protein could inhibit the antimicrobial activity of single, linear peptides, and that it may also inhibit the activity of our star antimicrobial peptide.</p><br />
<p><br />
SUMO as a fusion protein also has the benefit of leaving no residues at the C-terminal end of the cleavage site. This means that upon cleavage with the SUMO protease, the native protein can be recovered. In our case, this means that one of the arms of the star can be designed without the need to take into account the addition of any amino acid residues left behind by the protease.<br><br />
We used the SUMO peptide sequence reported by TU Delft. However, our construct contained the following unique features:</p><br />
<ol><br />
<li> Standardisation of the biobrick by substituting the T7 promoter and RBS (the origins of which are both not specified in the Delft documentation) with the standard T7 promoter and RBS BioBrick<a target="_blank" href="http://parts.igem.org/Part:BBa_K525998">BBa_K525998</a>. In addition to supporting the principle of standardization, using the well-characterized promoter BioBrick should help assure expression levels.<br><br />
</li><br />
<li>Biobrick BBa_K1022101 lacks a terminator sequence (this was presumably because the part was meant to be integrated into a larger genetic construct with a terminator). A terminator from the registry of standard parts was added (specifically, the wild type terminator from T7 bacteriophage,<a target="_blank" href="http://parts.igem.org/Part:BBa_K731721">BBa_K731721</a>).<br />
</li><br />
<li>The original biobrick BBa_K1022101 codes for three amino acid residues before the HIS-tag (ASM), which appeared to be redundant. Correspondence with the 2013 TU Delft team suggested that these residues were unnecessary and appear to be cleaved within the cell as part of the cells post-translational modifications. However, their presence complicates the addition of additional tags at the N-terminus of the protein (e.g. periplasmic export tags), and therefore were not included.<br><br />
</li><br />
<li>The SUMO sequence was codon optimised for E. coli during synthesis. As the Delft documentation did not specify whether the gene was codon optimal, codon optomisation was undertaken to potentially improve expression levels. The linear Magainin 1 construct, however, was not codon optimised in the SUMO region in order to provide a control condition.</li><br />
</ol><br />
<p>To summarise, the protein expression devices used in our project to the following form:</p><br />
<img src="https://static.igem.org/mediawiki/2014/d/d0/Gene_circuit_export.png" width="720" height="91" alt=""/></td><br />
<p>The protein coding region consisted of a 6x-HIS tag followed by the SUMO protein and the relevant star or linear peptide.</p><br />
<p>&nbsp;</p><br />
<h2>Plasmid preparation: Cloning and acquisition of the genetic constructs</h2><br />
<h3>Magainin 1 Star Peptide</h3><br />
<p>This construct was synthesised in the standard shipping plasmid from Life Technologies- plasmid pMK. We had originally planned to express our protein to the periplasm of E. coli, and therefore had included in the synthesis a periplasmic export tag, the TorTss signal sequence (Steiner et al., 2006).</p><br />
<p><br />
Cytoplasmic and periplasmic expression may both allow for disulfide bond formation in the cell. We decided, however, to focus on cytoplasmic expression. There are distinct advantages to cytoplasmic expression (e.g. the absence of several periplasmic proteases and potentially higher expression levels (Baneyx, 1999)).</p><br />
<p><br />
Another reason the cytoplasm was chosen was to allow us to test the effects of an oxidising versus reducing intracellular environment on disulfide bond formation. We planned to express the construct in both SHuffle T7 cells (oxidising cytoplasm) and BL21(DE3) (reducing cytoplasm) to probe whether there was a difference in disulfide bond formation. Therefore, we needed to remove the periplasmic export tag from the gene.</p><br />
<p><br />
To do this, we use the following cloning strategy (noting that the top row represents the gene initially synthesised in plasmid pMK):</p><br />
<img src="https://static.igem.org/mediawiki/2014/2/2c/Melbourne_cloning_strategy_diagram.jpg" width="900" height="437" alt=""/><br />
<p>The gene was inserted into plasmid pSB1C3 containing the T7 promoter and ribosome binding site, BBa_K525998. It was inserted after the promoter and before the BioBrick suffix. This was accomplished by digesting the destination vector with SpeI and PstI. At the same time, PCR was used to amplify the segment of the gene containing the SUMO fusion and the Magainin 1 Star peptide, adding XbaI and keeping the PstI site existing in the gene.</p><br />
<p><br />
Digestion of the PCR product then allowed for ligation of the insert and destination vector using the PstI sites and XbaI and SpeI (XbaI and SpeI have compatible sticky ends). Note that after the ligation, there will be a scar in the gene where the XbaI and the SpeI sites were ligated.</p><br />
<p><br />
The ligation appeared to be successful. The ligation mixture was transformed to DH5α competent cells and plated onto chloramphenicol-containing agar plates.</p><br />
<p><br />
Plasmid from 5 colonies (C1, C2, C3, C4, and C5) was cultured, extracted, and then digested with EcoRI and PstI.</p><br />
<p><br />
As evident in the figure below, at least 4 of the colonies appeared to contain the insert. The empty, linearised pSB1C3 backbone ran at approximately the correct size (2070 bps), as did the insert (740 bps).</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/9/95/Magainin_1_subcloning.png" width="353" height="400" alt=""/></center><br />
<p>N.B. MW marker is the 100 bp Ladder from Axygen. * indicates the colony picked for sequencing and eventual transformation to the expression cell lines.</p><br />
<p>The DNA from Colony C2 was confirmed using Sanger sequencing at the Australian Genome Research Facility. This DNA appears in the registry of standard parts as BioBrick <a target="_blank" href="http://parts.igem.org/Part:BBa_K1394000">BBa_K1394000</a>.</p><br />
<p><br />
For the USP peptide and the linear Magainin 1 peptide, the genes were synthesised by GenScript and delivered in pSB1C3. The expression vectors had identical gene regulatory elements to that used for the Magainin 1 Star peptide. They only differed in the codon optimisation used, and they also lacked the assembly scar described above.</p><br />
<br />
<h2><br />
Protein expression and characterisation<br />
</h2><br />
<p><br />
After acquiring our genes, the project moved to protein expression and purification. We expressed both star peptides (Magainin 1 Star Peptide and the USP peptide) in both the SHuffle T7 and BL21(DE3) cell lines, both sourced from New England Biolabs. As the Linear Magainin 1 peptide does not have any special disulfide bonding<br />
requirements, we expressed it in BL21(DE3).<br />
</p><br />
<p><br />
The plasmids were transformed to the expression cells, and a single colony was cultured and induced overnight at 17 °C. A whole cell sample both before<br />
IPTG induction (-IPTG) and after the induction period (+IPTG) were boiled in SDS-PAGE sample buffer and loaded on a 15% tris-glycine gel. The whole cell<br />
Coomassie stained gel is shown below alongside the NEB P7712S molecular weight marker.<br />
</p><br />
<br><br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/2/24/Whole_cell_CS.png" height="330" width="439"/><br />
</p><br />
<br><br />
<p><br />
From theoretical prediction of the peptide masses, we would expect the following distribution of molecular weights:<br />
</p><br />
<br><br />
<table border="1" cellpadding="0" cellspacing="0" align="center"><br />
<tbody><br />
<tr><br />
<td width="156"><br />
<p align="center"><br />
Magainin 1 Star Peptide (Mag1 Star)<br />
</p><br />
</td><br />
<td width="145"><br />
<p align="center"><br />
USP<br />
</p><br />
</td><br />
<td width="156"><br />
<p align="center"><br />
Linear Magainin 1 (Linear Mag1)<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="156"><br />
<p align="center"><br />
22.15 kDa<br />
</p><br />
</td><br />
<td width="145"><br />
<p align="center"><br />
29.3 kDa<br />
</p><br />
</td><br />
<td width="156"><br />
<p align="center"><br />
14.32 kDa<br />
</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<br><br />
<p><br />
We looked for bands corresponding to overexpression of the proteins. We observed a thick band, post IPTG around 32 kDa in the BL21(DE3) cells carrying the<br />
USP plasmid. However, we saw this same band appearing in the Linear Magainin 1 lane but the Linear Magainin 1 protein should have a much lower molecular<br />
weight than what was observed. Therefore, we did not assume that we had overexpression overexpression of the USP 1 protein.<br />
</p><br />
<p><br />
We also noted bands in all post-IPTG lanes around 17 kDa. This would be roughly consistent with both the Linear Magainin 1 and Magainin 1 Star Peptide,<br />
noting that small proteins may not run at their expected molecular weight. Again, the analysis is complicated by the fact that the band also appears in the<br />
lane corresponding to the larger molecular weight protein, USP I. Given this ambiguity, we decided to purify all the protein in the sample using Ni-NTA<br />
purification.<br />
</p><br />
<h3><br />
Purification and further characterisation<br />
</h3><br />
<p><br />
A small-scale purification was carried out according to our <a href="https://static.igem.org/mediawiki/2014/e/e1/MELBOURNE_D1V1_Column_Purification_of_His-Tagged_Proteins.pdf">protocol</a>. The cells were lysed with iodoacetamide, an alkylating agent, added to the lysis buffer. Iodoacetamide blocks all free cysteines on proteins with a short alkyl group. This was added<br />
because we wanted to determine if a disulphide bond had formed inside the cell. If we did not block free cysteines, then any bond that had formed could be<br />
attributed to spontaneous/air oxidation of disulfides outside the cell.<br />
</p><br />
<p><br />
After purification, we ran a concurrent Western Blot and Coomassie stain on both the pre-induction samples from above and the purified protein (namely, the<br />
first elution from the batch purification).<br />
</p><br />
<p><br />
The Western Blot used mouse monoclonal antibodies against the N-terminal HIS-tag as primary antibodies and anti-mouse secondary antibodies:<br />
</p><br />
<br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/5/54/Western_Blot_Melb.png" height="400" width="460" border="0"/><br />
</p><br />
<p><br />
The corresponding Coomassie stain is show below:<br />
</p><br />
<br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f0/Purified_CS.png" height="365" width="486" border="0"/><br />
</p><br />
<p><br />
There are several interesting aspects of the Western Blot. Firstly, there was a strong band between 25 and 32 kDa in most lanes. However, it<br />
appears in all of the pre-induction controls, suggesting non-specific binding of the anti-HIS antibodies.<br />
</p><br />
<p><br />
Secondly, we observed a band in most of the lanes around 17 kDa (with the exception being the USP protein in SHuffle cells). As these bands were not<br />
present in the pre-induction controls, we suspected they were a result of the induction.<br />
</p><br />
<h3><br />
Mass spectroscopy<br />
</h3><br />
<p><br />
In order to assess the identity of the bands, we performed an in-gel tryptic digestion on select bands. We digested bands in the Coomassie stain which<br />
corresponded to the HIS-tagged bands in the Western Blot. This was followed by mass spec analysis (LC MS/MS). We focused our analysis on the Magainin 1<br />
Star Peptide bands and the Linear Magainin 1 band. Our USP peptide was, by design, highly rich in basic residues, greatly reducing the likelihood that<br />
tryptic fragments would be detected by the mass spec.<br />
</p><br />
<p><br />
We found that the Linear Magainin 1 peptide appeared to be present in the approximately 17 kDa band, with the following detected tryptic fragments in the<br />
sequence below (bold; basic residues underlined):<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/1/1b/Tryptic_digest_linear_mag1.PNG" height="63" width="601" border="0"/><br />
</p><br />
<p><br />
Note that spaces have been added in the sequence to emphasise distinct domains in the protein (in this case, the fusion protein versus the Magainin 1<br />
peptide). Together with the fact that the protein runs close to the expected molecular weight, this seems to provide good evidence that the protein is<br />
being expressed.<br />
</p><br />
<p><br />
We then examined the bands corresponding to the Magainin 1 Star Peptide.<br />
</p><br />
<p><br />
Again, we digested bands in the Coomassie stained gel corresponding to the prominent HIS-tagged bands on the <em>Western Blot</em> (near 17 kDa).<br />
</p><br />
<p><br />
The mass spec coverage for the Magainin 1 Star Peptide expressed in SHuffle cells was as follows:<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/6/63/Tryptic_digest_Sample_A_Star_Mag_Shuffle.PNG" height="90" width="601" border="0"/><br />
</p><br />
<p><br />
The coverage for the same peptide expressed in BL21(DE3) was found to be:<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/d/db/Tryptic_digest_Sample_A_Star_Mag_BL21%28DE3%29.PNG" height="85" width="602" border="0"/><br />
</p><br />
<p><br />
Again, we can see that tryptic peptides from the expressed protein appear to present in the gel band under analysis.<br />
</p><br />
<p><br />
There is a relatively lower sequence coverage. This could be a limitation of our procedure: for example, improper destaining during the in gel digestion<br />
procedure can inhibit tryptic digestion.<br />
</p><br />
<p><br />
However, even if the digestion was complete, the mass spec will only detect fragments which ionize well. The Magainin 1 Star peptide consists of four<br />
repeats of the Magainin 1 sequence (corresponding to the arms of the star). If the two tryptic fragments within Magainin 1 do not ionize well, then indeed<br />
the entire peptide would not be read.<br />
</p><br />
<p><br />
Finally, it is possible that the protein has been cleaved or degraded. This would account for the slightly lower-than-expected mass on the SDS page gel.<br />
</p><br />
<p><br />
Nonetheless, it is a possibility that the peptide fragments are present but not detected. Replication of the in-gel mass spec would be useful<br />
in clarifying this point, or provided the purity of the sample is acceptable, it may be possible to run an intact mass spec. Digestion with alternative enzymes may also provide a more robust sequence coverage, particularly for the USP peptide.<br />
</p><br />
<h2><br />
Conclusions<br />
</h2><br />
<p><br />
We designed several novel star peptides based on several rational design criteria. Further, we began the optimisation process required to express these peptides in <em>E. coli</em>.<br />
</p><br />
<p><br />
To this end, we constructed an expression system based on the SUMO protein and standard BioBrick parts. The function of this system was confirmed, as it<br />
appears it does produce HIS-tagged SUMO fusion protein in <em>E. coli</em>, as expected. Nonetheless, we hope to continue to characterise our parts, likely through further purification and measurement of the mass. <br />
</p><br />
<p>Given the success of SUMO in the protein expression community, we hope our BioBrick<br />
will encourage further use of the fusion domain within iGEM. In addition, we hope that our ideas and concepts will inspire future<br />
iGEM efforts to explore the applications of synthetic biology for material science. We continue to believe that bacteria have great promise for the <em>in vivo</em><br />
construction of unique biomaterials, and we look forward to seeing how the synthetic biology community will develop in this area.<br />
</p><br />
<br />
<br />
<br />
<h2>References</h2><br />
<p>ADER, C., FREY, S., MAAS, W., SCHMIDT, H. B., GÖRLICH, D. &amp; BALDUS, M. 2010. Amyloid-like interactions within nucleoporin FG hydrogels. <em>Proceedings of the National Academy of Sciences,</em> 107<strong>,</strong> 6281-6285.</p><br />
<p><br />
BANEYX, F. 1999. Recombinant protein expression in Escherichia coli. <em>Current Opinion in Biotechnology,</em> 10<strong>,</strong> 411-421.</p><br />
<p><br />
BOMMARIUS, B., JENSSEN, H., ELLIOTT, M., KINDRACHUK, J., PASUPULETI, M., GIEREN, H., JAEGER, K. E., HANCOCK, R. E. W. &amp; KALMAN, D. 2010. Cost-effective expression and purification of antimicrobial and host defense peptides in Escherichia coli. <em>Peptides,</em> 31<strong>,</strong> 1957-1965.</p><br />
<p><br />
BROGDEN, K. A. 2005. Antimicrobial peptides: Pore formers or metabolic inhibitors in bacteria? <em>Nature Reviews Microbiology,</em> 3<strong>,</strong> 238-250.</p><br />
<p><br />
CHANG, C., CHHOR, G., CLANCY, S. & JOACHIMIAK, A. 2014. Crystal Structure of Glutathione S-transferase Domain Protein From Haliangium Ochraceum DSM 14365 [Online]. ''NCBI. Available: http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=4w66' [Accessed September 9 2014].</p><br />
<p><br />
CHEN, X., ZHU, F., CAO, Y. &amp; QIAO, S. 2009. Novel expression vector for secretion of cecropin AD in Bacillus subtilis with enhanced antimicrobial activity. <em>Antimicrobial agents and chemotherapy,</em> 53<strong>,</strong> 3683-3689.</p><br />
<p><br />
GLINEL, K., JONAS, A. M., JOUENNE, T., LEPRINCE, J., GALAS, L. & HUCK, W. T. 2008. Antibacterial and antifouling polymer brushes incorporating antimicrobial peptide. <em>Bioconjug Chem,</em> 20<strong>,</strong> 71-77.</p><br />
<p><br />
HUMBLOT, V., YALA, J.-F., THEBAULT, P., BOUKERMA, K., HÉQUET, A., BERJEAUD, J.-M. & PRADIER, C.-M. 2009. The antibacterial activity of Magainin I immobilized onto mixed thiols self-assembled monolayers. <em>Biomaterials,</em> 30<strong>,</strong> 3503-3512.</p><br />
<p><br />
DAWSON, P. E., MUIR, T. W., CLARK-LEWIS, I. &amp; KENT, S. 1994. Synthesis of proteins by native chemical ligation. <em>Science,</em> 266<strong>,</strong> 776-779.</p><br />
<p><br />
KADOKURA, H. &amp; BECKWITH, J. 2009. Detecting folding intermediates of a protein as it passes through the bacterial translocation channel. <em>Cell,</em> 138<strong>,</strong> 1164-1173.</p><br />
<p><br />
KADOKURA, H., KATZEN, F. &amp; BECKWITH, J. 2003. Protein disulfide bond formation in prokaryotes. <em>Annual review of biochemistry,</em> 72<strong>,</strong> 111-135.</p><br />
<p><br />
LI, Y. 2011. Recombinant production of antimicrobial peptides in&lt; i&gt; Escherichia coli&lt;/i&gt;: A review. <em>Protein Expression and Purification,</em> 80<strong>,</strong> 260-267.</p><br />
<p><br />
LOBSTEIN, J., EMRICH, C. A., JEANS, C., FAULKNER, M., RIGGS, P. &amp; BERKMEN, M. 2012. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. <em>Microb Cell Fact,</em> 11<strong>,</strong> 56-56.</p><br />
<p><br />
PROTEIN MODEL PORTAL. Query result, GST and DcmA [Online]. Available: http://www.proteinmodelportal.org/query/uniprot/P21161 [Accessed September 9 2014].</p><br />
<p><br />
STEINER, D., FORRER, P., STUMPP, M. T. &amp; PLUCKTHUN, A. 2006. Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display. <em>Nat Biotech,</em> 24<strong>,</strong> 823-831.</p><br />
<p><br />
SULISTIO, A., GURR, P. A., BLENCOWE, A. &amp; QIAO, G. G. 2012. Peptide-Based Star Polymers: The Rising Star in Functional Polymers. <em>Australian Journal of Chemistry,</em> 65<strong>,</strong> 978-984.</p><br />
<p><br />
SULISTIO, A., LOWENTHAL, J., BLENCOWE, A., BONGIOVANNI, M. N., ONG, L., GRAS, S. L., ZHANG, X. &amp; QIAO, G. G. 2011. Folic Acid Conjugated Amino Acid-Based Star Polymers for Active Targeting of Cancer Cells. <em>Biomacromolecules,</em> 12<strong>,</strong> 3469-3477.</p><br />
<p><br />
TANAKA, N., KUSAKABE, Y., ITO, K., YOSHIMOTO, T. & NAKAMURA, K. T. 2002. Crystal Structure of Formaldehyde Dehydrogenase from< i> Pseudomonas putida</i>: the Structural Origin of the Tightly Bound Cofactor in Nicotinoprotein Dehydrogenases. Journal of molecular biology, 324, 519-533.</p><br />
<p><br />
WIRADHARMA, N., LIU, S. Q. &amp; YANG, Y. Y. 2012. Branched and 4-Arm Starlike α-Helical Peptide Structures with Enhanced Antimicrobial Potency and Selectivity. <em>Small,</em> 8<strong>,</strong> 362-366.</p><br />
<p><br />
YU, Z., WANG, Q., MA, Q. &amp; ZHANG, R. 2013. Secretory expression of lacticin Q fused with SUMO in Bacillus subtilis. <em>Protein Expression and Purification,</em> 89<strong>,</strong> 51-55.</p><br />
<p><br />
ZASLOFF, M. 1987. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. <em>Proceedings of the National Academy of Sciences,</em> 84<strong>,</strong> 5449-5453.</p><br />
<p>&nbsp;</p><br />
<br />
<A NAME="Oxford"></A><br />
<h1>Collaboration with Oxford</h1><br />
<p>To strengthen the sense of iGEM community, we contacted <a target="_blank" href="https://2014.igem.org/Team:Oxford">University of Oxford iGEM team</a> to discuss the possibility of collaboration between our teams. The Oxford project aims to develop a system that can dispose of the carcinogenic, hazardous solvent dichloromethane (DCM). To do this, Oxford team has proposed the use of the DcmA enzyme. This enzyme degrades DCM to form a toxic intermediate which in turn is converted by another enzyme, FdhA, into a neutral molecule. Schematically this can be presented in the following way:</p><br />
<br />
<p><br />
Reaction 1: DCM +&nbsp;DcmA -> <strong>toxic intermediate</strong><br><br />
Reaction 2: <strong>toxic intermediate</strong> + FdhA -> neutral product.</p><br />
<p>The problem for their system lies in bringing the two enzymes together to ensure efficient reaction kinetics. An idea that we discussed with Oxford was to use the star peptide platform to link the two enzymes together, in a structure similar to the following: </p><br />
<img src="https://static.igem.org/mediawiki/2014/2/2e/Melbourne_Collab_1.png" width="500" height="325" alt=""/><br />
<p>We worked with Oxford to study this system from a theoretical standpoint. Our team studied the 3D structure of both enzymes involved to confirm that the enzyme could in theory be attached to a linker in this manner, while Oxford team did stochastic modelling to determine how reaction rate changes when the linker length, labeled D on the diagram, changes. </p><br />
<p><br />
As a first check, it was necessary to determine whether anchoring these enzymes to our star would be sterically possible. We studied the structures of FdhA and DcmA determined by crystallography using data from the protein data bank. As no crystallographic data was available in the databank for DcmA, GST was examined as a proxy, as GST is structurally homologous to DcmA. </p><br />
<p><br />
The crystalographically resolved structure of FdhA enzyme (Tanaka et al., 2002) from the protein data bank revealed the following image:</p><br />
<img src="https://static.igem.org/mediawiki/2014/d/dc/Melbourne_Collab_2.png" width="700" height="355" alt=""/><br />
<p>The crystalographically resolved structure of GST was as follows (Chang et al., 2014):</p><br />
<img src="https://static.igem.org/mediawiki/2014/e/e9/Melbourne_Collab_3.png" width="800" height="361" alt=""/><br />
<p>It can be seen that the amino- and carboxy-terminals are located away from the active site. This suggests that both enzymes, DcmA and FdhA, might be linked together via linkers that are used in our star peptide. That is, the active site will not be sterically hindered by attachment to the star peptide. That said, it is difficult to predict how anchoring the protein to the star will affect its tertiary structure. Further, it is difficult to predict how limiting the rotational degrees of freedom will affect enzyme activity.</p><br />
<br />
<p>The Oxford team modeled this system and found that, indeed, the star peptide as an enzyme linker was favourable to enzyme kinetics. Their work can be found <br />
<br />
<a href="https://2014.igem.org/Team:Oxford/alternatives_to_microcompartments#show2"><br />
here</a>.</div>Slowehttp://2014.igem.org/Team:Melbourne/TeamTeam:Melbourne/Team2014-11-25T05:24:31Z<p>Slowe: </p>
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<h1 >Team</h1><br />
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<h3>About The University of Melbourne</h3><br />
<p>Located in Melbourne, Victoria, The University of Melbourne was founded in 1853 and is Australia’s second oldest university. The university is consistently ranked among the leading universities in the world, with international rankings of world universities placing it at number 1 in Australia and number 34 in the world (Times Higher Education World University Ranking 2013-2014).</p><br />
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<h3>About The 2014 Melbourne iGEM Team</h3><br />
<p>The 2014 Melbourne iGEM team consists of 19 highly-motivated undergraduate students and 1 postgraduate student from a range of disciplines, including biochemistry and molecular biology, chemistry, chemical engineering and bioengineering. The team’s academic and research credentials are well-tested. This year, the team comprises three of The University of Melbourne’s Chancellor’s Scholars (top 0.1% of all Australian high school students) and several team members with research experience at Melbourne’s high-profile research institutes, such as the prestigious Walter and Eliza Hall Institute, The Peter MacCallum Cancer Centre, and The Baker IDI Heart and Diabetes Institute. The team is supervised by Dr Heung-Chin Cheng, Professor Paul Gooley, Dr Angus Johnson and Dr Neil O’Brien-Simpson.<br />
The Melbourne iGEM team was founded by a group of enthusiastic and motivated students in 2013. The 2013 team played a key role in establishing the foundations of this year’s team and generating this year’s project. Unfortunately many of the 2013 team members could not continue their work for iGEM in 2014, so credit must also be given to Pedro Avellar Costa, Michelle Tie, Georgina Panshem, Morgana Cerqueira, Jeong Yoon Esther Kim, Winnie Tan, Hannah Nguyen, Mary Teo, Cathy Pitt and Joyce Kant.</p><br />
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<h3>Faculty Supervisors</h3><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/d/d4/Melbourne_Cheng.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Heung-Chin Cheng (Supervisor)</strong></p><br />
An Associate Professor with the Department of Biochemistry and Molecular Biology, Heung-Chin has been studying the biochemical basis of the regulation of protein kinases and phosphatases since he was a graduate student at the University of California in 1982. His PhD project unravelled the active site structure of c-AMP-dependent protein kinase, how the kinase recognises its protein substrates and how the kinase is selectively inhibited by its endogenous inhibitor. Heung-Chin was the Melbourne iGEM team&rsquo;s supervisor and he kindly offered us space to work in his lab and assisted us when we had questions about practical procedures, troubleshooting or theory.</td><br />
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<td><p><strong>Paul Gooley (Advisor)</strong></p><br />
Associate Professor Paul Gooley directs a structural biology research group that focuses on the application of NMR spectroscopy to elucidate structure and protein interactions. He obtained his degrees at The University of New South Wales and spent 10 years in the USA, including 5 years at the pharmaceutical company Merk and Co. Over the last 10 years his group has conducted and published NMR structural and dynamical analyses on a number of protein domains and systems that have biological functions in stress and infection, in lipid transport, in protein and membrane trafficking, and in receptor signalling. Paul assisted the 2014 Melbourne iGEM team by providing advice, answering questions and brainstorming.</td><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/9/97/Melbourne_Angus.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Angus Johnston (Advisor)</strong></p><br />
Dr Angus Johnston is a researcher at the Monash Institute of Pharmaceutical Science. Angus is an ARC Future Fellow who’s work focuses on developing better ways to deliver drugs, making them more therapeutically active and limiting side effects. He has extensive knowledge and expertise in nanomaterials assembly, material characterisation, cellular interactions and advanced imaging techniques. Angus’ research interests include targeted vaccine therapy, sensors for cellular imaging, understanding cellular processing of nanoparticles, self assembling peptides as drug carrier and the toxicology of nanomaterials. Angus assisted the 2014 Melbourne iGEM team by providing advice, answering questions and brainstorming.</td><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/f/fd/Melbourne_Neil_O%E2%80%99Brien-Simpson.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Neil O’Brien-Simpson (Advisor)</strong></p><br />
Associate Professor Neil O’Brien-Simpson is a researcher at the Royal Dental Hospital in Melbourne and has an interdisciplinary background, combining organic and peptide chemistry with immunology to develop novel vaccines and therapeutics and investigating the immune response to pathogens. His research into vaccine design and periodontitis has resulted in Neil being awarded the Colgate Prize for Dental Research (1999), The IADR Hatton Award (2000) and the Oral Biology Award (2003). In 2004 he became program co-leader for the Novel Diagnostics, Vaccines and Pharmaceuticals Research Program in the Oral Health CRC. Neil assisted the 2014 Melbourne iGEM team by providing advice, answering questions and brainstorming.</td><br />
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<h3>Team Members</h3><br />
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<td><p><strong>Sean Lowe (Team Leader)</strong></p><br />
Sean started the 2014 Melbourne iGEM team while completing his Master of Engineering in chemical engineering. As the team leader, Sean was responsible for communicating with supervisors and sponsors, setting up the lab, and making sure the team ran smoothly. Sean is passionate about science and saw iGEM as an amazing opportunity to combine his interests in chemical engineering and biotechnology. He found that the best part of iGEM was the creative process, and thoroughly enjoyed discussing many great ideas with his bright and capable teammates. He looks forward to passing on the torch to the next generation of Melbourne Uni iGEMers.</td><br />
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<td><p><strong>Elizabeth Brookes</strong></p><br />
Elizabeth is in her second year of the Bachelor of Biomedicine degree and is majoring in Immunology. In the future Elizabeth hopes to continue to develop her passion for research while studying postgraduate medicine. Elizabeth was new to iGEM in 2014 and contributed to Melbourne’s iGEM project by working in the laboratory, developing protocols and performing various administrative duties. For Elizabeth, the most enjoyable aspect of iGEM was getting hands-on experience while meeting like-minded people. In her free time Elizabeth enjoys playing netball and discovering new restaurants with friends.</td><br />
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<td><p><strong>Peter Collins</strong></p><br />
Peter is in his final year of the Bachelor of Science at The Australian National University in Canberra and is majoring in Science Communications, as well as completing his first year of the Bachelor of Environments at The University of Melbourne. In the future, Peter aims to work on The Human Variome Project, develop the iGEM competition in Australia and work for UNESCO. Peter joined the Melbourne iGEM team in 2014 and was in charge of human practices and outreach, working to establish relations with high schools in Melbourne for on-going public outreach efforts in the future. As such, Peter has enjoyed getting an insight into managing science innovation teams and the tremendous array of challenges that accompany this. In his spare time, Peter enjoys learning about esoteric topics that may have unrealised commercial applications.</td><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/1/1a/Melbourne_Sheryl.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Sheryl Ding</strong></p><br />
Sheryl is in the first year of a Bachelor of Biomedicine degree and is hoping to major in Bioengineering Systems. In the future, Sheryl would like to work in biomedical research, preferably specialising in engineering or molecular and cellular biology. After joining the iGEM team in early 2014, Sheryl contributed to the laboratory work and some administrative tasks for the project. Sheryl enjoyed all aspects of this experience, but particularly the opportunity to gain practical insight into laboratory research and meet the people on the iGEM team. Outside of science, Sheryl enjoys visual art and watching TV shows.</td><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/6/66/Melbourne_Robyn.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Robyn Esterbauer</strong></p><br />
As a second year student of the Bachelor of Science with a major in Microbiology and Immunology, Robyn hopes to undertake a masters and PhD, specialising in infectious disease research. After joining the team in March 2014, Robyn contributed to the team with her work in the laboratory, research into cysteine bond formation and computational protein design analysis, and public outreach activities. Robyn has enjoyed experiencing a group research environment and learning some very useful practical laboratory skills. In her free time, Robyn loves to read cutting edge infectious disease research and playing soccer.</td><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/d/d4/Melbourne_Tobias.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Tobias Gustavsson</strong></p><br />
Tobias is in his second year of the Bachelor of Biomedicine degree and is majoring in Immunology. In the future Tobias hopes to study medicine and continue to pursue research in immunology. Tobias was new to iGEM in 2014 and contributed to the theoretical design and research of the team’s project. iGEM presented Tobias with the opportunity to finally partake in the laboratory work that he had heard so much about in lectures.</td><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/e/e8/Melbourne_Henry.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Henry Howard</strong></p><br />
Henry has just completed his Honours year for the Bachelor of Biomedicine and in the future he hopes to undertake a PhD and work in Melbourne’s bustling medical research sector. After joining the iGEM team in February 2013, Henry predominantly contributed to the theoretical design of the project. He most enoyed having access to resources that have allowed us to undertake serious scientific research of our own design. Outside of iGEM, Henry is a black belt in Tang Soo Tao and enjoys planting trees and completing jigsaw puzzles.</td><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/8/8f/Melbourne_Aishwarya.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Aishwarya Kulkarni</strong></p><br />
Aishwarya is in her second year of the Bachelor of Science and is majoring in Biochemistry and Molecular Biology. Her future plans include completing a PhD and research in the area of translational bioinformatics and personalised medicine. As part of the Melbourne iGEM 2014 team, Aishwarya contributed to laboratory and budgeting tasks. She enjoyed both the atmosphere of the team and the challenges with which we were confronted when trying to work independently to solve problems. Outside of iGEM, Aishwarya also enjoys playing tennis.</td><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/2/2a/Melbourne_Ranit.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Ranit Lahmy</strong></p><br />
Ranit is in her final year of the Bachelor of Science degree and is completing her major in Chemistry. Ranit joined the Melbourne iGEM team in early 2013 and has contributed to many facets of the project, including laboratory work, administration and human outreach. As a consequence, Ranit has really enjoyed the chance to meet new people while learning research and laboratory skills. When not working on iGEM, Ranit enjoys reading books, playing tennis and hanging out with friends.</td><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/b/bc/Melbourne_Jeffrey.JPG" width="150" height="196" alt=""/></td><br />
<td><p><strong>Jeffrey Lai</strong></p><br />
Jeffrey is in his first year of the Bachelor of Biomedicine at The University of Melbourne and in the future he would like to become a medical practitioner or researcher. After joining the Melbourne team in July 2014, Jeffrey helped out in the laboratory work and the iGEM website. Jeffrey found it incredibly rewarding when we finally saw some promising results after putting so much time and effort into the project. In his spare time, Jeffrey likes to play the piano and compose symphonic music.</td><br />
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<td><p><strong>Melissa Leckie</strong></p><br />
Melissa is in her final year of the Bachelor of Science and is completing a major in biochemistry and molecular biology. Competing in her first iGEM jamboree in 2008, Melissa joined the current team in 2013 and has contributed to project design, administration and human outreach. Through this experience Melissa has enjoyed making life long friends and facing the challenges of being part of a student run team. Melissa wishes to pursue a career in Medical Technology and in her spare time she enjoys reading and travelling.</td><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/3/3c/Melbourne_Keit.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Keit Loi</strong></p><br />
Keit is in his final year of the Bachelor of Biomedicine and is majoring in infection and immunity. Since joining the Melbourne iGEM team in early 2014, Keit has mainly contributed to the laboratory work for the project and he has enjoyed that this has given him a good understanding of what working in research would be like. In the future, Keit would like to pursue this passion and work towards a PhD and university lecturing. Outside of iGEM, Keit works on projects with high school students and enjoys chilling with friends and kicking back with some video games.</td><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/0/07/Melbourne_Darcy.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Darcy Marum</strong></p><br />
Darcy is in his second year of the Bachelor of Science degree and is majoring in Microbiology and Immunology. Darcy hopes to pursue research in these exciting fields after completing an honours year. After joining the team in early 2014, Darcy predominantly worked on laboratory work for the project and enjoyed the practical experience that he gained.</td><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/2/2e/Melbourne_Gayle.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Gayle Pereira</strong></p><br />
Gayle is in her final year of the Bachelor of Science and is majoring in Pathology. In the future Gayle hopes to pursue a career either as a clinical pathologist or secondary school teacher. Gayle joined the iGEM team in 2013 and was the senior laboratory leader for the group, tirelessly working and teaching others in the laboratory. Working on the project, Gayle appreciated gaining the laboratory experience that she requires for her future ambitions. In her free time Gayle enjoys supporting her team in the Australian Football League and doing handicraft-based hobbies.</td><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/b/b1/Melbourne_Lumi.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Lumi Tomren</strong></p><br />
Lumi is in her first year of the Bachelor of Biomedicine at The University of Melbourne and is planning on majoring in Microbiology and Immunology. With a desire to learn more about synthetic biology and the microscopic world around us, Lumi joined the team in Spring 2014. Lumi has contributed to the team by performing lab work and she has really enjoyed learning a lot of new practical techniques. She has also enjoyed discovering many of the possibilities of synthetic biology by researching past projects undertaken by iGEM teams. Outside of iGEM and science, Lumi enjoys visual art.</td><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/d/d6/Melbourne_Michael.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Michael Wei</strong></p><br />
Michael is in his first year of the Bachelor of Biomedicine and plans on majoring in neuroscience. Michael was new to iGEM in 2014 and worked primarily in the laboratory and designing primers. Michael is interested in working towards a career in neurosurgery or cancer tumour research, looking specifically at the epigenetic mechanisms underlying cancer regulation. As part of the iGEM team, Michael has enjoyed meeting like-minded individuals who share similar interests. Michael is fascinated by marine life and enjoys reading in his leisure time.</td><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/0/07/Melbourne_Stan.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Stanislav Yatskevich</strong></p><br />
Although Stanislav started studying his first year of a Bachelor of Biomedicine at The University of Melbourne, he has now commenced study at The University of Oxford reading Biochemistry. Stanislav joined the Melbourne iGEM team in March 2014 and contributed greatly to the theoretical project design and laboratory work. After moving to Oxford, Stanislav also facilitated communication between our team and the Oxford iGEM team. In the future, Stanislav would like to pursue research in Biochemistry and he has appreciated the amount of experience he was able to gain in such a short time while taking part in iGEM. Outside of Biochemistry, Stanislav enjoys playing football and procrastinating his study.</td><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/d/dc/Melbourne_Nicholas.JPG" width="150" height="196" alt=""/></td><br />
<td><p><strong>Nicholas Yee</strong></p><br />
Nicholas has just completed the Honours year of his Bachelor of Biomedicine degree, studying novel pathway inhibitors to elucidate mechanisms of brain tumours. He plans to continue his research into brain tumours by undertaking a PhD and potentially studying medicine. After joining the team in 2014, Nicholas enjoyed helping the younger students in the team by teaching them new laboratory techniques and troubleshooting their experiments. When not in the laboratory, Nicholas enjoys hanging out with friends and playing badminton.</td><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/1/1b/Melbourne_Michelle.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Michelle Zheng</strong></p><br />
Michelle is in her second year of the Bachelor of Science and is undertaking the Biochemistry major. At the completion of her degree, Michelle seeks to pursue further research through the honours or masters programmes. Michelle joined the Melbourne iGEM team in 2014 and completed many hours of laboratory work contributing to the project. Being part of a team dedicated to science, the laboratory work and the challenge of overcoming troubleshooting problems were the aspects of iGEM that excited Michelle most. In her free time she enjoys playing the violin.</td><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/0/0f/Melbourne_Wayne.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Wayne Zheng</strong></p><br />
Wayne is in his first year of the Bachelor of Biomedicine and hopes to eventually major in Human Structure and Function. In the future, Wayne would like to continue his involvement in biomedical research while also studying and practising medicine. In contributing to the 2014 Melbourne iGEM team Wayne performed laboratory work as well as some theoretical research. Wayne loved learning new techniques in the laboratory and reading into a wide range of research topics.</td><br />
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<h3>Advisors</h3><br />
<p>A special mention must also be made to our Honours, Masters and PhD student advisors in the Cheng Lab who tirelessly and patiently assisted with our troubleshooting and planning of experimental procedures. These students were: <br />
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Ashfaqul Hoque (PhD student), Gahana Advani (PhD student), George Cao (Masters student), Ken Ang (Honours student) and David Zula (Honours student).</p><br />
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<td width="152" ><img src="https://static.igem.org/mediawiki/2014/5/58/Melbourne_Ashfaqul.jpg" width="150" height="196" alt=""/></td><br />
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<td width="152" ><img src="https://static.igem.org/mediawiki/2014/a/aa/Melbourne_Ken.jpg" width="150" height="196" alt=""/></td><br />
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<td width="152" ><img src="https://static.igem.org/mediawiki/2014/9/9c/Melbourne_Dave.jpg" width="150" height="196" alt=""/></td><br />
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<td align="center"><strong>Ashfaqul Hoque</strong></td><br />
<td></td><br />
<td align="center"> <strong>Gahana Advani</strong></td><br />
<td></td><br />
<td align="center"> <strong>George Cao</strong></td><br />
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<td align="center"><strong>Ken Ang</strong></td><br />
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<td align="center"><strong>David Zula</strong></td><br />
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<p>&nbsp;</p><br />
<p>Many thanks also go to <strong>Sze-Ting Bong</strong> and <strong>Anna Gakusurudoi</strong>, Masters students in the Cheng lab whose assistance with practical methods was extremely helpful.<br />
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<div id="intro"><br />
<img src="https://static.igem.org/mediawiki/2014/7/73/Site_logo_invert.png" alt="Mel Uni iGEM Logo" width="706" height="175"><br />
<br />
<p>Welcome to 2014 University of Melbourne iGEM Wiki. At the University of Melbourne, we’ve been developing systems for expressing star peptides in E. coli. We welcome you to step into our scientific stratosphere to learn more about star peptides and the Melbourne Uni iGEM team.</p><br />
<br />
</div><br />
<br />
<br />
<br />
<table width="100%" style="text-transform: uppercase; font-family: 'Lato', sans-serif;"><br />
<br />
<tr height="10px"><br />
<br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne"style="color:#000000"><br />
Home</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Team"style="color:#000000"><br />
Team</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Project"style="color:#000000"><br />
Project</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Human_Practices"style="color:#000000"> <br />
Human Practices</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Achievements"style="color:#000000"><br />
Achievements</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Notebook"style="color:#000000"><br />
Notebook</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Protocols"style=" color:#000000"> <br />
Protocols</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Safety"style="color:#000000"><br />
Safety</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Sponsors"style="color:#000000"><br />
Sponsors</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Attributions"style="color:#000000"><br />
Attributions</a></td><br />
<br />
<br />
<td align="right"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="46px"></a> </td><br />
</tr><br />
</table><br />
<br />
<h1 >University of Melbourne<br>iGEM team </h1><br />
<h3>Star peptides are an exciting type of peptide architecture, with the many different types of star peptides forming a constellation of useful molecules. Watch the video below to find out more.</h3><br />
<br />
<table align="center"><br />
<tr><br />
<td align="center"><br />
<iframe width="828" height="465" src="//www.youtube.com/embed/3g2MlA2py8c" frameborder="0" allowfullscreen></iframe></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<p>Direct download of the video is available on the iGEM server <a href="https://2014.igem.org/File:University_of_Melbourne_intro_video.mov">here</a>.</p><br />
<br />
<h3>Browse through our website to learn more details of our star peptide design concept here at the University of Melbourne.</h3><br />
<p>&nbsp;</p><br />
<h4>Also visit us on Facebook at <a href="https://www.facebook.com/MelbourneUniIGem"style="color:#000000"><br />
https://www.facebook.com/MelbourneUniIGem</a> or get in touch at MelbourneUniIgem@gmail.com.</h4><br />
<br />
<br><br />
<br />
<table width="100%" height="1" border="1"><br />
<tr><td></td></tr></table><br />
<p>&nbsp;</p><br />
<h3>Sponsors:</h3><br />
<p>&nbsp;</p><br />
<table width="889"><br />
<tr><br />
<td width="364" ><p><img src="https://static.igem.org/mediawiki/2014/4/4c/Melbourne_DSI.jpg" width="363" height="128" alt=""/></p></td><br />
<td width="167"><p><img src="https://static.igem.org/mediawiki/2014/e/e8/Melbourne_Bio21_Institute.jpg" width="115" height="196" alt=""/></p></td><br />
<td width="342" ><p><img src="https://static.igem.org/mediawiki/2014/6/61/Melbourne_Department_of_biochemistry.png" width="252" height="185" alt=""/></p></td><br />
</tr><br />
</table><br />
<table width="800"><br />
<tr><br />
<td width="365"><table width="800"><br />
<tr><br />
<td width="80"><img src="https://static.igem.org/mediawiki/2014/5/58/Melbourne_Genesearch.png" width="170" height="53" alt=""/></td><br />
<td width="78"><img src="https://static.igem.org/mediawiki/2014/d/df/Melbourne_GenScript.png" width="183" height="56" alt=""/></td><br />
<td width="109"><img src="https://static.igem.org/mediawiki/2014/7/71/Melbourne_New_England_bio_labs.png" width="171" height="67" alt=""/></td><br />
<td width="365"><img src="https://static.igem.org/mediawiki/2014/a/af/Melbourne_Mettler_Toledo.png" width="188" height="111" alt=""/></td><br />
<td width="365"><img src="https://static.igem.org/mediawiki/2014/9/9c/Melbounre_IndieMosh.png" width="149" height="40" alt=""/></td><br />
</tr><br />
</table></td><br />
</tr><br />
</table><br />
<br />
</html></div>Slowehttp://2014.igem.org/Team:Melbourne/ProjectTeam:Melbourne/Project2014-11-21T00:31:14Z<p>Slowe: </p>
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<h1>Project and results</h1><br />
<br />
<p>Read about our experimental work below, or jump to our theoretical collaboration with the <A HREF="#Oxford">University of Oxford</A><br />
<br />
<h2>Introduction and theory</h2><br />
<p>Synthetic peptide chemists have long produced peptide-based materials <em>in vitro</em>. Star-shaped peptides are a promising type of biomaterial being explored in the field of nanomedicine (Sulistio et al., 2012). Star peptides can have several biomedical uses, such as acting as drug delivery vehicles (Sulistio et al., 2011) or linkers for other biomacromolecules. Star peptides generally take the form of several linear peptide arms linked together in a central core. One way of linking these linear peptide arms together is to used covalent bonds such as those in disulfides. Typically, disulfide bonds are formed synthetically by taking several linear arms and treating them with an oxidant <em>in vitro</em>. Here, we introduce a new approach to forming star peptides using <em>E. coli</em> and synthetic biology. Thus, we aimed to show how the peptides synthesis and disulfide bond forming machinery of <em>E. coli</em> can be used to form disulfide linked star peptides and key star peptide precursors.</p><br />
<p>&nbsp;</p><br />
<h2>Synthesis approach</h2><br />
<p><em>E. coli</em> naturally possesses the capacity to form disulfide bonds. In native strains, disulfide bonds are naturally formed by an array of enzymes which are part of the Dsb family (e.g. DsbA and DsbC) (Kadokura et al., 2003, Kadokura and Beckwith, 2009). Normally, these enzymes are found in the oxidizing periplasm of the cell. However, several new strains of <em>E. coli</em> have recently been engineered which contain an oxidizing cytoplasm conducive to disulfide bond formation. One example of this is the SHuffle cell line (Lobstein et al., 2012). The cell line contains mutations to key enzymes responsible for the reducing nature of the cytoplasm, namely thioredoxin reductase (<em>trxB</em>) and glutathione reductase (<em>gor</em>). Furthermore, the Shuffle cell line over expresses the disulfide bond isomerase DsbC to the cytoplasm. Together, these mutations allow SHuffle to fold disulfide-bonded proteins in the cytoplasm at a higher success rate compared to non-mutants. We aimed to take advantage of the disulfide bond forming capabilities of this strain of <em>E. coli</em> to synthesize star peptides in cells. As shown in the figure below, the synthesis steps may proceed as follows:</p><br />
<p>&nbsp;</p><br />
<ol><br />
<li> Express a short peptide containing two cysteine residues either to the <em>E. coli</em> periplasm or the cytoplasm of a <em>trxB gor </em>mutant.</li><br />
<li><em>E. coli</em> disulfide bond forming enzymes fold the peptide into a hairpin loop structure.<br />
</li><br />
<li>Cut the loop at a protease recognition site engineered into the peptide. This may be done by extracting the folded peptide from the cell and treating it with the protease in vitro. Alternatively, the protease may be co-expressed in the cell to allow for in vivo cleavage.<br />
</li><br />
</ol><br />
<p>&nbsp;</p><br />
<table align="center"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9d/Project_concept-v2.png" width="480" height="600" alt=""/></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<p>This synthesis approach has several benefits over purely <em>in vitro</em> approaches. Firstly, the exact peptide sequence can be precisely programmed into <em>E. coli</em> using recombinant DNA synthesis. Secondly, by performing the disulfide bond formation in cells and optionally the proteolytic cleavage, several synthesis steps which would need to be performed <em>in vitro</em> are eliminated. From a scale up perspective, this would eliminate entire unit operations which would otherwise be required to produce this product. Given these benefits, in the current study, we aimed to express a star peptide precursor to the cytoplasm of SHuffle cells, which would later be extracted and externally digested with the star-forming protease. In order to achieve this, we first designed several star peptides which might be amenable to this synthetic strategy. These peptides are described below.</p><br />
<p>&nbsp;</p><br />
<h2>Rationally designed peptides</h2><br />
<p>There are two approaches to functionalising star peptides. In the first, the identity of the arms can be chosen to be bioactive peptide molecules. This is the simplest approach to producing a biologically-relevant star. In the second, the arms can be functionalised by ligating molecules to them at any point. We used both of these approaches to design two separate peptides.</p><br />
<h3>Magainin 1 star and linear peptides</h3><br />
<p>Our first strategy was to make a star peptide using antimicrobial peptides(AMPs) as building blocks. AMPs are small, approximately 50 residue peptides secreted by some bacterial and eukaryotic cells which selectively kill microbial cells. It is thought that AMPs work by forming pores in the membrane of prokaryotic cells (Brogden, 2005). AMPs have been recombinantly expressed in a number of organisms, including <em>E. coli</em> (for a review, see Li, 2011) and <em>B. Subtilis</em> (Chen et al., 2009, Yu et al., 2013).</p><br />
<p>Our concept was to design a star peptide with antimicrobial peptide arms. Wiradharma et al. (2012) first showed that placing linear antimicrobial peptides in a star configuration could lead to enhanced antimicrobial activity and decreased hemolytic activity. Although it is unclear why this is the case, it may be due to the ability of neighbouring antimicrobial peptide arms to interact with each other to synergistically rupture the membrane.<br><br />
While Wiradharma used a synthetic peptide sequence, we designed a peptide using the naturally occurring AMP, Magainin 1 (Zasloff, 1987). Magainin 1 peptides will be placed to the ends of each star arm.</p><br />
<br />
<p>Magainin 1 was chosen because the Magainins are one of the major classes of antimicrobial peptides, being well studied and characterised. In addition, we were concerned that tethering the antimicrobial peptide to the star peptide might interfere with its antimicrobial activity. Magainin 1, however, has previously been tethered to surfaces, where it has imparted the surfaces with microbicidal properties (Glinel et al., 2008; Humblot et al., 2009). We surmised that if Magainin 1 maintained its activity while anchored to surfaces, it may also maintain its activity while anchored to a star peptide.</p><br />
<p>The sequence for Magainin 1 is: GIGKFLHSAGKFGKAFVGEIMKS.</p><br />
<p>When attached to a star peptide it will have the following structure:</p><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/MAGAININ_1_peptide.png" width="720" height="300" alt=""/><br />
<p>There are several design elements to note:</p><br />
<ul><br />
<li><br />
The antimicrobial peptide star will be expressed with a SUMO fusion protein. This is because without the fusion, it is likely that the peptide would be toxic to the host cell.</li><br />
<li>The peptide includes a Factor X cutting site between the two cysteines for eventual proteolytic cleavage and formation of the star peptide.</li><br />
</ul><br />
<p>In addition to the star peptide Magainin 1, we synthesised a gene for a linear Magainin 1 peptide as well. This is identical to the construct above, except that there is only one Magainin 1 peptide attached to the SUMO fusion.</p><br />
<br />
<h3>Unstructured Peptide (USP) Construct</h3><br />
<p>In the second approach, we designed a peptide which can be functionalised using chemical approaches. This peptide was designed to have flexible, unstructured arms and was termed the USP construct. Unlike the Magainin 1 star, the arms of this peptide serve not as active peptides themselves, but as inert structural linkers.</p><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/USPI_Peptide.png" width="720" height="216" alt=""/><br />
<p>The arms were designed with the following elements in mind:</p><br />
<ul><br />
<li><br />
<b>Lack of structure.</b> The arms were designed with a bioinspired approach, using the FxFG motif of nucleoporins (where x is a variable amino acid residue). Such segments naturally repeat in nucleoporins and are thought to lead to disorder/lack of stable secondary structure. Nucleoporins are found in mammalian cells, serving as flexible brushes around nuclear pores (Ader et al., 2010).<br />
</li><br />
<li><b>Water-soluble.</b> The arms were designed with several charged amino acids to improve solubility.<br />
</li><br />
<li><b>Designed to form a disulfide bond.</b> Although it is difficult to rationally ensure that the disulfide bond will form between two cysteines in our peptide, we incorporated a beta turn between the two cysteines which may encourage the peptide to fold at the apex of the hairpin loop. This may bring the cysteines into closer proximity, providing more probable bond formation.</li></ul><br />
<br />
<p>The ultimate utility of this peptide lies in its ability to be functionalised with other biomacromolecules. For example, the technique of Native Chemical Ligation(NCL) can be used to join peptides, proteins, and other ligands to the arms (Dawson et al., 1994). The idea of attaching enzymes to the star peptide was explored by the University of Oxford iGEM 2014 team in a collaborative effort between our two teams (See Supplementary Project Work at the end of this page). </p><br />
<br />
<p>&nbsp;</p><br />
<h2>Expression system</h2><br />
<p>In order to successfully express our constructs, we designed our protein expression vectors to include a fusion protein. The fusion protein was necessary for two reasons. First, some of our constructs are very small (e.g. the non-star Magainin 1), and expression levels of very small peptides can be difficult without a fusion partner. Second, two of our constructs code for antimicrobial peptides. Without a fusion partner, it is likely that these genes would be toxic to their hosts upon induction.</p><br />
<p>To find a suitable expression system, we looked towards the Registry of Standard Parts. We used the SUMO protein expression system designed by TU Delft 2014 (for example, see<a target="_blank" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1022101">http://parts.igem.org/wiki/index.php?title=Part:BBa_K1022101</a>). This system essentially consists of a N-terminal HIS-tag followed by the SUMO protein (also known as UlpI).</p><br />
<p><br />
The strategy of expressing toxic AMPs using SUMO has been successfully reported in the literature (Bommarius et al., 2010). We surmise that the SUMO protein could inhibit the antimicrobial activity of single, linear peptides, and that it may also inhibit the activity of our star antimicrobial peptide.</p><br />
<p><br />
SUMO as a fusion protein also has the benefit of leaving no residues at the C-terminal end of the cleavage site. This means that upon cleavage with the SUMO protease, the native protein can be recovered. In our case, this means that one of the arms of the star can be designed without the need to take into account the addition of any amino acid residues left behind by the protease.<br><br />
We used the SUMO peptide sequence reported by TU Delft. However, our construct contained the following unique features:</p><br />
<ol><br />
<li> Standardisation of the biobrick by substituting the T7 promoter and RBS (the origins of which are both not specified in the Delft documentation) with the standard T7 promoter and RBS BioBrick<a target="_blank" href="http://parts.igem.org/Part:BBa_K525998">BBa_K525998</a>. In addition to supporting the principle of standardization, using the well-characterized promoter BioBrick should help assure expression levels.<br><br />
</li><br />
<li>Biobrick BBa_K1022101 lacks a terminator sequence (this was presumably because the part was meant to be integrated into a larger genetic construct with a terminator). A terminator from the registry of standard parts was added (specifically, the wild type terminator from T7 bacteriophage,<a target="_blank" href="http://parts.igem.org/Part:BBa_K731721">BBa_K731721</a>).<br />
</li><br />
<li>The original biobrick BBa_K1022101 codes for three amino acid residues before the HIS-tag (ASM), which appeared to be redundant. Correspondence with the 2013 TU Delft team suggested that these residues were unnecessary and appear to be cleaved within the cell as part of the cells post-translational modifications. However, their presence complicates the addition of additional tags at the N-terminus of the protein (e.g. periplasmic export tags), and therefore were not included.<br><br />
</li><br />
<li>The SUMO sequence was codon optimised for E. coli during synthesis. As the Delft documentation did not specify whether the gene was codon optimal, codon optomisation was undertaken to potentially improve expression levels. The linear Magainin 1 construct, however, was not codon optimised in the SUMO region in order to provide a control condition.</li><br />
</ol><br />
<p>To summarise, the protein expression devices used in our project to the following form:</p><br />
<img src="https://static.igem.org/mediawiki/2014/d/d0/Gene_circuit_export.png" width="720" height="91" alt=""/></td><br />
<p>The protein coding region consisted of a 6x-HIS tag followed by the SUMO protein and the relevant star or linear peptide.</p><br />
<p>&nbsp;</p><br />
<h2>Plasmid preparation: Cloning and acquisition of the genetic constructs</h2><br />
<h3>Magainin 1 Star Peptide</h3><br />
<p>This construct was synthesised in the standard shipping plasmid from Life Technologies- plasmid pMK. We had originally planned to express our protein to the periplasm of E. coli, and therefore had included in the synthesis a periplasmic export tag, the TorTss signal sequence (Steiner et al., 2006).</p><br />
<p><br />
Cytoplasmic and periplasmic expression may both allow for disulfide bond formation in the cell. We decided, however, to focus on cytoplasmic expression. There are distinct advantages to cytoplasmic expression (e.g. the absence of several periplasmic proteases and potentially higher expression levels (Baneyx, 1999)).</p><br />
<p><br />
Another reason the cytoplasm was chosen was to allow us to test the effects of an oxidising versus reducing intracellular environment on disulfide bond formation. We planned to express the construct in both SHuffle T7 cells (oxidising cytoplasm) and BL21(DE3) (reducing cytoplasm) to probe whether there was a difference in disulfide bond formation. Therefore, we needed to remove the periplasmic export tag from the gene.</p><br />
<p><br />
To do this, we use the following cloning strategy (noting that the top row represents the gene initially synthesised in plasmid pMK):</p><br />
<img src="https://static.igem.org/mediawiki/2014/2/2c/Melbourne_cloning_strategy_diagram.jpg" width="900" height="437" alt=""/><br />
<p>The gene was inserted into plasmid pSB1C3 containing the T7 promoter and ribosome binding site, BBa_K525998. It was inserted after the promoter and before the BioBrick suffix. This was accomplished by digesting the destination vector with SpeI and PstI. At the same time, PCR was used to amplify the segment of the gene containing the SUMO fusion and the Magainin 1 Star peptide, adding XbaI and keeping the PstI site existing in the gene.</p><br />
<p><br />
Digestion of the PCR product then allowed for ligation of the insert and destination vector using the PstI sites and XbaI and SpeI (XbaI and SpeI have compatible sticky ends). Note that after the ligation, there will be a scar in the gene where the XbaI and the SpeI sites were ligated.</p><br />
<p><br />
The ligation appeared to be successful. The ligation mixture was transformed to DH5α competent cells and plated onto chloramphenicol-containing agar plates.</p><br />
<p><br />
Plasmid from 5 colonies (C1, C2, C3, C4, and C5) was cultured, extracted, and then digested with EcoRI and PstI.</p><br />
<p><br />
As evident in the figure below, at least 4 of the colonies appeared to contain the insert. The empty, linearised pSB1C3 backbone ran at approximately the correct size (2070 bps), as did the insert (740 bps).</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/9/95/Magainin_1_subcloning.png" width="353" height="400" alt=""/></center><br />
<p>N.B. MW marker is the 100 bp Ladder from Axygen. * indicates the colony picked for sequencing and eventual transformation to the expression cell lines.</p><br />
<p>The DNA from Colony C2 was confirmed using Sanger sequencing at the Australian Genome Research Facility. This DNA appears in the registry of standard parts as BioBrick <a target="_blank" href="http://parts.igem.org/Part:BBa_K1394000">BBa_K1394000</a>.</p><br />
<p><br />
For the USP peptide and the linear Magainin 1 peptide, the genes were synthesised by GenScript and delivered in pSB1C3. The expression vectors had identical gene regulatory elements to that used for the Magainin 1 Star peptide. They only differed in the codon optimisation used, and they also lacked the assembly scar described above.</p><br />
<br />
<h2><br />
Protein expression and characterisation<br />
</h2><br />
<p><br />
After acquiring our genes, the project moved to protein expression and purification. We expressed both star peptides (Magainin 1 Star Peptide and the USP peptide) in both the SHuffle T7 and BL21(DE3) cell lines, both sourced from New England Biolabs. As the Linear Magainin 1 peptide does not have any special disulfide bonding<br />
requirements, we expressed it in BL21(DE3).<br />
</p><br />
<p><br />
The plasmids were transformed to the expression cells, and a single colony was cultured and induced overnight at 17 °C. A whole cell sample both before<br />
IPTG induction (-IPTG) and after the induction period (+IPTG) were boiled in SDS-PAGE sample buffer and loaded on a 15% tris-glycine gel. The whole cell<br />
Coomassie stained gel is shown below alongside the NEB P7712S molecular weight marker.<br />
</p><br />
<br><br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/2/24/Whole_cell_CS.png" height="330" width="439"/><br />
</p><br />
<br><br />
<p><br />
From theoretical prediction of the peptide masses, we would expect the following distribution of molecular weights:<br />
</p><br />
<br><br />
<table border="1" cellpadding="0" cellspacing="0" align="center"><br />
<tbody><br />
<tr><br />
<td width="156"><br />
<p align="center"><br />
Magainin 1 Star Peptide (Mag1 Star)<br />
</p><br />
</td><br />
<td width="145"><br />
<p align="center"><br />
USP<br />
</p><br />
</td><br />
<td width="156"><br />
<p align="center"><br />
Linear Magainin 1 (Linear Mag1)<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="156"><br />
<p align="center"><br />
22.15 kDa<br />
</p><br />
</td><br />
<td width="145"><br />
<p align="center"><br />
29.3 kDa<br />
</p><br />
</td><br />
<td width="156"><br />
<p align="center"><br />
14.32 kDa<br />
</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<br><br />
<p><br />
We looked for bands corresponding to overexpression of the proteins. We observed a thick band, post IPTG around 32 kDa in the BL21(DE3) cells carrying the<br />
USP plasmid. However, we saw this same band appearing in the Linear Magainin 1 lane but the Linear Magainin 1 protein should have a much lower molecular<br />
weight than what was observed. Therefore, we did not assume that we had overexpression overexpression of the USP 1 protein.<br />
</p><br />
<p><br />
We also noted bands in all post-IPTG lanes around 17 kDa. This would be roughly consistent with both the Linear Magainin 1 and Magainin 1 Star Peptide,<br />
noting that small proteins may not run at their expected molecular weight. Again, the analysis is complicated by the fact that the band also appears in the<br />
lane corresponding to the larger molecular weight protein, USP I. Given this ambiguity, we decided to purify all the protein in the sample using Ni-NTA<br />
purification.<br />
</p><br />
<h3><br />
Purification and further characterisation<br />
</h3><br />
<p><br />
A small-scale purification was carried out according to our <a href="https://static.igem.org/mediawiki/2014/e/e1/MELBOURNE_D1V1_Column_Purification_of_His-Tagged_Proteins.pdf">protocol</a>. The cells were lysed with iodoacetamide, an alkylating agent, added to the lysis buffer. Iodoacetamide blocks all free cysteines on proteins with a short alkyl group. This was added<br />
because we wanted to determine if a disulphide bond had formed inside the cell. If we did not block free cysteines, then any bond that had formed could be<br />
attributed to spontaneous/air oxidation of disulfides outside the cell.<br />
</p><br />
<p><br />
After purification, we ran a concurrent Western Blot and Coomassie stain on both the pre-induction samples from above and the purified protein (namely, the<br />
first elution from the batch purification).<br />
</p><br />
<p><br />
The Western Blot used mouse monoclonal antibodies against the N-terminal HIS-tag as primary antibodies and anti-mouse secondary antibodies:<br />
</p><br />
<br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/5/54/Western_Blot_Melb.png" height="400" width="460" border="0"/><br />
</p><br />
<p><br />
The corresponding Coomassie stain is show below:<br />
</p><br />
<br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f0/Purified_CS.png" height="365" width="486" border="0"/><br />
</p><br />
<p><br />
There are several interesting aspects of the Western Blot. Firstly, there was a strong band between 25 and 32 kDa in most lanes. However, it<br />
appears in all of the pre-induction controls, suggesting non-specific binding of the anti-HIS antibodies.<br />
</p><br />
<p><br />
Secondly, we observed a band in most of the lanes around 17 kDa (with the exception being the USP protein in SHuffle cells). As these bands were not<br />
present in the pre-induction controls, we suspected they were a result of the induction.<br />
</p><br />
<h3><br />
Mass spectroscopy<br />
</h3><br />
<p><br />
In order to assess the identity of the bands, we performed an in-gel tryptic digestion on select bands. We digested bands in the Coomassie stain which<br />
corresponded to the HIS-tagged bands in the Western Blot. This was followed by mass spec analysis (LC MS/MS). We focused our analysis on the Magainin 1<br />
Star Peptide bands and the Linear Magainin 1 band. Our USP peptide was, by design, highly rich in basic residues, greatly reducing the likelihood that<br />
tryptic fragments would be detected by the mass spec.<br />
</p><br />
<p><br />
We found that the Linear Magainin 1 peptide appeared to be present in the approximately 17 kDa band, with the following detected tryptic fragments in the<br />
sequence below (bold; basic residues underlined):<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/1/1b/Tryptic_digest_linear_mag1.PNG" height="63" width="601" border="0"/><br />
</p><br />
<p><br />
Note that spaces have been added in the sequence to emphasise distinct domains in the protein (in this case, the fusion protein versus the Magainin 1<br />
peptide). Together with the fact that the protein runs close to the expected molecular weight, this seems to provide good evidence that the protein is<br />
being expressed.<br />
</p><br />
<p><br />
We then examined the bands corresponding to the Magainin 1 Star Peptide.<br />
</p><br />
<p><br />
Again, we digested bands in the Coomassie stained gel corresponding to the prominent HIS-tagged bands on the <em>Western Blot</em> (near 17 kDa).<br />
</p><br />
<p><br />
The mass spec coverage for the Magainin 1 Star Peptide expressed in SHuffle cells was as follows:<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/6/63/Tryptic_digest_Sample_A_Star_Mag_Shuffle.PNG" height="90" width="601" border="0"/><br />
</p><br />
<p><br />
The coverage for the same peptide expressed in BL21(DE3) was found to be:<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/d/db/Tryptic_digest_Sample_A_Star_Mag_BL21%28DE3%29.PNG" height="85" width="602" border="0"/><br />
</p><br />
<p><br />
Again, we can see that tryptic peptides from the expressed protein appear to present in the gel band under analysis.<br />
</p><br />
<p><br />
There is a relatively lower sequence coverage. This could be a limitation of our procedure: for example, improper destaining during the in gel digestion<br />
procedure can inhibit tryptic digestion.<br />
</p><br />
<p><br />
However, even if the digestion was complete, the mass spec will only detect fragments which ionize well. The Magainin 1 Star peptide consists of four<br />
repeats of the Magainin 1 sequence (corresponding to the arms of the star). If the two tryptic fragments within Magainin 1 do not ionize well, then indeed<br />
the entire peptide would not be read.<br />
</p><br />
<p><br />
Finally, it is possible that the protein has been cleaved or degraded. This would account for the slightly lower-than-expected mass on the SDS page gel.<br />
</p><br />
<p><br />
Nonetheless, it is a possibility that the peptide fragments are present but not detected. Replication of the in-gel mass spec would be useful<br />
in clarifying this point, or provided the purity of the sample is acceptable, it may be possible to run an intact mass spec. Digestion with alternative enzymes may also provide a more robust sequence coverage, particularly for the USP peptide.<br />
</p><br />
<h2><br />
Conclusions<br />
</h2><br />
<p><br />
We designed several novel star peptides based on several rational design criteria. Further, we began the optimisation process required to express these peptides in <em>E. coli</em>.<br />
</p><br />
<p><br />
To this end, we constructed an expression system based on the SUMO protein and standard BioBrick parts. The function of this system was confirmed, as it<br />
appears it does produce HIS-tagged SUMO fusion protein in <em>E. coli</em>, as expected. Nonetheless, we hope to continue to characterise our parts, likely through further purification and measurement of the mass. <br />
</p><br />
<p>Given the success of SUMO in the protein expression community, we hope our BioBrick<br />
will encourage further use of the fusion domain within iGEM. In addition, we hope that our ideas and concepts will inspire future<br />
iGEM efforts to explore the applications of synthetic biology for material science. We continue to believe that bacteria have great promise for the <em>in vivo</em><br />
construction of unique biomaterials, and we look forward to seeing how the synthetic biology community will develop in this area.<br />
</p><br />
<br />
<br />
<br />
<h2>References</h2><br />
<p>ADER, C., FREY, S., MAAS, W., SCHMIDT, H. B., GÖRLICH, D. &amp; BALDUS, M. 2010. Amyloid-like interactions within nucleoporin FG hydrogels. <em>Proceedings of the National Academy of Sciences,</em> 107<strong>,</strong> 6281-6285.</p><br />
<p><br />
BANEYX, F. 1999. Recombinant protein expression in Escherichia coli. <em>Current Opinion in Biotechnology,</em> 10<strong>,</strong> 411-421.</p><br />
<p><br />
BOMMARIUS, B., JENSSEN, H., ELLIOTT, M., KINDRACHUK, J., PASUPULETI, M., GIEREN, H., JAEGER, K. E., HANCOCK, R. E. W. &amp; KALMAN, D. 2010. Cost-effective expression and purification of antimicrobial and host defense peptides in Escherichia coli. <em>Peptides,</em> 31<strong>,</strong> 1957-1965.</p><br />
<p><br />
BROGDEN, K. A. 2005. Antimicrobial peptides: Pore formers or metabolic inhibitors in bacteria? <em>Nature Reviews Microbiology,</em> 3<strong>,</strong> 238-250.</p><br />
<p><br />
CHANG, C., CHHOR, G., CLANCY, S. & JOACHIMIAK, A. 2014. Crystal Structure of Glutathione S-transferase Domain Protein From Haliangium Ochraceum DSM 14365 [Online]. ''NCBI. Available: http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=4w66' [Accessed September 9 2014].</p><br />
<p><br />
CHEN, X., ZHU, F., CAO, Y. &amp; QIAO, S. 2009. Novel expression vector for secretion of cecropin AD in Bacillus subtilis with enhanced antimicrobial activity. <em>Antimicrobial agents and chemotherapy,</em> 53<strong>,</strong> 3683-3689.</p><br />
<p><br />
GLINEL, K., JONAS, A. M., JOUENNE, T., LEPRINCE, J., GALAS, L. & HUCK, W. T. 2008. Antibacterial and antifouling polymer brushes incorporating antimicrobial peptide. <em>Bioconjug Chem,</em> 20<strong>,</strong> 71-77.</p><br />
<p><br />
HUMBLOT, V., YALA, J.-F., THEBAULT, P., BOUKERMA, K., HÉQUET, A., BERJEAUD, J.-M. & PRADIER, C.-M. 2009. The antibacterial activity of Magainin I immobilized onto mixed thiols self-assembled monolayers. <em>Biomaterials,</em> 30<strong>,</strong> 3503-3512.</p><br />
<p><br />
DAWSON, P. E., MUIR, T. W., CLARK-LEWIS, I. &amp; KENT, S. 1994. Synthesis of proteins by native chemical ligation. <em>Science,</em> 266<strong>,</strong> 776-779.</p><br />
<p><br />
KADOKURA, H. &amp; BECKWITH, J. 2009. Detecting folding intermediates of a protein as it passes through the bacterial translocation channel. <em>Cell,</em> 138<strong>,</strong> 1164-1173.</p><br />
<p><br />
KADOKURA, H., KATZEN, F. &amp; BECKWITH, J. 2003. Protein disulfide bond formation in prokaryotes. <em>Annual review of biochemistry,</em> 72<strong>,</strong> 111-135.</p><br />
<p><br />
LI, Y. 2011. Recombinant production of antimicrobial peptides in&lt; i&gt; Escherichia coli&lt;/i&gt;: A review. <em>Protein Expression and Purification,</em> 80<strong>,</strong> 260-267.</p><br />
<p><br />
LOBSTEIN, J., EMRICH, C. A., JEANS, C., FAULKNER, M., RIGGS, P. &amp; BERKMEN, M. 2012. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. <em>Microb Cell Fact,</em> 11<strong>,</strong> 56-56.</p><br />
<p><br />
PROTEIN MODEL PORTAL. Query result, GST and DcmA [Online]. Available: http://www.proteinmodelportal.org/query/uniprot/P21161 [Accessed September 9 2014].</p><br />
<p><br />
STEINER, D., FORRER, P., STUMPP, M. T. &amp; PLUCKTHUN, A. 2006. Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display. <em>Nat Biotech,</em> 24<strong>,</strong> 823-831.</p><br />
<p><br />
SULISTIO, A., GURR, P. A., BLENCOWE, A. &amp; QIAO, G. G. 2012. Peptide-Based Star Polymers: The Rising Star in Functional Polymers. <em>Australian Journal of Chemistry,</em> 65<strong>,</strong> 978-984.</p><br />
<p><br />
SULISTIO, A., LOWENTHAL, J., BLENCOWE, A., BONGIOVANNI, M. N., ONG, L., GRAS, S. L., ZHANG, X. &amp; QIAO, G. G. 2011. Folic Acid Conjugated Amino Acid-Based Star Polymers for Active Targeting of Cancer Cells. <em>Biomacromolecules,</em> 12<strong>,</strong> 3469-3477.</p><br />
<p><br />
TANAKA, N., KUSAKABE, Y., ITO, K., YOSHIMOTO, T. & NAKAMURA, K. T. 2002. Crystal Structure of Formaldehyde Dehydrogenase from< i> Pseudomonas putida</i>: the Structural Origin of the Tightly Bound Cofactor in Nicotinoprotein Dehydrogenases. Journal of molecular biology, 324, 519-533.</p><br />
<p><br />
WIRADHARMA, N., LIU, S. Q. &amp; YANG, Y. Y. 2012. Branched and 4-Arm Starlike α-Helical Peptide Structures with Enhanced Antimicrobial Potency and Selectivity. <em>Small,</em> 8<strong>,</strong> 362-366.</p><br />
<p><br />
YU, Z., WANG, Q., MA, Q. &amp; ZHANG, R. 2013. Secretory expression of lacticin Q fused with SUMO in Bacillus subtilis. <em>Protein Expression and Purification,</em> 89<strong>,</strong> 51-55.</p><br />
<p><br />
ZASLOFF, M. 1987. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. <em>Proceedings of the National Academy of Sciences,</em> 84<strong>,</strong> 5449-5453.</p><br />
<p>&nbsp;</p><br />
<br />
<A NAME="Oxford"></A><br />
<h1>Collaboration with Oxford</h1><br />
<p>To strengthen the sense of iGEM community, we contacted <a target="_blank" href="https://2014.igem.org/Team:Oxford">University of Oxford iGEM team</a> to discuss the possibility of collaboration between our teams. The Oxford project aims to develop a system that can dispose of the carcinogenic, hazardous solvent dichloromethane (DCM). To do this, Oxford team has proposed the use of the DcmA enzyme. This enzyme degrades DCM to form a toxic intermediate which in turn is converted by another enzyme, FdhA, into a neutral molecule. Schematically this can be presented in the following way:</p><br />
<br />
<p><br />
Reaction 1: DCM +&nbsp;DcmA <strong>toxic intermediate</strong><br><br />
Reaction 2: <strong>toxic intermediate</strong> + FdhA neutral product.</p><br />
<p>The problem for their system lies in bringing the two enzymes together to ensure efficient reaction kinetics. An idea that we discussed with Oxford was to use the star peptide platform to link the two enzymes together, in a structure similar to the following: </p><br />
<img src="https://static.igem.org/mediawiki/2014/2/2e/Melbourne_Collab_1.png" width="500" height="325" alt=""/><br />
<p>We worked with Oxford to study this system from a theoretical standpoint. Our team studied the 3D structure of both enzymes involved to confirm that the enzyme could in theory be attached to a linker in this manner, while Oxford team did stochastic modelling to determine how reaction rate changes when the linker length, labeled D on the diagram, changes. </p><br />
<p><br />
As a first check, it was necessary to determine whether anchoring these enzymes to our star would be sterically possible. We studied the structures of FdhA and DcmA determined by crystallography using data from the protein data bank. As no crystallographic data was available in the databank for DcmA, GST was examined as a proxy, as GST is structurally homologous to DcmA. </p><br />
<p><br />
The crystalographically resolved structure of FdhA enzyme (Tanaka et al., 2002) from the protein data bank revealed the following image:</p><br />
<img src="https://static.igem.org/mediawiki/2014/d/dc/Melbourne_Collab_2.png" width="700" height="355" alt=""/><br />
<p>The crystalographically resolved structure of GST was as follows (Chang et al., 2014):</p><br />
<img src="https://static.igem.org/mediawiki/2014/e/e9/Melbourne_Collab_3.png" width="800" height="361" alt=""/><br />
<p>It can be seen that the amino- and carboxy-terminals are located away from the active site. This suggests that both enzymes, DcmA and FdhA, might be linked together via linkers that are used in our star peptide. That is, the active site will not be sterically hindered by attachment to the star peptide. That said, it is difficult to predict how anchoring the protein to the star will affect its tertiary structure. Further, it is difficult to predict how limiting the rotational degrees of freedom will affect enzyme activity.</p><br />
<br />
<p>The Oxford team modeled this system and found that, indeed, the star peptide as an enzyme linker was favourable to enzyme kinetics. Their work can be found <br />
<br />
<a href="https://2014.igem.org/Team:Oxford/alternatives_to_microcompartments#show2"><br />
here</a>.</div>Slowehttp://2014.igem.org/Team:Melbourne/Human_PracticesTeam:Melbourne/Human Practices2014-10-18T03:57:31Z<p>Slowe: </p>
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<img src="https://static.igem.org/mediawiki/2014/9/9b/Melbourne_Banner.png" alt="Banner" height="225" width="1000"><br />
<br />
<br />
<table width="100%" style="text-transform: uppercase; font-family: 'Lato', sans-serif;"><br />
<br />
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<br />
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<a href="https://2014.igem.org/Team:Melbourne"style="color:#000000"><br />
Home</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Team"style="color:#000000"><br />
Team</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Project"style="color:#000000"><br />
Project</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Human_Practices"style="color:#000000"> <br />
Human Practices</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Achievements"style="color:#000000"><br />
Achievements</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Notebook"style="color:#000000"><br />
Notebook</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Protocols"style=" color:#000000"> <br />
Protocols</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Safety"style="color:#000000"><br />
Safety</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Sponsors"style="color:#000000"><br />
Sponsors</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Attributions"style="color:#000000"><br />
Attributions</a></td><br />
<br />
<br />
<td align="right"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="46px"></a> </td><br />
</tr><br />
</table><br />
<br />
<br />
<h1>Public Outreach</h1><br />
<h2>&lsquo;Synthetic biology for kids and adults and everyone in between&rsquo; </h2><br />
<p>The University of Melbourne took up the challenge of teaching synthetic biology to all ages, embracing a &lsquo;science for everyone&rsquo; approach. In so doing, we engaged in education outreach activities with kindergarten students from three childcare centres as well as both university and high school students from across Australia.</p><br />
<p>&nbsp; </p><br />
<h3>For kindergarten students </h3><br />
<table width="100%"><br />
<tr><br />
<td width="195" ><img src="https://static.igem.org/mediawiki/2014/4/4b/Melbourne_Adventure_of_E_coli.jpg" width="176" height="264" alt="Adventure of E coli"/></td><br />
<td width="594"><p>We decided to write the first in a series of children&rsquo;s books for pre-schoolers aged 4-6 themed in the spirit of iGEM entitled &lsquo;The Adventures of E. coli&rsquo; for a number of reasons:</p><br />
<ul><br />
<li> Children aged 4-6 are in a key learning stage, during which their interest in science can significantly influence their long-term science education outcomes (Bowman, Donovan, &amp; Burns, 2001, pp. 8-9). </li><br />
<li>There are lots of resources about science for older kids but not so many for kids at such a young age. </li><br />
<li>Picture books provide a springboard for further classroom activities </li><br />
</ul><br />
<p>With the assistance of IndieMosh (Publishers link is here: <A href="http://www.indiemosh.com.au/author-42-iGEM-Melbourne-childrens-biology-science" rel="nofollow">http://www.indiemosh.com.au/author-42-iGEM-Melbourne-childrens-biology-science</A>) – a self-publishing facilitator – we managed to get our book in print and on Amazon, please see <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/</A></p><br />
<p>After we successfully published our book, it was time to take it to the audience. We approached childcare centres across Melbourne and negotiated for our book to be read to pre-schoolers and for our team members to come along and teach the kids about science.</p></td><br />
<br />
</tr><br />
</table><p>The book reading was well received and was followed by playing some science-related games, and having a chat about science in groups. Please check out the video we made about the day: </p><br />
<br />
<iframe class="centervid" width="560" height="315" src="//www.youtube.com/embed/RNV_lPKcrSc" frameborder="0" allowfullscreen></iframe><br />
<br />
<p> A big thanks to Kids Time Early Learning Centre (Moorabin), Kids Time Early Learning Centre (Bentleigh East) and Coco's Early Learning Centre for inviting us to present our picture book to the kids. </p><br />
<br />
<p>We believe that through this book, young children can learn about basic concepts and the language of biology, forming a scientific foundation to be built on in future years. Moreover, it is our hope that this book will inspire children to continue to learn about science and to appreciate all the wonders that it holds. </p><br />
<p>The Kindle book is available from here: <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon-ebook/dp/B00OJ691DI/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon-ebook/dp/B00OJ691DI/</A></p><br />
<p>Paperback available on Amazon here: <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/</A></p><br />
<p>Fixed layout epub (for iPad and Android tablets, phones etc) available on Smashwords here: <A href="http://www.smashwords.com/books/view/484305" rel="nofollow">http://www.smashwords.com/books/view/484305</A></p><br />
<br />
<br />
<p> <strong> References: </strong> </p><br />
<p> Bowman, Barbara T.; Donovan, M. Suzanne; & Burns, M. Susan (Eds.). (2001). Eager to learn: Educating our preschoolers. Washington, DC: National Academy Press. </p><br />
<br />
<h3>&nbsp;</h3><br />
<h3>For high school students </h3><br />
<p>In an effort to broaden the accessibility of the iGEM competition to those who appeared to be currently underrepresented in the competition (e.g. disadvantaged students, females and Australian high school students), we approached a private high school in Victoria with the following aim: </p><br />
<p>To find a well-resourced, private high school to partner with an iGEM team (e.g. UoM) to field a team consisting of half private high school students/half underrepresented students – who would have their flights to Boston funded by fundraising done primarily by the private high school. </p><br />
<p>After extensive email &amp; phone correspondence spanning several months, the school decided it was not currently in the position to go ahead with the iGEM team proposal due to upcoming infrastructure developments, though would be interested in fielding a team after these are completed (scheduled for 2016). Nonetheless, the school did encourage us that other APS schools would likely be interested in hearing about iGEM and our team concept. </p><br />
<p>Whilst we didn&rsquo;t achieve our ultimate goal, this activity did raise awareness of the iGEM competition with all the key science staff members at the school which requested not to be named on this wiki (for further details please contact our team leader Sean). </p><br />
<p>Finally, we would encourage all Australian teams to work on the following goals: </p><br />
<ul><br />
<li>improve iGEM's accessibility to those of disadvantaged backgrounds </li><br />
<li>increase female representation in science &amp; iGEM </li><br />
<li>increase the participation of Australian/international high school students in the iGEM competition. </li><br />
</ul><br />
<p><BR><br />
In addition to our negotiations with the private high school, we also aimed at widening the participation of high school students in the iGEM competition by collaborating with the University of Sydney on The Strange Nature Writing competition. </p><br />
<p>We spruiked the competition not only on our project flyers which described the iGEM competition, our project and the Strange Nature Writing Competition, but also through our development of a promotional video designed to be shown in high school assemblies as a way of promoting both iGEM and the Strange Nature Writing competition. Please see the video below: </p><br />
<br />
<iframe class="centervid" width="560" height="315" src="//www.youtube.com/embed/ZuSoO3HVMjo" frameborder="0" allowfullscreen></iframe><br />
<br />
<p>Furthermore, we wrote and published articles on Comet.is which is aimed at high school students, university students and recent university graduates, please see 'For university students' for a list of the articles we wrote. </p><br />
<h3>&nbsp;</h3><br />
<h3>For university students </h3><br />
<p>Our main form of outreach toward university students was in writing and publishing articles on a burgeoning and government sponsored website Comet.is which is aimed at high school students, university students and recent graduates. Our articles covered a range of topics from our iGEM experience to considerations of ethical issues of synthetic biology and the potential of synthetic biology. </p><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/d/d5/Comet.jpg" width="640" height="400" alt="Adventure of E coli"/><br />
<br />
<br />
</p> <strong>Here are some excerpts:</strong> </p><br />
<br />
<blockquote></p> "All this leads me to refer to synthetic biology as a ménage à trois of a discipline, utilizing engineering, biology and computer science to produce a fantastically creative activity." </p></blockquote><br />
<br />
<blockquote></p> "Given assistance in the form of grants or human resources, entrepreneurial forums along with student competitions like InnovationACT and iGEM have the chance to inspire entrepreneurialism in students. It is in these opportunities for students to go on entrepreneurial adventures, replete with late nights, red bulls and a shot at winning a grand prize – that the next generation of entrepreneurs is born." </p></blockquote><br />
<br />
<blockquote></p> "A prime example of this is in the International Genetically Engineered Machine (iGEM) competition, which I am a team member of for the University of Melbourne this year. Our team stretches across many disciplines; from telecommunications and business to chemical engineering and science communication, even information technology - we are practically the UN of fields and backgrounds." </p></blockquote><br />
<br />
<blockquote></p> "Participating in IGEM can definitely be categorized as having an experience and not just joining a team. The competition brings together all the components of completing a project for undergraduate students to be part of, a little like a trial run of a Masters or Doctorate course." </p></blockquote><br />
<br />
<p>Here are the links to our articles: </p><br />
<p><A href="https://comet.is/article/2960" rel="nofollow">https://comet.is/article/2960</A> <A href="https://comet.is/article/3499/" rel="nofollow">https://comet.is/article/3499/</A> <A href="https://comet.is/article/3381/" rel="nofollow">https://comet.is/article/3381/</A> <A href="https://comet.is/article/3561/" rel="nofollow">https://comet.is/article/3561/</A> <A href="https://comet.is/article/3562/" rel="nofollow">https://comet.is/article/3562/</A> <A href="https://comet.is/article/3694/" rel="nofollow">https://comet.is/article/3694/</A> <A href="http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/" rel="nofollow">http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/</A> <A href="http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/" rel="nofollow">http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/</A></p><br />
<p>Furthermore, as part of our outreach directed at university students, we combined our recruitment campaign for an IT whiz to help with our wiki, with a presentation on what iGEM was all about. We lecture bashed over several weeks, presenting over 11 times. </p><br />
<br />
<h3><BR><br />
</h3><br />
<h3>For general public </h3><br />
<p>After promoting the iGEM competition to the Victorian State Chair of National Science Week (NSW) - Nick Besley, our team decided to field a stall in &lsquo;Market of the Mind&rsquo; – a NSW event aimed at the general public located in a high foot traffic area alongside the Yarra River in Southbank across the river from Flinders St Station. Luckily, our stall was located next to the bar, which meant there was a lot of foot traffic directed to our happy team members wearing our iGEM team t-shirts.</p><br />
<img class="centervid" src="https://static.igem.org/mediawiki/2014/d/d7/10641184_10203547262856505_6097351474732015309_n.jpg" width="400" height="300" /><br />
<p> Using a true/false quiz, armed with Ferrero Rochers as rewards for getting all the answers correct, we had over 30 quiz contestants engage with us on the first night and 35+ contestants on the second day. Please see the accompanying video made by the City of Melbourne, which shows our stall/team members from 0:45 </p><br />
<br />
<p><iframe class="centervid" width="560" height="315" src="//www.youtube.com/embed/ahFI3aFBdNc" frameborder="0" allowfullscreen></iframe><p><br />
<br />
<br />
<p> <strong> Our team consulted: </strong> </p><br />
<p>IndieMosh</p><br />
<p>Kids Time Early Learning Centre (Bentleigh East)</p><br />
<p>Kids Time Early Learning Centre (Moorabin)</p><br />
<p>Coco's Early Learning Centre</p><br />
<p>A Victorian private school (one of the 11 Associated Public Schools)</p><br />
<p>USyd-Australia</p><br />
<p>University of Melbourne IT Faculty</p><br />
<p>University of Melbourne Education Faculty</p><br />
<p> University of Melbourne Media and Public Relations Department </p><br />
<p>Victorian State Chair of National Science Week (NSW) - Nick Besley</p><br />
<p> Teachers' Association of Australia (TAA) </p><br />
<br />
<br />
<br />
<h1>Intellectual Property </h1><br />
<br />
<p> <strong> Our team considered the question of ‘how should we proceed if we want to retain IP rights on our project’? </strong> </p><br />
<br />
<p> Before attempting to answer this pertinent IP question, our team leader decided to recruit two more team members who had some intellectual property experience to complement the learning on patent law that a few early team members had acquired in their biotechnology undergraduate degrees. Both the new team members had previously filed provisional patents; one had filed some in Russia and the other in Australia, offering two perspectives on this question of IP. </p><br />
<br />
<p> In answering this question, we approached our universities’ tech transfer office to gain clarity over our team’s discussions and brainstorm sessions on whether our project was patentable. </p><br />
<br />
<p> The tech transfer office advised us that our project may have commercial applications, however it was probably too soon to act on the idea and convert it into a provisional patent, so we decided to hold off and wait till we had a more substantiated grasp on our commercial applications. We were also advised that if at any point we wanted to file for IP protection on the project, it would be really important to not have disclosed the details of our idea to anyone else, as this could result in us losing the right to our IP. </p><br />
<br />
<p> After doing some research on the various law firms who could offer us a provisional patent, we discovered the estimated cost would be in the vicinity of $3500-$6500 depending on the level of detail we wanted to cover e.g. the number and extent of the claims, the full description of the idea, diagrams and experimental protocols as well as the filing & service fees associated. </p><br />
<br />
<p> Due to the relatively high estimates we received, we looked into other options for filing a provisional, including recruiting a law student to join the team and contribute their knowledge to our IP protection. This avenue did not eventuate as our ideas on what we would like to patent continuously evolved over the course of the project and we could not reach a sufficient degree of clarity to bring on a lawyer for the filing of a provisional patent. </p><br />
<br />
<p> Nonetheless, we proceeded with caution, ensuring to follow the advice of the University of Melbourne’s tech transfer office and not disclose our ideas without ensuring our disclosure was protected. An instance when we had to implement this protection was during our scientific collaboration with Oxford, were we ensured Non Disclosure Agreements were signed prior to the disclosure of our project to them. They indicated their interest in using our project as a case study in their ‘iGEM intellectual property guide’, however we felt that this might put our IP rights in jeopardy, so we decided not to proceed as a case study. </p><br />
<br />
<p> At this time, our team is looking to produce at least one provisional patent following the iGEM 2014 competition on at least one of the various techniques we designed during the project. </p><br />
<br />
<br />
<p> <strong> Our team consulted: </strong> </p><br />
<br />
<p> University of Melbourne Technology Transfer Office </p><br />
<p> University of Melbourne Technology Commercialisation Team </p><br />
<p> UoM Commercial </p><br />
<p> Stanislauv Yatskevich (iGEM team member with Russian patent experience) </p><br />
<p> Peter Collins (iGEM team member with Australian patent experience) </p><br />
<br />
<br />
<p> <strong> References: </strong> </p><br />
<br />
<p> http://www.ipaustralia.gov.au/.../factsheet-experimental.../ </p><br />
<br />
<p>http://www.google.com.au/url?sa=t&rct=j&q=&esrc=s...</p></div>Slowehttp://2014.igem.org/Team:Melbourne/Human_PracticesTeam:Melbourne/Human Practices2014-10-18T03:56:58Z<p>Slowe: </p>
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<img src="https://static.igem.org/mediawiki/2014/9/9b/Melbourne_Banner.png" alt="Banner" height="225" width="1000"><br />
<br />
<br />
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<a href="https://2014.igem.org/Team:Melbourne"style="color:#000000"><br />
Home</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Team"style="color:#000000"><br />
Team</a> </td><br />
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Project</a> </td><br />
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Human Practices</a></td><br />
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Achievements</a></td><br />
<br />
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Notebook</a></td><br />
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<a href="https://2014.igem.org/Team:Melbourne/Protocols"style=" color:#000000"> <br />
Protocols</a></td><br />
<br />
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<a href="https://2014.igem.org/Team:Melbourne/Safety"style="color:#000000"><br />
Safety</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Sponsors"style="color:#000000"><br />
Sponsors</a></td><br />
<br />
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<a href="https://2014.igem.org/Team:Melbourne/Attributions"style="color:#000000"><br />
Attributions</a></td><br />
<br />
<br />
<td align="right"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="46px"></a> </td><br />
</tr><br />
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<br />
<br />
<h1>Public Outreach</h1><br />
<h2>&lsquo;Synthetic biology for kids and adults and everyone in between&rsquo; </h2><br />
<p>The University of Melbourne took up the challenge of teaching synthetic biology to all ages, embracing a &lsquo;science for everyone&rsquo; approach. In so doing, we engaged in education outreach activities with kindergarten students from three childcare centres as well as both university and high school students from across Australia.</p><br />
<p>&nbsp; </p><br />
<h3>For kindergarten students </h3><br />
<table width="100%"><br />
<tr><br />
<td width="195" ><img src="https://static.igem.org/mediawiki/2014/4/4b/Melbourne_Adventure_of_E_coli.jpg" width="176" height="264" alt="Adventure of E coli"/></td><br />
<td width="594"><p>We decided to write the first in a series of children&rsquo;s books for pre-schoolers aged 4-6 themed in the spirit of iGEM entitled &lsquo;The Adventures of E. coli&rsquo; for a number of reasons:</p><br />
<ul><br />
<li> Children aged 4-6 are in a key learning stage, during which their interest in science can significantly influence their long-term science education outcomes (Bowman, Donovan, &amp; Burns, 2001, pp. 8-9). </li><br />
<li>There are lots of resources about science for older kids but not so many for kids at such a young age. </li><br />
<li>Picture books provide a springboard for further classroom activities </li><br />
</ul><br />
<p>With the assistance of IndieMosh (Publishers link is here: <A href="http://www.indiemosh.com.au/author-42-iGEM-Melbourne-childrens-biology-science" rel="nofollow">http://www.indiemosh.com.au/author-42-iGEM-Melbourne-childrens-biology-science</A>) – a self-publishing facilitator – we managed to get our book in print and on Amazon, please see <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/</A></p><br />
<p>After we successfully published our book, it was time to take it to the audience. We approached childcare centres across Melbourne and negotiated for our book to be read to pre-schoolers and for our team members to come along and teach the kids about science.</p></td><br />
<br />
</tr><br />
</table><p>The book reading was well received and was followed by playing some science-related games, and having a chat about science in groups. Please check out the video we made about the day: </p><br />
<br />
<iframe class="centervid" width="560" height="315" src="//www.youtube.com/embed/RNV_lPKcrSc" frameborder="0" allowfullscreen></iframe><br />
<br />
<p> A big thanks to Kids Time Early Learning Centre (Moorabin), Kids Time Early Learning Centre (Bentleigh East) and Coco's Early Learning Centre for inviting us to present our picture book to the kids. </p><br />
<br />
<p>We believe that through this book, young children can learn about basic concepts and the language of biology, forming a scientific foundation to be built on in future years. Moreover, it is our hope that this book will inspire children to continue to learn about science and to appreciate all the wonders that it holds. </p><br />
<p>The Kindle book is available from here: <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon-ebook/dp/B00OJ691DI/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon-ebook/dp/B00OJ691DI/</A></p><br />
<p>Paperback available on Amazon here: <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/</A></p><br />
<p>Fixed layout epub (for iPad and Android tablets, phones etc) available on Smashwords here: <A href="http://www.smashwords.com/books/view/484305" rel="nofollow">http://www.smashwords.com/books/view/484305</A></p><br />
<br />
<br />
<p> <strong> References: </strong> </p><br />
<p> Bowman, Barbara T.; Donovan, M. Suzanne; & Burns, M. Susan (Eds.). (2001). Eager to learn: Educating our preschoolers. Washington, DC: National Academy Press. </p><br />
<br />
<h3>&nbsp;</h3><br />
<h3>For high school students </h3><br />
<p>In an effort to broaden the accessibility of the iGEM competition to those who appeared to be currently underrepresented in the competition (e.g. disadvantaged students, females and Australian high school students), we approached a private high school in Victoria with the following aim: </p><br />
<p>To find a well-resourced, private high school to partner with an iGEM team (e.g. UoM) to field a team consisting of half private high school students/half underrepresented students – who would have their flights to Boston funded by fundraising done primarily by the private high school. </p><br />
<p>After extensive email &amp; phone correspondence spanning several months, the school decided it was not currently in the position to go ahead with the iGEM team proposal due to upcoming infrastructure developments, though would be interested in fielding a team after these are completed (scheduled for 2016). Nonetheless, the school did encourage us that other APS schools would likely be interested in hearing about iGEM and our team concept. </p><br />
<p>Whilst we didn&rsquo;t achieve our ultimate goal, this activity did raise awareness of the iGEM competition with all the key science staff members at the school which requested not to be named on this wiki (for further details please contact our team leader Sean). </p><br />
<p>Finally, we would encourage all Australian teams to work on the following goals: </p><br />
<ul><br />
<li>improve iGEM's accessibility to those of disadvantaged backgrounds </li><br />
<li>increase female representation in science &amp; iGEM </li><br />
<li>increase the participation of Australian/international high school students in the iGEM competition. </li><br />
</ul><br />
<p><BR><br />
In addition to our negotiations with the private high school, we also aimed at widening the participation of high school students in the iGEM competition by collaborating with the University of Sydney on The Strange Nature Writing competition. </p><br />
<p>We spruiked the competition not only on our project flyers which described the iGEM competition, our project and the Strange Nature Writing Competition, but also through our development of a promotional video designed to be shown in high school assemblies as a way of promoting both iGEM and the Strange Nature Writing competition. Please see the video below: </p><br />
<br />
<iframe class="centervid" width="560" height="315" src="//www.youtube.com/embed/ZuSoO3HVMjo" frameborder="0" allowfullscreen></iframe><br />
<br />
<p>Furthermore, we wrote and published articles on Comet.is which is aimed at high school students, university students and recent university graduates, please see 'For university students' for a list of the articles we wrote. </p><br />
<h3>&nbsp;</h3><br />
<h3>For university students </h3><br />
<p>Our main form of outreach toward university students was in writing and publishing articles on a burgeoning and government sponsored website Comet.is which is aimed at high school students, university students and recent graduates. Our articles covered a range of topics from our iGEM experience to considerations of ethical issues of synthetic biology and the potential of synthetic biology. </p><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/d/d5/Comet.jpg" width="640" height="400" alt="Adventure of E coli"/><br />
<br />
<br />
</p> <strong>Here are some excerpts:</strong> </p><br />
<br />
<blockquote></p> "All this leads me to refer to synthetic biology as a ménage à trois of a discipline, utilizing engineering, biology and computer science to produce a fantastically creative activity." </p></blockquote><br />
<br />
<blockquote></p> "Given assistance in the form of grants or human resources, entrepreneurial forums along with student competitions like InnovationACT and iGEM have the chance to inspire entrepreneurialism in students. It is in these opportunities for students to go on entrepreneurial adventures, replete with late nights, red bulls and a shot at winning a grand prize – that the next generation of entrepreneurs is born." </p></blockquote><br />
<br />
<blockquote></p> "A prime example of this is in the International Genetically Engineered Machine (iGEM) competition, which I am a team member of for the University of Melbourne this year. Our team stretches across many disciplines; from telecommunications and business to chemical engineering and science communication, even information technology - we are practically the UN of fields and backgrounds." </p></blockquote><br />
<br />
<blockquote></p> "Participating in IGEM can definitely be categorized as having an experience and not just joining a team. The competition brings together all the components of completing a project for undergraduate students to be part of, a little like a trial run of a Masters or Doctorate course." </p></blockquote><br />
<br />
<p>Here are the links to our articles: </p><br />
<p><A href="https://comet.is/article/2960" rel="nofollow">https://comet.is/article/2960</A> <A href="https://comet.is/article/3499/" rel="nofollow">https://comet.is/article/3499/</A> <A href="https://comet.is/article/3381/" rel="nofollow">https://comet.is/article/3381/</A> <A href="https://comet.is/article/3561/" rel="nofollow">https://comet.is/article/3561/</A> <A href="https://comet.is/article/3562/" rel="nofollow">https://comet.is/article/3562/</A> <A href="https://comet.is/article/3694/" rel="nofollow">https://comet.is/article/3694/</A> <A href="http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/" rel="nofollow">http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/</A> <A href="http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/" rel="nofollow">http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/</A></p><br />
<p>Furthermore, as part of our outreach directed at university students, we combined our recruitment campaign for an IT whiz to help with our wiki, with a presentation on what iGEM was all about. We lecture bashed over several weeks, presenting over 11 times. </p><br />
<br />
<h3><BR><br />
</h3><br />
<h3>For general public </h3><br />
<p>After promoting the iGEM competition to the Victorian State Chair of National Science Week (NSW) - Nick Besley, our team decided to field a stall in &lsquo;Market of the Mind&rsquo; – a NSW event aimed at the general public located in a high foot traffic area alongside the Yarra River in Southbank across the river from Flinders St Station. Luckily, our stall was located next to the bar, which meant there was a lot of foot traffic directed to our happy team members wearing our iGEM team t-shirts.</p><br />
<img src="https://static.igem.org/mediawiki/2014/d/d7/10641184_10203547262856505_6097351474732015309_n.jpg" width="400" height="300" /><br />
<p> Using a true/false quiz, armed with Ferrero Rochers as rewards for getting all the answers correct, we had over 30 quiz contestants engage with us on the first night and 35+ contestants on the second day. Please see the accompanying video made by the City of Melbourne, which shows our stall/team members from 0:45 </p><br />
<br />
<p><iframe class="centervid" width="560" height="315" src="//www.youtube.com/embed/ahFI3aFBdNc" frameborder="0" allowfullscreen></iframe><p><br />
<br />
<br />
<p> <strong> Our team consulted: </strong> </p><br />
<p>IndieMosh</p><br />
<p>Kids Time Early Learning Centre (Bentleigh East)</p><br />
<p>Kids Time Early Learning Centre (Moorabin)</p><br />
<p>Coco's Early Learning Centre</p><br />
<p>A Victorian private school (one of the 11 Associated Public Schools)</p><br />
<p>USyd-Australia</p><br />
<p>University of Melbourne IT Faculty</p><br />
<p>University of Melbourne Education Faculty</p><br />
<p> University of Melbourne Media and Public Relations Department </p><br />
<p>Victorian State Chair of National Science Week (NSW) - Nick Besley</p><br />
<p> Teachers' Association of Australia (TAA) </p><br />
<br />
<br />
<br />
<h1>Intellectual Property </h1><br />
<br />
<p> <strong> Our team considered the question of ‘how should we proceed if we want to retain IP rights on our project’? </strong> </p><br />
<br />
<p> Before attempting to answer this pertinent IP question, our team leader decided to recruit two more team members who had some intellectual property experience to complement the learning on patent law that a few early team members had acquired in their biotechnology undergraduate degrees. Both the new team members had previously filed provisional patents; one had filed some in Russia and the other in Australia, offering two perspectives on this question of IP. </p><br />
<br />
<p> In answering this question, we approached our universities’ tech transfer office to gain clarity over our team’s discussions and brainstorm sessions on whether our project was patentable. </p><br />
<br />
<p> The tech transfer office advised us that our project may have commercial applications, however it was probably too soon to act on the idea and convert it into a provisional patent, so we decided to hold off and wait till we had a more substantiated grasp on our commercial applications. We were also advised that if at any point we wanted to file for IP protection on the project, it would be really important to not have disclosed the details of our idea to anyone else, as this could result in us losing the right to our IP. </p><br />
<br />
<p> After doing some research on the various law firms who could offer us a provisional patent, we discovered the estimated cost would be in the vicinity of $3500-$6500 depending on the level of detail we wanted to cover e.g. the number and extent of the claims, the full description of the idea, diagrams and experimental protocols as well as the filing & service fees associated. </p><br />
<br />
<p> Due to the relatively high estimates we received, we looked into other options for filing a provisional, including recruiting a law student to join the team and contribute their knowledge to our IP protection. This avenue did not eventuate as our ideas on what we would like to patent continuously evolved over the course of the project and we could not reach a sufficient degree of clarity to bring on a lawyer for the filing of a provisional patent. </p><br />
<br />
<p> Nonetheless, we proceeded with caution, ensuring to follow the advice of the University of Melbourne’s tech transfer office and not disclose our ideas without ensuring our disclosure was protected. An instance when we had to implement this protection was during our scientific collaboration with Oxford, were we ensured Non Disclosure Agreements were signed prior to the disclosure of our project to them. They indicated their interest in using our project as a case study in their ‘iGEM intellectual property guide’, however we felt that this might put our IP rights in jeopardy, so we decided not to proceed as a case study. </p><br />
<br />
<p> At this time, our team is looking to produce at least one provisional patent following the iGEM 2014 competition on at least one of the various techniques we designed during the project. </p><br />
<br />
<br />
<p> <strong> Our team consulted: </strong> </p><br />
<br />
<p> University of Melbourne Technology Transfer Office </p><br />
<p> University of Melbourne Technology Commercialisation Team </p><br />
<p> UoM Commercial </p><br />
<p> Stanislauv Yatskevich (iGEM team member with Russian patent experience) </p><br />
<p> Peter Collins (iGEM team member with Australian patent experience) </p><br />
<br />
<br />
<p> <strong> References: </strong> </p><br />
<br />
<p> http://www.ipaustralia.gov.au/.../factsheet-experimental.../ </p><br />
<br />
<p>http://www.google.com.au/url?sa=t&rct=j&q=&esrc=s...</p></div>Slowehttp://2014.igem.org/Team:Melbourne/ProjectTeam:Melbourne/Project2014-10-18T03:54:03Z<p>Slowe: </p>
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<a href="https://2014.igem.org/Team:Melbourne"style="color:#000000"><br />
Home</a> </td><br />
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Team</a> </td><br />
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Project</a> </td><br />
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Human Practices</a></td><br />
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<a href="https://2014.igem.org/Team:Melbourne/Achievements"style="color:#000000"><br />
Achievements</a></td><br />
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Notebook</a></td><br />
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<a href="https://2014.igem.org/Team:Melbourne/Protocols"style=" color:#000000"> <br />
Protocols</a></td><br />
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<a href="https://2014.igem.org/Team:Melbourne/Safety"style="color:#000000"><br />
Safety</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Sponsors"style="color:#000000"><br />
Sponsors</a></td><br />
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<a href="https://2014.igem.org/Team:Melbourne/Attributions"style="color:#000000"><br />
Attributions</a></td><br />
<br />
<br />
<td align="right"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="46px"></a> </td><br />
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<h1>Project and results</h1><br />
<br />
<p>Read about our experimental work below, or jump to our theoretical collaboration with the <A HREF="#Oxford">University of Oxford</A><br />
<br />
<h2>Introduction and theory</h2><br />
<p>Synthetic peptide chemists have long produced peptide-based materials <em>in vitro</em>. Star-shaped peptides are a promising type of biomaterial being explored in the field of nanomedicine (Sulistio et al., 2012). Star peptides can have several biomedical uses, such as acting as drug delivery vehicles (Sulistio et al., 2011) or linkers for other biomacromolecules. Star peptides generally take the form of several linear peptide arms linked together in a central core. One way of linking these linear peptide arms together is to used covalent bonds such as those in disulfides. Typically, disulfide bonds are formed synthetically by taking several linear arms and treating them with an oxidant <em>in vitro</em>. Here, we introduce a new approach to forming star peptides using <em>E. coli</em> and synthetic biology. Thus, we aimed to show how the peptides synthesis and disulfide bond forming machinery of <em>E. coli</em> can be used to form disulfide linked star peptides and key star peptide precursors.</p><br />
<p>&nbsp;</p><br />
<h2>Synthesis approach</h2><br />
<p><em>E. coli</em> naturally possesses the capacity to form disulfide bonds. In native strains, disulfide bonds are naturally formed by an array of enzymes which are part of the Dsb family (e.g. DsbA and DsbC) (Kadokura et al., 2003, Kadokura and Beckwith, 2009). Normally, these enzymes are found in the oxidizing periplasm of the cell. However, several new strains of <em>E. coli</em> have recently been engineered which contain an oxidizing cytoplasm conducive to disulfide bond formation. One example of this is the SHuffle cell line (Lobstein et al., 2012). The cell line contains mutations to key enzymes responsible for the reducing nature of the cytoplasm, namely thioredoxin reductase (<em>trxB</em>) and glutathione reductase (<em>gor</em>). Furthermore, the Shuffle cell line over expresses the disulfide bond isomerase DsbC to the cytoplasm. Together, these mutations allow SHuffle to fold disulfide-bonded proteins in the cytoplasm at a higher success rate compared to non-mutants. We aimed to take advantage of the disulfide bond forming capabilities of this strain of <em>E. coli</em> to synthesize star peptides in cells. As shown in the figure below, the synthesis steps may proceed as follows:</p><br />
<p>&nbsp;</p><br />
<ol><br />
<li> Express a short peptide containing two cysteine residues either to the <em>E. coli</em> periplasm or the cytoplasm of a <em>trxB gor </em>mutant.</li><br />
<li><em>E. coli</em> disulfide bond forming enzymes fold the peptide into a hairpin loop structure.<br />
</li><br />
<li>Cut the loop at a protease recognition site engineered into the peptide. This may be done by extracting the folded peptide from the cell and treating it with the protease in vitro. Alternatively, the protease may be co-expressed in the cell to allow for in vivo cleavage.<br />
</li><br />
</ol><br />
<p>&nbsp;</p><br />
<table align="center"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9d/Project_concept-v2.png" width="480" height="600" alt=""/></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<p>This synthesis approach has several benefits over purely <em>in vitro</em> approaches. Firstly, the exact peptide sequence can be precisely programmed into <em>E. coli</em> using recombinant DNA synthesis. Secondly, by performing the disulfide bond formation in cells and optionally the proteolytic cleavage, several synthesis steps which would need to be performed <em>in vitro</em> are eliminated. From a scale up perspective, this would eliminate entire unit operations which would otherwise be required to produce this product. Given these benefits, in the current study, we aimed to express a star peptide precursor to the cytoplasm of SHuffle cells, which would later be extracted and externally digested with the star-forming protease. In order to achieve this, we first designed several star peptides which might be amenable to this synthetic strategy. These peptides are described below.</p><br />
<p>&nbsp;</p><br />
<h2>Rationally designed peptides</h2><br />
<p>There are two approaches to functionalising star peptides. In the first, the identity of the arms can be chosen to be bioactive peptide molecules. This is the simplest approach to producing a biologically-relevant star. In the second, the arms can be functionalised by ligating molecules to them at any point. We used both of these approaches to design two separate peptides.</p><br />
<h3>Magainin 1 star and linear peptides</h3><br />
<p>Our first strategy was to make a star peptide using antimicrobial peptides(AMPs) as building blocks. AMPs are small, approximately 50 residue peptides secreted by some bacterial and eukaryotic cells which selectively kill microbial cells. It is thought that AMPs work by forming pores in the membrane of prokaryotic cells (Brogden, 2005). AMPs have been recombinantly expressed in a number of organisms, including <em>E. coli</em> (for a review, see Li, 2011) and <em>B. Subtilis</em> (Chen et al., 2009, Yu et al., 2013).</p><br />
<p>Our concept was to design a star peptide with antimicrobial peptide arms. Wiradharma et al. (2012) first showed that placing linear antimicrobial peptides in a star configuration could lead to enhanced antimicrobial activity and decreased hemolytic activity. Although it is unclear why this is the case, it may be due to the ability of neighbouring antimicrobial peptide arms to interact with each other to synergistically rupture the membrane.<br><br />
While Wiradharma used a synthetic peptide sequence, we designed a peptide using the naturally occurring AMP, Magainin 1 (Zasloff, 1987). Magainin 1 peptides will be placed to the ends of each star arm.</p><br />
<br />
<p>Magainin 1 was chosen because the Magainins are one of the major classes of antimicrobial peptides, being well studied and characterised. In addition, we were concerned that tethering the antimicrobial peptide to the star peptide might interfere with its antimicrobial activity. Magainin 1, however, has previously been tethered to surfaces, where it has imparted the surfaces with microbicidal properties (Glinel et al., 2008; Humblot et al., 2009). We surmised that if Magainin 1 maintained its activity while anchored to surfaces, it may also maintain its activity while anchored to a star peptide.</p><br />
<p>The sequence for Magainin 1 is: GIGKFLHSAGKFGKAFVGEIMKS.</p><br />
<p>When attached to a star peptide it will have the following structure:</p><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/MAGAININ_1_peptide.png" width="720" height="300" alt=""/><br />
<p>There are several design elements to note:</p><br />
<ul><br />
<li><br />
The antimicrobial peptide star will be expressed with a SUMO fusion protein. This is because without the fusion, it is likely that the peptide would be toxic to the host cell.</li><br />
<li>The peptide includes a Factor X cutting site between the two cysteines for eventual proteolytic cleavage and formation of the star peptide.</li><br />
</ul><br />
<p>In addition to the star peptide Magainin 1, we synthesised a gene for a linear Magainin 1 peptide as well. This is identical to the construct above, except that there is only one Magainin 1 peptide attached to the SUMO fusion.</p><br />
<br />
<h3>Unstructured Peptide (USP) Construct</h3><br />
<p>In the second approach, we designed a peptide which can be functionalised using chemical approaches. This peptide was designed to have flexible, unstructured arms and was termed the USP construct. Unlike the Magainin 1 star, the arms of this peptide serve not as active peptides themselves, but as inert structural linkers.</p><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/USPI_Peptide.png" width="720" height="216" alt=""/><br />
<p>The arms were designed with the following elements in mind:</p><br />
<ul><br />
<li><br />
<b>Lack of structure.</b> The arms were designed with a bioinspired approach, using the FxFG motif of nucleoporins (where x is a variable amino acid residue). Such segments naturally repeat in nucleoporins and are thought to lead to disorder/lack of stable secondary structure. Nucleoporins are found in mammalian cells, serving as flexible brushes around nuclear pores (Ader et al., 2010).<br />
</li><br />
<li><b>Water-soluble.</b> The arms were designed with several charged amino acids to improve solubility.<br />
</li><br />
<li><b>Designed to form a disulfide bond.</b> Although it is difficult to rationally ensure that the disulfide bond will form between two cysteines in our peptide, we incorporated a beta turn between the two cysteines which may encourage the peptide to fold at the apex of the hairpin loop. This may bring the cysteines into closer proximity, providing more probable bond formation.</li></ul><br />
<br />
<p>The ultimate utility of this peptide lies in its ability to be functionalised with other biomacromolecules. For example, the technique of Native Chemical Ligation(NCL) can be used to join peptides, proteins, and other ligands to the arms (Dawson et al., 1994). The idea of attaching enzymes to the star peptide was explored by the University of Oxford iGEM 2014 team in a collaborative effort between our two teams (See Supplementary Project Work at the end of this page). </p><br />
<br />
<p>&nbsp;</p><br />
<h2>Expression system</h2><br />
<p>In order to successfully express our constructs, we designed our protein expression vectors to include a fusion protein. The fusion protein was necessary for two reasons. First, some of our constructs are very small (e.g. the non-star Magainin 1), and expression levels of very small peptides can be difficult without a fusion partner. Second, two of our constructs code for antimicrobial peptides. Without a fusion partner, it is likely that these genes would be toxic to their hosts upon induction.</p><br />
<p>To find a suitable expression system, we looked towards the Registry of Standard Parts. We used the SUMO protein expression system designed by TU Delft 2014 (for example, see<a target="_blank" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1022101">http://parts.igem.org/wiki/index.php?title=Part:BBa_K1022101</a>). This system essentially consists of a N-terminal HIS-tag followed by the SUMO protein (also known as UlpI).</p><br />
<p><br />
The strategy of expressing toxic AMPs using SUMO has been successfully reported in the literature (Bommarius et al., 2010). We surmise that the SUMO protein could inhibit the antimicrobial activity of single, linear peptides, and that it may also inhibit the activity of our star antimicrobial peptide.</p><br />
<p><br />
SUMO as a fusion protein also has the benefit of leaving no residues at the C-terminal end of the cleavage site. This means that upon cleavage with the SUMO protease, the native protein can be recovered. In our case, this means that one of the arms of the star can be designed without the need to take into account the addition of any amino acid residues left behind by the protease.<br><br />
We used the SUMO peptide sequence reported by TU Delft. However, our construct contained the following unique features:</p><br />
<ol><br />
<li> Standardisation of the biobrick by substituting the T7 promoter and RBS (the origins of which are both not specified in the Delft documentation) with the standard T7 promoter and RBS BioBrick<a target="_blank" href="http://parts.igem.org/Part:BBa_K525998">BBa_K525998</a>. In addition to supporting the principle of standardization, using the well-characterized promoter BioBrick should help assure expression levels.<br><br />
</li><br />
<li>Biobrick BBa_K1022101 lacks a terminator sequence (this was presumably because the part was meant to be integrated into a larger genetic construct with a terminator). A terminator from the registry of standard parts was added (specifically, the wild type terminator from T7 bacteriophage,<a target="_blank" href="http://parts.igem.org/Part:BBa_K731721">BBa_K731721</a>).<br />
</li><br />
<li>The original biobrick BBa_K1022101 codes for three amino acid residues before the HIS-tag (ASM), which appeared to be redundant. Correspondence with the 2013 TU Delft team suggested that these residues were unnecessary and appear to be cleaved within the cell as part of the cells post-translational modifications. However, their presence complicates the addition of additional tags at the N-terminus of the protein (e.g. periplasmic export tags), and therefore were not included.<br><br />
</li><br />
<li>The SUMO sequence was codon optimised for E. coli during synthesis. As the Delft documentation did not specify whether the gene was codon optimal, codon optomisation was undertaken to potentially improve expression levels. The linear Magainin 1 construct, however, was not codon optimised in the SUMO region in order to provide a control condition.</li><br />
</ol><br />
<p>To summarise, the protein expression devices used in our project to the following form:</p><br />
<img src="https://static.igem.org/mediawiki/2014/d/d0/Gene_circuit_export.png" width="720" height="91" alt=""/></td><br />
<p>The protein coding region consisted of a 6x-HIS tag followed by the SUMO protein and the relevant star or linear peptide.</p><br />
<p>&nbsp;</p><br />
<h2>Plasmid preparation: Cloning and acquisition of the genetic constructs</h2><br />
<h3>Magainin 1 Star Peptide</h3><br />
<p>This construct was synthesised in the standard shipping plasmid from Life Technologies- plasmid pMK. We had originally planned to express our protein to the periplasm of E. coli, and therefore had included in the synthesis a periplasmic export tag, the TorTss signal sequence (Steiner et al., 2006).</p><br />
<p><br />
Cytoplasmic and periplasmic expression may both allow for disulfide bond formation in the cell. We decided, however, to focus on cytoplasmic expression. There are distinct advantages to cytoplasmic expression (e.g. the absence of several periplasmic proteases and potentially higher expression levels (Baneyx, 1999)).</p><br />
<p><br />
Another reason the cytoplasm was chosen was to allow us to test the effects of an oxidising versus reducing intracellular environment on disulfide bond formation. We planned to express the construct in both SHuffle T7 cells (oxidising cytoplasm) and BL21(DE3) (reducing cytoplasm) to probe whether there was a difference in disulfide bond formation. Therefore, we needed to remove the periplasmic export tag from the gene.</p><br />
<p><br />
To do this, we use the following cloning strategy (noting that the top row represents the gene initially synthesised in plasmid pMK):</p><br />
<img src="https://static.igem.org/mediawiki/2014/2/2c/Melbourne_cloning_strategy_diagram.jpg" width="900" height="437" alt=""/><br />
<p>The gene was inserted into plasmid pSB1C3 containing the T7 promoter and ribosome binding site, BBa_K525998. It was inserted after the promoter and before the BioBrick suffix. This was accomplished by digesting the destination vector with SpeI and PstI. At the same time, PCR was used to amplify the segment of the gene containing the SUMO fusion and the Magainin 1 Star peptide, adding XbaI and keeping the PstI site existing in the gene.</p><br />
<p><br />
Digestion of the PCR product then allowed for ligation of the insert and destination vector using the PstI sites and XbaI and SpeI (XbaI and SpeI have compatible sticky ends). Note that after the ligation, there will be a scar in the gene where the XbaI and the SpeI sites were ligated.</p><br />
<p><br />
The ligation appeared to be successful. The ligation mixture was transformed to DH5α competent cells and plated onto chloramphenicol-containing agar plates.</p><br />
<p><br />
Plasmid from 5 colonies (C1, C2, C3, C4, and C5) was cultured, extracted, and then digested with EcoRI and PstI.</p><br />
<p><br />
As evident in the figure below, at least 4 of the colonies appeared to contain the insert. The empty, linearised pSB1C3 backbone ran at approximately the correct size (2070 bps), as did the insert (740 bps).</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/9/95/Magainin_1_subcloning.png" width="353" height="400" alt=""/></center><br />
<p>N.B. MW marker is the 100 bp Ladder from Axygen. * indicates the colony picked for sequencing and eventual transformation to the expression cell lines.</p><br />
<p>The DNA from Colony C2 was confirmed using Sanger sequencing at the Australian Genome Research Facility. This DNA appears in the registry of standard parts as BioBrick <a target="_blank" href="http://parts.igem.org/Part:BBa_K1394000">BBa_K1394000</a>.</p><br />
<p><br />
For the USP peptide and the linear Magainin 1 peptide, the genes were synthesised by GenScript and delivered in pSB1C3. The expression vectors had identical gene regulatory elements to that used for the Magainin 1 Star peptide. They only differed in the codon optimisation used, and they also lacked the assembly scar described above.</p><br />
<br />
<h2><br />
Protein expression and characterisation<br />
</h2><br />
<p><br />
After acquiring our genes, the project moved to protein expression and purification. We expressed both star peptides (Magainin 1 Star Peptide and the USP peptide) in both the SHuffle T7 and BL21(DE3) cell lines, both sourced from New England Biolabs. As the Linear Magainin 1 peptide does not have any special disulfide bonding<br />
requirements, we expressed it in BL21(DE3).<br />
</p><br />
<p><br />
The plasmids were transformed to the expression cells, and a single colony was cultured and induced overnight at 17 °C. A whole cell sample both before<br />
IPTG induction (-IPTG) and after the induction period (+IPTG) were boiled in SDS-PAGE sample buffer and loaded on a 15% tris-glycine gel. The whole cell<br />
Coomassie stained gel is shown below alongside the NEB P7712S molecular weight marker.<br />
</p><br />
<br><br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/2/24/Whole_cell_CS.png" height="330" width="439"/><br />
</p><br />
<br><br />
<p><br />
From theoretical prediction of the peptide masses, we would expect the following distribution of molecular weights:<br />
</p><br />
<br><br />
<table border="1" cellpadding="0" cellspacing="0" align="center"><br />
<tbody><br />
<tr><br />
<td width="156"><br />
<p align="center"><br />
Magainin 1 Star Peptide (Mag1 Star)<br />
</p><br />
</td><br />
<td width="145"><br />
<p align="center"><br />
USP<br />
</p><br />
</td><br />
<td width="156"><br />
<p align="center"><br />
Linear Magainin 1 (Linear Mag1)<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="156"><br />
<p align="center"><br />
22.15 kDa<br />
</p><br />
</td><br />
<td width="145"><br />
<p align="center"><br />
29.3 kDa<br />
</p><br />
</td><br />
<td width="156"><br />
<p align="center"><br />
14.32 kDa<br />
</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<br><br />
<p><br />
We looked for bands corresponding to overexpression of the proteins. We observed a thick band, post IPTG around 32 kDa in the BL21(DE3) cells carrying the<br />
USP plasmid. However, we saw this same band appearing in the Linear Magainin 1 lane but the Linear Magainin 1 protein should have a much lower molecular<br />
weight than what was observed. Therefore, we did not assume that we had overexpression overexpression of the USP 1 protein.<br />
</p><br />
<p><br />
We also noted bands in all post-IPTG lanes around 17 kDa. This would be roughly consistent with both the Linear Magainin 1 and Magainin 1 Star Peptide,<br />
noting that small proteins may not run at their expected molecular weight. Again, the analysis is complicated by the fact that the band also appears in the<br />
lane corresponding to the larger molecular weight protein, USP I. Given this ambiguity, we decided to purify all the protein in the sample using Ni-NTA<br />
purification.<br />
</p><br />
<h3><br />
Purification and further characterisation<br />
</h3><br />
<p><br />
A small-scale purification was carried out according to our <a href="https://static.igem.org/mediawiki/2014/e/e1/MELBOURNE_D1V1_Column_Purification_of_His-Tagged_Proteins.pdf">protocol</a>. The cells were lysed with iodoacetamide, an alkylating agent, added to the lysis buffer. Iodoacetamide blocks all free cysteines on proteins with a short alkyl group. This was added<br />
because we wanted to determine if a disulphide bond had formed inside the cell. If we did not block free cysteines, then any bond that had formed could be<br />
attributed to spontaneous/air oxidation of disulfides outside the cell.<br />
</p><br />
<p><br />
After purification, we ran a concurrent Western Blot and Coomassie stain on both the pre-induction samples from above and the purified protein (namely, the<br />
first elution from the batch purification).<br />
</p><br />
<p><br />
The Western Blot used mouse monoclonal antibodies against the N-terminal HIS-tag as primary antibodies and anti-mouse secondary antibodies:<br />
</p><br />
<br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/5/54/Western_Blot_Melb.png" height="400" width="460" border="0"/><br />
</p><br />
<p><br />
The corresponding Coomassie stain is show below:<br />
</p><br />
<br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f0/Purified_CS.png" height="365" width="486" border="0"/><br />
</p><br />
<p><br />
There are several interesting aspects of the Western Blot. Firstly, there was a strong band between 25 and 32 kDa in most lanes. However, it<br />
appears in all of the pre-induction controls, suggesting non-specific binding of the anti-HIS antibodies.<br />
</p><br />
<p><br />
Secondly, we observed a band in most of the lanes around 17 kDa (with the exception being the USP protein in SHuffle cells). As these bands were not<br />
present in the pre-induction controls, we suspected they were a result of the induction.<br />
</p><br />
<h3><br />
Mass spectroscopy<br />
</h3><br />
<p><br />
In order to assess the identity of the bands, we performed an in-gel tryptic digestion on select bands. We digested bands in the Coomassie stain which<br />
corresponded to the HIS-tagged bands in the Western Blot. This was followed by mass spec analysis (LC MS/MS). We focused our analysis on the Magainin 1<br />
Star Peptide bands and the Linear Magainin 1 band. Our USP peptide was, by design, highly rich in basic residues, greatly reducing the likelihood that<br />
tryptic fragments would be detected by the mass spec.<br />
</p><br />
<p><br />
We found that the Linear Magainin 1 peptide appeared to be present in the approximately 17 kDa band, with the following detected tryptic fragments in the<br />
sequence below (bold; basic residues underlined):<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/1/1b/Tryptic_digest_linear_mag1.PNG" height="63" width="601" border="0"/><br />
</p><br />
<p><br />
Note that spaces have been added in the sequence to emphasise distinct domains in the protein (in this case, the fusion protein versus the Magainin 1<br />
peptide). Together with the fact that the protein runs close to the expected molecular weight, this seems to provide good evidence that the protein is<br />
being expressed.<br />
</p><br />
<p><br />
We then examined the bands corresponding to the Magainin 1 Star Peptide.<br />
</p><br />
<p><br />
Again, we digested bands in the Coomassie stained gel corresponding to the prominent HIS-tagged bands on the <em>Western Blot</em> (near 17 kDa).<br />
</p><br />
<p><br />
The mass spec coverage for the Magainin 1 Star Peptide expressed in SHuffle cells was as follows:<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/6/63/Tryptic_digest_Sample_A_Star_Mag_Shuffle.PNG" height="90" width="601" border="0"/><br />
</p><br />
<p><br />
The coverage for the same peptide expressed in BL21(DE3) was found to be:<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/d/db/Tryptic_digest_Sample_A_Star_Mag_BL21%28DE3%29.PNG" height="85" width="602" border="0"/><br />
</p><br />
<p><br />
Again, we can see that tryptic peptides from the expressed protein appear to present in the gel band under analysis.<br />
</p><br />
<p><br />
There is a relatively lower sequence coverage. This could be a limitation of our procedure: for example, improper destaining during the in gel digestion<br />
procedure can inhibit tryptic digestion.<br />
</p><br />
<p><br />
However, even if the digestion was complete, the mass spec will only detect fragments which ionize well. The Magainin 1 Star peptide consists of four<br />
repeats of the Magainin 1 sequence (corresponding to the arms of the star). If the two tryptic fragments within Magainin 1 do not ionize well, then indeed<br />
the entire peptide would not be read.<br />
</p><br />
<p><br />
Finally, it is possible that the protein has been cleaved or degraded. This would account for the slightly lower-than-expected mass on the SDS page gel.<br />
</p><br />
<p><br />
Nonetheless, it is a possibility that the peptide fragments are present but not detected. Replication of the in-gel mass spec would be useful<br />
in clarifying this point, or provided the purity of the sample is acceptable, it may be possible to run an intact mass spec. Digestion with alternative enzymes may also provide a more robust sequence coverage, particularly for the USP peptide.<br />
</p><br />
<h2><br />
Conclusions<br />
</h2><br />
<p><br />
We designed several novel star peptides based on several rational design criteria. Further, we began the optimisation process required to express these peptides in <em>E. coli</em>.<br />
</p><br />
<p><br />
To this end, we constructed an expression system based on the SUMO protein and standard BioBrick parts. The function of this system was confirmed, as it<br />
appears it does produce HIS-tagged SUMO fusion protein in <em>E. coli</em>, as expected. Nonetheless, we hope to continue to characterise our parts, likely through further purification and measurement of the mass. <br />
</p><br />
<p>Given the success of SUMO in the protein expression community, we hope our BioBrick<br />
will encourage further use of the fusion domain within iGEM. In addition, we hope that our ideas and concepts will inspire future<br />
iGEM efforts to explore the applications of synthetic biology for material science. We continue to believe that bacteria have great promise for the <em>in vivo</em><br />
construction of unique biomaterials, and we look forward to seeing how the synthetic biology community will develop in this area.<br />
</p><br />
<br />
<br />
<br />
<h2>References</h2><br />
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<p><br />
BANEYX, F. 1999. Recombinant protein expression in Escherichia coli. <em>Current Opinion in Biotechnology,</em> 10<strong>,</strong> 411-421.</p><br />
<p><br />
BOMMARIUS, B., JENSSEN, H., ELLIOTT, M., KINDRACHUK, J., PASUPULETI, M., GIEREN, H., JAEGER, K. E., HANCOCK, R. E. W. &amp; KALMAN, D. 2010. Cost-effective expression and purification of antimicrobial and host defense peptides in Escherichia coli. <em>Peptides,</em> 31<strong>,</strong> 1957-1965.</p><br />
<p><br />
BROGDEN, K. A. 2005. Antimicrobial peptides: Pore formers or metabolic inhibitors in bacteria? <em>Nature Reviews Microbiology,</em> 3<strong>,</strong> 238-250.</p><br />
<p><br />
CHANG, C., CHHOR, G., CLANCY, S. & JOACHIMIAK, A. 2014. Crystal Structure of Glutathione S-transferase Domain Protein From Haliangium Ochraceum DSM 14365 [Online]. ''NCBI. Available: http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=4w66' [Accessed September 9 2014].</p><br />
<p><br />
CHEN, X., ZHU, F., CAO, Y. &amp; QIAO, S. 2009. Novel expression vector for secretion of cecropin AD in Bacillus subtilis with enhanced antimicrobial activity. <em>Antimicrobial agents and chemotherapy,</em> 53<strong>,</strong> 3683-3689.</p><br />
<p><br />
GLINEL, K., JONAS, A. M., JOUENNE, T., LEPRINCE, J., GALAS, L. & HUCK, W. T. 2008. Antibacterial and antifouling polymer brushes incorporating antimicrobial peptide. <em>Bioconjug Chem,</em> 20<strong>,</strong> 71-77.</p><br />
<p><br />
HUMBLOT, V., YALA, J.-F., THEBAULT, P., BOUKERMA, K., HÉQUET, A., BERJEAUD, J.-M. & PRADIER, C.-M. 2009. The antibacterial activity of Magainin I immobilized onto mixed thiols self-assembled monolayers. <em>Biomaterials,</em> 30<strong>,</strong> 3503-3512.</p><br />
<p><br />
DAWSON, P. E., MUIR, T. W., CLARK-LEWIS, I. &amp; KENT, S. 1994. Synthesis of proteins by native chemical ligation. <em>Science,</em> 266<strong>,</strong> 776-779.</p><br />
<p><br />
KADOKURA, H. &amp; BECKWITH, J. 2009. Detecting folding intermediates of a protein as it passes through the bacterial translocation channel. <em>Cell,</em> 138<strong>,</strong> 1164-1173.</p><br />
<p><br />
KADOKURA, H., KATZEN, F. &amp; BECKWITH, J. 2003. Protein disulfide bond formation in prokaryotes. <em>Annual review of biochemistry,</em> 72<strong>,</strong> 111-135.</p><br />
<p><br />
LI, Y. 2011. Recombinant production of antimicrobial peptides in&lt; i&gt; Escherichia coli&lt;/i&gt;: A review. <em>Protein Expression and Purification,</em> 80<strong>,</strong> 260-267.</p><br />
<p><br />
LOBSTEIN, J., EMRICH, C. A., JEANS, C., FAULKNER, M., RIGGS, P. &amp; BERKMEN, M. 2012. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. <em>Microb Cell Fact,</em> 11<strong>,</strong> 56-56.</p><br />
<p><br />
PROTEIN MODEL PORTAL. Query result, GST and DcmA [Online]. Available: http://www.proteinmodelportal.org/query/uniprot/P21161 [Accessed September 9 2014].</p><br />
<p><br />
STEINER, D., FORRER, P., STUMPP, M. T. &amp; PLUCKTHUN, A. 2006. Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display. <em>Nat Biotech,</em> 24<strong>,</strong> 823-831.</p><br />
<p><br />
SULISTIO, A., GURR, P. A., BLENCOWE, A. &amp; QIAO, G. G. 2012. Peptide-Based Star Polymers: The Rising Star in Functional Polymers. <em>Australian Journal of Chemistry,</em> 65<strong>,</strong> 978-984.</p><br />
<p><br />
SULISTIO, A., LOWENTHAL, J., BLENCOWE, A., BONGIOVANNI, M. N., ONG, L., GRAS, S. L., ZHANG, X. &amp; QIAO, G. G. 2011. Folic Acid Conjugated Amino Acid-Based Star Polymers for Active Targeting of Cancer Cells. <em>Biomacromolecules,</em> 12<strong>,</strong> 3469-3477.</p><br />
<p><br />
TANAKA, N., KUSAKABE, Y., ITO, K., YOSHIMOTO, T. & NAKAMURA, K. T. 2002. Crystal Structure of Formaldehyde Dehydrogenase from< i> Pseudomonas putida</i>: the Structural Origin of the Tightly Bound Cofactor in Nicotinoprotein Dehydrogenases. Journal of molecular biology, 324, 519-533.</p><br />
<p><br />
WIRADHARMA, N., LIU, S. Q. &amp; YANG, Y. Y. 2012. Branched and 4-Arm Starlike α-Helical Peptide Structures with Enhanced Antimicrobial Potency and Selectivity. <em>Small,</em> 8<strong>,</strong> 362-366.</p><br />
<p><br />
YU, Z., WANG, Q., MA, Q. &amp; ZHANG, R. 2013. Secretory expression of lacticin Q fused with SUMO in Bacillus subtilis. <em>Protein Expression and Purification,</em> 89<strong>,</strong> 51-55.</p><br />
<p><br />
ZASLOFF, M. 1987. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. <em>Proceedings of the National Academy of Sciences,</em> 84<strong>,</strong> 5449-5453.</p><br />
<p>&nbsp;</p><br />
<br />
<A NAME="Oxford"></A><br />
<h1>Collaboration with Oxford</h1><br />
<p>To strengthen the sense of iGEM community, we contacted <a target="_blank" href="https://2014.igem.org/Team:Oxford">University of Oxford iGEM team</a> to discuss the possibility of collaboration between our teams. The Oxford project aims to develop a system that can dispose of the carcinogenic, hazardous solvent dichloromethane (DCM). To do this, Oxford team has proposed the use of the DcmA enzyme. This enzyme degrades DCM to form a toxic intermediate which in turn is converted by another enzyme, FdhA, into a neutral molecule. Schematically this can be presented in the following way:</p><br />
<br />
<p><br />
Reaction 1: DCM +&nbsp;DcmA <strong>toxic intermediate</strong><br><br />
Reaction 2: <strong>toxic intermediate</strong> + FdhA neutral product.</p><br />
<p>The problem for their system lies in bringing the two enzymes together to ensure efficient reaction kinetics. An idea that we discussed with Oxford was to use the star peptide platform to link the two enzymes together, in a structure similar to the following: </p><br />
<img src="https://static.igem.org/mediawiki/2014/2/2e/Melbourne_Collab_1.png" width="500" height="325" alt=""/><br />
<p>We worked with Oxford to study this system from a theoretical standpoint. Our team studied the 3D structure of both enzymes involved to confirm that the enzyme could in theory be attached to a linker in this manner, while Oxford team did stochastic modelling to determine how reaction rate changes when the linker length, labeled D on the diagram, changes. </p><br />
<p><br />
As a first check, it was necessary to determine whether anchoring these enzymes to our star would be sterically possible. We studied the structures of FdhA and DcmA determined by crystallography using data from the protein data bank. As no crystallographic data was available in the databank for DcmA, GST was examined as a proxy, as GST is structurally homologous to DcmA. </p><br />
<p><br />
The crystalographically resolved structure of FdhA enzyme (Tanaka et al., 2002) from the protein data bank revealed the following image:</p><br />
<img src="https://static.igem.org/mediawiki/2014/d/dc/Melbourne_Collab_2.png" width="700" height="355" alt=""/><br />
<p>The crystalographically resolved structure of GST was as follows (Chang et al., 2014):</p><br />
<img src="https://static.igem.org/mediawiki/2014/e/e9/Melbourne_Collab_3.png" width="800" height="361" alt=""/><br />
<p>It can be seen that the amino- and carboxy-terminals are located away from the active site. This suggests that both enzymes, DcmA and FdhA, might be linked together via linkers that are used in our star peptide. That is, the active site will not be sterically hindered by attachment to the star peptide. That said, it is difficult to predict how anchoring the protein to the star</div>Slowehttp://2014.igem.org/Team:Melbourne/ProjectTeam:Melbourne/Project2014-10-18T03:53:36Z<p>Slowe: </p>
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<h1>Project and results</h1><br />
<br />
<p>Read about our experimental work below, or jump to our theoretical collaboration with the <A HREF="#Oxford">University of Oxford</A><br />
<br />
<h2>Introduction and theory</h2><br />
<p>Synthetic peptide chemists have long produced peptide-based materials <em>in vitro</em>. Star-shaped peptides are a promising type of biomaterial being explored in the field of nanomedicine (Sulistio et al., 2012). Star peptides can have several biomedical uses, such as acting as drug delivery vehicles (Sulistio et al., 2011) or linkers for other biomacromolecules. Star peptides generally take the form of several linear peptide arms linked together in a central core. One way of linking these linear peptide arms together is to used covalent bonds such as those in disulfides. Typically, disulfide bonds are formed synthetically by taking several linear arms and treating them with an oxidant <em>in vitro</em>. Here, we introduce a new approach to forming star peptides using <em>E. coli</em> and synthetic biology. Thus, we aimed to show how the peptides synthesis and disulfide bond forming machinery of <em>E. coli</em> can be used to form disulfide linked star peptides and key star peptide precursors.</p><br />
<p>&nbsp;</p><br />
<h2>Synthesis approach</h2><br />
<p><em>E. coli</em> naturally possesses the capacity to form disulfide bonds. In native strains, disulfide bonds are naturally formed by an array of enzymes which are part of the Dsb family (e.g. DsbA and DsbC) (Kadokura et al., 2003, Kadokura and Beckwith, 2009). Normally, these enzymes are found in the oxidizing periplasm of the cell. However, several new strains of <em>E. coli</em> have recently been engineered which contain an oxidizing cytoplasm conducive to disulfide bond formation. One example of this is the SHuffle cell line (Lobstein et al., 2012). The cell line contains mutations to key enzymes responsible for the reducing nature of the cytoplasm, namely thioredoxin reductase (<em>trxB</em>) and glutathione reductase (<em>gor</em>). Furthermore, the Shuffle cell line over expresses the disulfide bond isomerase DsbC to the cytoplasm. Together, these mutations allow SHuffle to fold disulfide-bonded proteins in the cytoplasm at a higher success rate compared to non-mutants. We aimed to take advantage of the disulfide bond forming capabilities of this strain of <em>E. coli</em> to synthesize star peptides in cells. As shown in the figure below, the synthesis steps may proceed as follows:</p><br />
<p>&nbsp;</p><br />
<ol><br />
<li> Express a short peptide containing two cysteine residues either to the <em>E. coli</em> periplasm or the cytoplasm of a <em>trxB gor </em>mutant.</li><br />
<li><em>E. coli</em> disulfide bond forming enzymes fold the peptide into a hairpin loop structure.<br />
</li><br />
<li>Cut the loop at a protease recognition site engineered into the peptide. This may be done by extracting the folded peptide from the cell and treating it with the protease in vitro. Alternatively, the protease may be co-expressed in the cell to allow for in vivo cleavage.<br />
</li><br />
</ol><br />
<p>&nbsp;</p><br />
<table align="center"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9d/Project_concept-v2.png" width="480" height="600" alt=""/></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<p>This synthesis approach has several benefits over purely <em>in vitro</em> approaches. Firstly, the exact peptide sequence can be precisely programmed into <em>E. coli</em> using recombinant DNA synthesis. Secondly, by performing the disulfide bond formation in cells and optionally the proteolytic cleavage, several synthesis steps which would need to be performed <em>in vitro</em> are eliminated. From a scale up perspective, this would eliminate entire unit operations which would otherwise be required to produce this product. Given these benefits, in the current study, we aimed to express a star peptide precursor to the cytoplasm of SHuffle cells, which would later be extracted and externally digested with the star-forming protease. In order to achieve this, we first designed several star peptides which might be amenable to this synthetic strategy. These peptides are described below.</p><br />
<p>&nbsp;</p><br />
<h2>Rationally designed peptides</h2><br />
<p>There are two approaches to functionalising star peptides. In the first, the identity of the arms can be chosen to be bioactive peptide molecules. This is the simplest approach to producing a biologically-relevant star. In the second, the arms can be functionalised by ligating molecules to them at any point. We used both of these approaches to design two separate peptides.</p><br />
<h3>Magainin 1 star and linear peptides</h3><br />
<p>Our first strategy was to make a star peptide using antimicrobial peptides(AMPs) as building blocks. AMPs are small, approximately 50 residue peptides secreted by some bacterial and eukaryotic cells which selectively kill microbial cells. It is thought that AMPs work by forming pores in the membrane of prokaryotic cells (Brogden, 2005). AMPs have been recombinantly expressed in a number of organisms, including <em>E. coli</em> (for a review, see Li, 2011) and <em>B. Subtilis</em> (Chen et al., 2009, Yu et al., 2013).</p><br />
<p>Our concept was to design a star peptide with antimicrobial peptide arms. Wiradharma et al. (2012) first showed that placing linear antimicrobial peptides in a star configuration could lead to enhanced antimicrobial activity and decreased hemolytic activity. Although it is unclear why this is the case, it may be due to the ability of neighbouring antimicrobial peptide arms to interact with each other to synergistically rupture the membrane.<br><br />
While Wiradharma used a synthetic peptide sequence, we designed a peptide using the naturally occurring AMP, Magainin 1 (Zasloff, 1987). Magainin 1 peptides will be placed to the ends of each star arm.</p><br />
<br />
<p>Magainin 1 was chosen because the Magainins are one of the major classes of antimicrobial peptides, being well studied and characterised. In addition, we were concerned that tethering the antimicrobial peptide to the star peptide might interfere with its antimicrobial activity. Magainin 1, however, has previously been tethered to surfaces, where it has imparted the surfaces with microbicidal properties (Glinel et al., 2008; Humblot et al., 2009). We surmised that if Magainin 1 maintained its activity while anchored to surfaces, it may also maintain its activity while anchored to a star peptide.</p><br />
<p>The sequence for Magainin 1 is: GIGKFLHSAGKFGKAFVGEIMKS.</p><br />
<p>When attached to a star peptide it will have the following structure:</p><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/MAGAININ_1_peptide.png" width="720" height="300" alt=""/><br />
<p>There are several design elements to note:</p><br />
<ul><br />
<li><br />
The antimicrobial peptide star will be expressed with a SUMO fusion protein. This is because without the fusion, it is likely that the peptide would be toxic to the host cell.</li><br />
<li>The peptide includes a Factor X cutting site between the two cysteines for eventual proteolytic cleavage and formation of the star peptide.</li><br />
</ul><br />
<p>In addition to the star peptide Magainin 1, we synthesised a gene for a linear Magainin 1 peptide as well. This is identical to the construct above, except that there is only one Magainin 1 peptide attached to the SUMO fusion.</p><br />
<br />
<h3>Unstructured Peptide (USP) Construct</h3><br />
<p>In the second approach, we designed a peptide which can be functionalised using chemical approaches. This peptide was designed to have flexible, unstructured arms and was termed the USP construct. Unlike the Magainin 1 star, the arms of this peptide serve not as active peptides themselves, but as inert structural linkers.</p><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/USPI_Peptide.png" width="720" height="216" alt=""/><br />
<p>The arms were designed with the following elements in mind:</p><br />
<ul><br />
<li><br />
<b>Lack of structure.</b> The arms were designed with a bioinspired approach, using the FxFG motif of nucleoporins (where x is a variable amino acid residue). Such segments naturally repeat in nucleoporins and are thought to lead to disorder/lack of stable secondary structure. Nucleoporins are found in mammalian cells, serving as flexible brushes around nuclear pores (Ader et al., 2010).<br />
</li><br />
<li><b>Water-soluble.</b> The arms were designed with several charged amino acids to improve solubility.<br />
</li><br />
<li><b>Designed to form a disulfide bond.</b> Although it is difficult to rationally ensure that the disulfide bond will form between two cysteines in our peptide, we incorporated a beta turn between the two cysteines which may encourage the peptide to fold at the apex of the hairpin loop. This may bring the cysteines into closer proximity, providing more probable bond formation.</li></ul><br />
<br />
<p>The ultimate utility of this peptide lies in its ability to be functionalised with other biomacromolecules. For example, the technique of Native Chemical Ligation(NCL) can be used to join peptides, proteins, and other ligands to the arms (Dawson et al., 1994). The idea of attaching enzymes to the star peptide was explored by the University of Oxford iGEM 2014 team in a collaborative effort between our two teams (See Supplementary Project Work at the end of this page). </p><br />
<br />
<p>&nbsp;</p><br />
<h2>Expression system</h2><br />
<p>In order to successfully express our constructs, we designed our protein expression vectors to include a fusion protein. The fusion protein was necessary for two reasons. First, some of our constructs are very small (e.g. the non-star Magainin 1), and expression levels of very small peptides can be difficult without a fusion partner. Second, two of our constructs code for antimicrobial peptides. Without a fusion partner, it is likely that these genes would be toxic to their hosts upon induction.</p><br />
<p>To find a suitable expression system, we looked towards the Registry of Standard Parts. We used the SUMO protein expression system designed by TU Delft 2014 (for example, see<a target="_blank" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1022101">http://parts.igem.org/wiki/index.php?title=Part:BBa_K1022101</a>). This system essentially consists of a N-terminal HIS-tag followed by the SUMO protein (also known as UlpI).</p><br />
<p><br />
The strategy of expressing toxic AMPs using SUMO has been successfully reported in the literature (Bommarius et al., 2010). We surmise that the SUMO protein could inhibit the antimicrobial activity of single, linear peptides, and that it may also inhibit the activity of our star antimicrobial peptide.</p><br />
<p><br />
SUMO as a fusion protein also has the benefit of leaving no residues at the C-terminal end of the cleavage site. This means that upon cleavage with the SUMO protease, the native protein can be recovered. In our case, this means that one of the arms of the star can be designed without the need to take into account the addition of any amino acid residues left behind by the protease.<br><br />
We used the SUMO peptide sequence reported by TU Delft. However, our construct contained the following unique features:</p><br />
<ol><br />
<li> Standardisation of the biobrick by substituting the T7 promoter and RBS (the origins of which are both not specified in the Delft documentation) with the standard T7 promoter and RBS BioBrick<a target="_blank" href="http://parts.igem.org/Part:BBa_K525998">BBa_K525998</a>. In addition to supporting the principle of standardization, using the well-characterized promoter BioBrick should help assure expression levels.<br><br />
</li><br />
<li>Biobrick BBa_K1022101 lacks a terminator sequence (this was presumably because the part was meant to be integrated into a larger genetic construct with a terminator). A terminator from the registry of standard parts was added (specifically, the wild type terminator from T7 bacteriophage,<a target="_blank" href="http://parts.igem.org/Part:BBa_K731721">BBa_K731721</a>).<br />
</li><br />
<li>The original biobrick BBa_K1022101 codes for three amino acid residues before the HIS-tag (ASM), which appeared to be redundant. Correspondence with the 2013 TU Delft team suggested that these residues were unnecessary and appear to be cleaved within the cell as part of the cells post-translational modifications. However, their presence complicates the addition of additional tags at the N-terminus of the protein (e.g. periplasmic export tags), and therefore were not included.<br><br />
</li><br />
<li>The SUMO sequence was codon optimised for E. coli during synthesis. As the Delft documentation did not specify whether the gene was codon optimal, codon optomisation was undertaken to potentially improve expression levels. The linear Magainin 1 construct, however, was not codon optimised in the SUMO region in order to provide a control condition.</li><br />
</ol><br />
<p>To summarise, the protein expression devices used in our project to the following form:</p><br />
<img src="https://static.igem.org/mediawiki/2014/d/d0/Gene_circuit_export.png" width="720" height="91" alt=""/></td><br />
<p>The protein coding region consisted of a 6x-HIS tag followed by the SUMO protein and the relevant star or linear peptide.</p><br />
<p>&nbsp;</p><br />
<h2>Plasmid preparation: Cloning and acquisition of the genetic constructs</h2><br />
<h3>Magainin 1 Star Peptide</h3><br />
<p>This construct was synthesised in the standard shipping plasmid from Life Technologies- plasmid pMK. We had originally planned to express our protein to the periplasm of E. coli, and therefore had included in the synthesis a periplasmic export tag, the TorTss signal sequence (Steiner et al., 2006).</p><br />
<p><br />
Cytoplasmic and periplasmic expression may both allow for disulfide bond formation in the cell. We decided, however, to focus on cytoplasmic expression. There are distinct advantages to cytoplasmic expression (e.g. the absence of several periplasmic proteases and potentially higher expression levels (Baneyx, 1999)).</p><br />
<p><br />
Another reason the cytoplasm was chosen was to allow us to test the effects of an oxidising versus reducing intracellular environment on disulfide bond formation. We planned to express the construct in both SHuffle T7 cells (oxidising cytoplasm) and BL21(DE3) (reducing cytoplasm) to probe whether there was a difference in disulfide bond formation. Therefore, we needed to remove the periplasmic export tag from the gene.</p><br />
<p><br />
To do this, we use the following cloning strategy (noting that the top row represents the gene initially synthesised in plasmid pMK):</p><br />
<img src="https://static.igem.org/mediawiki/2014/2/2c/Melbourne_cloning_strategy_diagram.jpg" width="900" height="437" alt=""/><br />
<p>The gene was inserted into plasmid pSB1C3 containing the T7 promoter and ribosome binding site, BBa_K525998. It was inserted after the promoter and before the BioBrick suffix. This was accomplished by digesting the destination vector with SpeI and PstI. At the same time, PCR was used to amplify the segment of the gene containing the SUMO fusion and the Magainin 1 Star peptide, adding XbaI and keeping the PstI site existing in the gene.</p><br />
<p><br />
Digestion of the PCR product then allowed for ligation of the insert and destination vector using the PstI sites and XbaI and SpeI (XbaI and SpeI have compatible sticky ends). Note that after the ligation, there will be a scar in the gene where the XbaI and the SpeI sites were ligated.</p><br />
<p><br />
The ligation appeared to be successful. The ligation mixture was transformed to DH5α competent cells and plated onto chloramphenicol-containing agar plates.</p><br />
<p><br />
Plasmid from 5 colonies (C1, C2, C3, C4, and C5) was cultured, extracted, and then digested with EcoRI and PstI.</p><br />
<p><br />
As evident in the figure below, at least 4 of the colonies appeared to contain the insert. The empty, linearised pSB1C3 backbone ran at approximately the correct size (2070 bps), as did the insert (740 bps).</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/9/95/Magainin_1_subcloning.png" width="353" height="400" alt=""/></center><br />
<p>N.B. MW marker is the 100 bp Ladder from Axygen. * indicates the colony picked for sequencing and eventual transformation to the expression cell lines.</p><br />
<p>The DNA from Colony C2 was confirmed using Sanger sequencing at the Australian Genome Research Facility. This DNA appears in the registry of standard parts as BioBrick <a target="_blank" href="http://parts.igem.org/Part:BBa_K1394000">BBa_K1394000</a>.</p><br />
<p><br />
For the USP peptide and the linear Magainin 1 peptide, the genes were synthesised by GenScript and delivered in pSB1C3. The expression vectors had identical gene regulatory elements to that used for the Magainin 1 Star peptide. They only differed in the codon optimisation used, and they also lacked the assembly scar described above.</p><br />
<br />
<h2><br />
Protein expression and characterisation<br />
</h2><br />
<p><br />
After acquiring our genes, the project moved to protein expression and purification. We expressed both star peptides (Magainin 1 Star Peptide and the USP peptide) in both the SHuffle T7 and BL21(DE3) cell lines, both sourced from New England Biolabs. As the Linear Magainin 1 peptide does not have any special disulfide bonding<br />
requirements, we expressed it in BL21(DE3).<br />
</p><br />
<p><br />
The plasmids were transformed to the expression cells, and a single colony was cultured and induced overnight at 17 °C. A whole cell sample both before<br />
IPTG induction (-IPTG) and after the induction period (+IPTG) were boiled in SDS-PAGE sample buffer and loaded on a 15% tris-glycine gel. The whole cell<br />
Coomassie stained gel is shown below alongside the NEB P7712S molecular weight marker.<br />
</p><br />
<br><br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/2/24/Whole_cell_CS.png" height="330" width="439"/><br />
</p><br />
<br><br />
<p><br />
From theoretical prediction of the peptide masses, we would expect the following distribution of molecular weights:<br />
</p><br />
<br><br />
<table border="1" cellpadding="0" cellspacing="0" align="center"><br />
<tbody><br />
<tr><br />
<td width="156"><br />
<p align="center"><br />
Magainin 1 Star Peptide (Mag1 Star)<br />
</p><br />
</td><br />
<td width="145"><br />
<p align="center"><br />
USP<br />
</p><br />
</td><br />
<td width="156"><br />
<p align="center"><br />
Linear Magainin 1 (Linear Mag1)<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="156"><br />
<p align="center"><br />
22.15 kDa<br />
</p><br />
</td><br />
<td width="145"><br />
<p align="center"><br />
29.3 kDa<br />
</p><br />
</td><br />
<td width="156"><br />
<p align="center"><br />
14.32 kDa<br />
</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<br><br />
<p><br />
We looked for bands corresponding to overexpression of the proteins. We observed a thick band, post IPTG around 32 kDa in the BL21(DE3) cells carrying the<br />
USP plasmid. However, we saw this same band appearing in the Linear Magainin 1 lane but the Linear Magainin 1 protein should have a much lower molecular<br />
weight than what was observed. Therefore, we did not assume that we had overexpression overexpression of the USP 1 protein.<br />
</p><br />
<p><br />
We also noted bands in all post-IPTG lanes around 17 kDa. This would be roughly consistent with both the Linear Magainin 1 and Magainin 1 Star Peptide,<br />
noting that small proteins may not run at their expected molecular weight. Again, the analysis is complicated by the fact that the band also appears in the<br />
lane corresponding to the larger molecular weight protein, USP I. Given this ambiguity, we decided to purify all the protein in the sample using Ni-NTA<br />
purification.<br />
</p><br />
<h3><br />
Purification and further characterisation<br />
</h3><br />
<p><br />
A small-scale purification was carried out according to our <a href="https://static.igem.org/mediawiki/2014/e/e1/MELBOURNE_D1V1_Column_Purification_of_His-Tagged_Proteins.pdf">protocol</a>. The cells were lysed with iodoacetamide, an alkylating agent, added to the lysis buffer. Iodoacetamide blocks all free cysteines on proteins with a short alkyl group. This was added<br />
because we wanted to determine if a disulphide bond had formed inside the cell. If we did not block free cysteines, then any bond that had formed could be<br />
attributed to spontaneous/air oxidation of disulfides outside the cell.<br />
</p><br />
<p><br />
After purification, we ran a concurrent Western Blot and Coomassie stain on both the pre-induction samples from above and the purified protein (namely, the<br />
first elution from the batch purification).<br />
</p><br />
<p><br />
The Western Blot used mouse monoclonal antibodies against the N-terminal HIS-tag as primary antibodies and anti-mouse secondary antibodies:<br />
</p><br />
<br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/5/54/Western_Blot_Melb.png" height="400" width="460" border="0"/><br />
</p><br />
<p><br />
The corresponding Coomassie stain is show below:<br />
</p><br />
<br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f0/Purified_CS.png" height="365" width="486" border="0"/><br />
</p><br />
<p><br />
There are several interesting aspects of the Western Blot. Firstly, there was a strong band between 25 and 32 kDa in most lanes. However, it<br />
appears in all of the pre-induction controls, suggesting non-specific binding of the anti-HIS antibodies.<br />
</p><br />
<p><br />
Secondly, we observed a band in most of the lanes around 17 kDa (with the exception being the USP protein in SHuffle cells). As these bands were not<br />
present in the pre-induction controls, we suspected they were a result of the induction.<br />
</p><br />
<h3><br />
Mass spectroscopy<br />
</h3><br />
<p><br />
In order to assess the identity of the bands, we performed an in-gel tryptic digestion on select bands. We digested bands in the Coomassie stain which<br />
corresponded to the HIS-tagged bands in the Western Blot. This was followed by mass spec analysis (LC MS/MS). We focused our analysis on the Magainin 1<br />
Star Peptide bands and the Linear Magainin 1 band. Our USP peptide was, by design, highly rich in basic residues, greatly reducing the likelihood that<br />
tryptic fragments would be detected by the mass spec.<br />
</p><br />
<p><br />
We found that the Linear Magainin 1 peptide appeared to be present in the approximately 17 kDa band, with the following detected tryptic fragments in the<br />
sequence below (bold; basic residues underlined):<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/1/1b/Tryptic_digest_linear_mag1.PNG" height="63" width="601" border="0"/><br />
</p><br />
<p><br />
Note that spaces have been added in the sequence to emphasise distinct domains in the protein (in this case, the fusion protein versus the Magainin 1<br />
peptide). Together with the fact that the protein runs close to the expected molecular weight, this seems to provide good evidence that the protein is<br />
being expressed.<br />
</p><br />
<p><br />
We then examined the bands corresponding to the Magainin 1 Star Peptide.<br />
</p><br />
<p><br />
Again, we digested bands in the Coomassie stained gel corresponding to the prominent HIS-tagged bands on the <em>Western Blot</em> (near 17 kDa).<br />
</p><br />
<p><br />
The mass spec coverage for the Magainin 1 Star Peptide expressed in SHuffle cells was as follows:<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/6/63/Tryptic_digest_Sample_A_Star_Mag_Shuffle.PNG" height="90" width="601" border="0"/><br />
</p><br />
<p><br />
The coverage for the same peptide expressed in BL21(DE3) was found to be:<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/d/db/Tryptic_digest_Sample_A_Star_Mag_BL21%28DE3%29.PNG" height="85" width="602" border="0"/><br />
</p><br />
<p><br />
Again, we can see that tryptic peptides from the expressed protein appear to present in the gel band under analysis.<br />
</p><br />
<p><br />
There is a relatively lower sequence coverage. This could be a limitation of our procedure: for example, improper destaining during the in gel digestion<br />
procedure can inhibit tryptic digestion.<br />
</p><br />
<p><br />
However, even if the digestion was complete, the mass spec will only detect fragments which ionize well. The Magainin 1 Star peptide consists of four<br />
repeats of the Magainin 1 sequence (corresponding to the arms of the star). If the two tryptic fragments within Magainin 1 do not ionize well, then indeed<br />
the entire peptide would not be read.<br />
</p><br />
<p><br />
Finally, it is possible that the protein has been cleaved or degraded. This would account for the slightly lower-than-expected mass on the SDS page gel.<br />
</p><br />
<p><br />
Nonetheless, it is a possibility that the peptide fragments are present but not detected. Replication of the in-gel mass spec would be useful<br />
in clarifying this point, or provided the purity of the sample is acceptable, it may be possible to run an intact mass spec. Digestion with alternative enzymes may also provide a more robust sequence coverage, particularly for the USP peptide.<br />
</p><br />
<h2><br />
Conclusions<br />
</h2><br />
<p><br />
We designed several novel star peptides based on several rational design criteria. Further, we began the optimisation process required to express these peptides in <em>E. coli</em>.<br />
</p><br />
<p><br />
To this end, we constructed an expression system based on the SUMO protein and standard BioBrick parts. The function of this system was confirmed, as it<br />
appears it does produce HIS-tagged SUMO fusion protein in <em>E. coli</em>, as expected. Nonetheless, we hope to continue to characterise our parts, likely through further purification and measurement of the molecular weight. <br />
</p><br />
<p>Given the success of SUMO in the protein expression community, we hope our BioBrick<br />
will encourage further use of the fusion domain within iGEM. In addition, we hope that our ideas and concepts will inspire future<br />
iGEM efforts to explore the applications of synthetic biology for material science. We continue to believe that bacteria have great promise for the <em>in vivo</em><br />
construction of unique biomaterials, and we look forward to seeing how the synthetic biology community will develop in this area.<br />
</p><br />
<br />
<br />
<br />
<h2>References</h2><br />
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<p><br />
BANEYX, F. 1999. Recombinant protein expression in Escherichia coli. <em>Current Opinion in Biotechnology,</em> 10<strong>,</strong> 411-421.</p><br />
<p><br />
BOMMARIUS, B., JENSSEN, H., ELLIOTT, M., KINDRACHUK, J., PASUPULETI, M., GIEREN, H., JAEGER, K. E., HANCOCK, R. E. W. &amp; KALMAN, D. 2010. Cost-effective expression and purification of antimicrobial and host defense peptides in Escherichia coli. <em>Peptides,</em> 31<strong>,</strong> 1957-1965.</p><br />
<p><br />
BROGDEN, K. A. 2005. Antimicrobial peptides: Pore formers or metabolic inhibitors in bacteria? <em>Nature Reviews Microbiology,</em> 3<strong>,</strong> 238-250.</p><br />
<p><br />
CHANG, C., CHHOR, G., CLANCY, S. & JOACHIMIAK, A. 2014. Crystal Structure of Glutathione S-transferase Domain Protein From Haliangium Ochraceum DSM 14365 [Online]. ''NCBI. Available: http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=4w66' [Accessed September 9 2014].</p><br />
<p><br />
CHEN, X., ZHU, F., CAO, Y. &amp; QIAO, S. 2009. Novel expression vector for secretion of cecropin AD in Bacillus subtilis with enhanced antimicrobial activity. <em>Antimicrobial agents and chemotherapy,</em> 53<strong>,</strong> 3683-3689.</p><br />
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GLINEL, K., JONAS, A. M., JOUENNE, T., LEPRINCE, J., GALAS, L. & HUCK, W. T. 2008. Antibacterial and antifouling polymer brushes incorporating antimicrobial peptide. <em>Bioconjug Chem,</em> 20<strong>,</strong> 71-77.</p><br />
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HUMBLOT, V., YALA, J.-F., THEBAULT, P., BOUKERMA, K., HÉQUET, A., BERJEAUD, J.-M. & PRADIER, C.-M. 2009. The antibacterial activity of Magainin I immobilized onto mixed thiols self-assembled monolayers. <em>Biomaterials,</em> 30<strong>,</strong> 3503-3512.</p><br />
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DAWSON, P. E., MUIR, T. W., CLARK-LEWIS, I. &amp; KENT, S. 1994. Synthesis of proteins by native chemical ligation. <em>Science,</em> 266<strong>,</strong> 776-779.</p><br />
<p><br />
KADOKURA, H. &amp; BECKWITH, J. 2009. Detecting folding intermediates of a protein as it passes through the bacterial translocation channel. <em>Cell,</em> 138<strong>,</strong> 1164-1173.</p><br />
<p><br />
KADOKURA, H., KATZEN, F. &amp; BECKWITH, J. 2003. Protein disulfide bond formation in prokaryotes. <em>Annual review of biochemistry,</em> 72<strong>,</strong> 111-135.</p><br />
<p><br />
LI, Y. 2011. Recombinant production of antimicrobial peptides in&lt; i&gt; Escherichia coli&lt;/i&gt;: A review. <em>Protein Expression and Purification,</em> 80<strong>,</strong> 260-267.</p><br />
<p><br />
LOBSTEIN, J., EMRICH, C. A., JEANS, C., FAULKNER, M., RIGGS, P. &amp; BERKMEN, M. 2012. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. <em>Microb Cell Fact,</em> 11<strong>,</strong> 56-56.</p><br />
<p><br />
PROTEIN MODEL PORTAL. Query result, GST and DcmA [Online]. Available: http://www.proteinmodelportal.org/query/uniprot/P21161 [Accessed September 9 2014].</p><br />
<p><br />
STEINER, D., FORRER, P., STUMPP, M. T. &amp; PLUCKTHUN, A. 2006. Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display. <em>Nat Biotech,</em> 24<strong>,</strong> 823-831.</p><br />
<p><br />
SULISTIO, A., GURR, P. A., BLENCOWE, A. &amp; QIAO, G. G. 2012. Peptide-Based Star Polymers: The Rising Star in Functional Polymers. <em>Australian Journal of Chemistry,</em> 65<strong>,</strong> 978-984.</p><br />
<p><br />
SULISTIO, A., LOWENTHAL, J., BLENCOWE, A., BONGIOVANNI, M. N., ONG, L., GRAS, S. L., ZHANG, X. &amp; QIAO, G. G. 2011. Folic Acid Conjugated Amino Acid-Based Star Polymers for Active Targeting of Cancer Cells. <em>Biomacromolecules,</em> 12<strong>,</strong> 3469-3477.</p><br />
<p><br />
TANAKA, N., KUSAKABE, Y., ITO, K., YOSHIMOTO, T. & NAKAMURA, K. T. 2002. Crystal Structure of Formaldehyde Dehydrogenase from< i> Pseudomonas putida</i>: the Structural Origin of the Tightly Bound Cofactor in Nicotinoprotein Dehydrogenases. Journal of molecular biology, 324, 519-533.</p><br />
<p><br />
WIRADHARMA, N., LIU, S. Q. &amp; YANG, Y. Y. 2012. Branched and 4-Arm Starlike α-Helical Peptide Structures with Enhanced Antimicrobial Potency and Selectivity. <em>Small,</em> 8<strong>,</strong> 362-366.</p><br />
<p><br />
YU, Z., WANG, Q., MA, Q. &amp; ZHANG, R. 2013. Secretory expression of lacticin Q fused with SUMO in Bacillus subtilis. <em>Protein Expression and Purification,</em> 89<strong>,</strong> 51-55.</p><br />
<p><br />
ZASLOFF, M. 1987. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. <em>Proceedings of the National Academy of Sciences,</em> 84<strong>,</strong> 5449-5453.</p><br />
<p>&nbsp;</p><br />
<br />
<A NAME="Oxford"></A><br />
<h1>Collaboration with Oxford</h1><br />
<p>To strengthen the sense of iGEM community, we contacted <a target="_blank" href="https://2014.igem.org/Team:Oxford">University of Oxford iGEM team</a> to discuss the possibility of collaboration between our teams. The Oxford project aims to develop a system that can dispose of the carcinogenic, hazardous solvent dichloromethane (DCM). To do this, Oxford team has proposed the use of the DcmA enzyme. This enzyme degrades DCM to form a toxic intermediate which in turn is converted by another enzyme, FdhA, into a neutral molecule. Schematically this can be presented in the following way:</p><br />
<br />
<p><br />
Reaction 1: DCM +&nbsp;DcmA <strong>toxic intermediate</strong><br><br />
Reaction 2: <strong>toxic intermediate</strong> + FdhA neutral product.</p><br />
<p>The problem for their system lies in bringing the two enzymes together to ensure efficient reaction kinetics. An idea that we discussed with Oxford was to use the star peptide platform to link the two enzymes together, in a structure similar to the following: </p><br />
<img src="https://static.igem.org/mediawiki/2014/2/2e/Melbourne_Collab_1.png" width="500" height="325" alt=""/><br />
<p>We worked with Oxford to study this system from a theoretical standpoint. Our team studied the 3D structure of both enzymes involved to confirm that the enzyme could in theory be attached to a linker in this manner, while Oxford team did stochastic modelling to determine how reaction rate changes when the linker length, labeled D on the diagram, changes. </p><br />
<p><br />
As a first check, it was necessary to determine whether anchoring these enzymes to our star would be sterically possible. We studied the structures of FdhA and DcmA determined by crystallography using data from the protein data bank. As no crystallographic data was available in the databank for DcmA, GST was examined as a proxy, as GST is structurally homologous to DcmA. </p><br />
<p><br />
The crystalographically resolved structure of FdhA enzyme (Tanaka et al., 2002) from the protein data bank revealed the following image:</p><br />
<img src="https://static.igem.org/mediawiki/2014/d/dc/Melbourne_Collab_2.png" width="700" height="355" alt=""/><br />
<p>The crystalographically resolved structure of GST was as follows (Chang et al., 2014):</p><br />
<img src="https://static.igem.org/mediawiki/2014/e/e9/Melbourne_Collab_3.png" width="800" height="361" alt=""/><br />
<p>It can be seen that the amino- and carboxy-terminals are located away from the active site. This suggests that both enzymes, DcmA and FdhA, might be linked together via linkers that are used in our star peptide. That is, the active site will not be sterically hindered by attachment to the star peptide. That said, it is difficult to predict how anchoring the protein to the star</div>Slowehttp://2014.igem.org/Team:Melbourne/ProjectTeam:Melbourne/Project2014-10-18T02:54:51Z<p>Slowe: </p>
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<h1>Project and results</h1><br />
<br />
<p>Read about our experimental work below, or jump to our theoretical collaboration with the <A HREF="#Oxford">University of Oxford</A><br />
<br />
<h2>Introduction and theory</h2><br />
<p>Synthetic peptide chemists have long produced peptide-based materials <em>in vitro</em>. Star-shaped peptides are a promising type of biomaterial being explored in the field of nanomedicine (Sulistio et al., 2012). Star peptides can have several biomedical uses, such as acting as drug delivery vehicles (Sulistio et al., 2011) or linkers for other biomacromolecules. Star peptides generally take the form of several linear peptide arms linked together in a central core. One way of linking these linear peptide arms together is to used covalent bonds such as those in disulfides. Typically, disulfide bonds are formed synthetically by taking several linear arms and treating them with an oxidant <em>in vitro</em>. Here, we introduce a new approach to forming star peptides using <em>E. coli</em> and synthetic biology. Thus, we aimed to show how the peptides synthesis and disulfide bond forming machinery of <em>E. coli</em> can be used to form disulfide linked star peptides and key star peptide precursors.</p><br />
<p>&nbsp;</p><br />
<h2>Synthesis approach</h2><br />
<p><em>E. coli</em> naturally possesses the capacity to form disulfide bonds. In native strains, disulfide bonds are naturally formed by an array of enzymes which are part of the Dsb family (e.g. DsbA and DsbC) (Kadokura et al., 2003, Kadokura and Beckwith, 2009). Normally, these enzymes are found in the oxidizing periplasm of the cell. However, several new strains of <em>E. coli</em> have recently been engineered which contain an oxidizing cytoplasm conducive to disulfide bond formation. One example of this is the SHuffle cell line (Lobstein et al., 2012). The cell line contains mutations to key enzymes responsible for the reducing nature of the cytoplasm, namely thioredoxin reductase (<em>trxB</em>) and glutathione reductase (<em>gor</em>). Furthermore, the Shuffle cell line over expresses the disulfide bond isomerase DsbC to the cytoplasm. Together, these mutations allow SHuffle to fold disulfide-bonded proteins in the cytoplasm at a higher success rate compared to non-mutants. We aimed to take advantage of the disulfide bond forming capabilities of this strain of <em>E. coli</em> to synthesize star peptides in cells. As shown in the figure below, the synthesis steps may proceed as follows:</p><br />
<p>&nbsp;</p><br />
<ol><br />
<li> Express a short peptide containing two cysteine residues either to the <em>E. coli</em> periplasm or the cytoplasm of a <em>trxB gor </em>mutant.</li><br />
<li><em>E. coli</em> disulfide bond forming enzymes fold the peptide into a hairpin loop structure.<br />
</li><br />
<li>Cut the loop at a protease recognition site engineered into the peptide. This may be done by extracting the folded peptide from the cell and treating it with the protease in vitro. Alternatively, the protease may be co-expressed in the cell to allow for in vivo cleavage.<br />
</li><br />
</ol><br />
<p>&nbsp;</p><br />
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<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9d/Project_concept-v2.png" width="480" height="600" alt=""/></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<p>This synthesis approach has several benefits over purely <em>in vitro</em> approaches. Firstly, the exact peptide sequence can be precisely programmed into <em>E. coli</em> using recombinant DNA synthesis. Secondly, by performing the disulfide bond formation in cells and optionally the proteolytic cleavage, several synthesis steps which would need to be performed <em>in vitro</em> are eliminated. From a scale up perspective, this would eliminate entire unit operations which would otherwise be required to produce this product. Given these benefits, in the current study, we aimed to express a star peptide precursor to the cytoplasm of SHuffle cells, which would later be extracted and externally digested with the star-forming protease. In order to achieve this, we first designed several star peptides which might be amenable to this synthetic strategy. These peptides are described below.</p><br />
<p>&nbsp;</p><br />
<h2>Rationally designed peptides</h2><br />
<p>There are two approaches to functionalising star peptides. In the first, the identity of the arms can be chosen to be bioactive peptide molecules. This is the simplest approach to producing a biologically-relevant star. In the second, the arms can be functionalised by ligating molecules to them at any point. We used both of these approaches to design two separate peptides.</p><br />
<h3>Magainin 1 star and linear peptides</h3><br />
<p>Our first strategy was to make a star peptide using antimicrobial peptides(AMPs) as building blocks. AMPs are small, approximately 50 residue peptides secreted by some bacterial and eukaryotic cells which selectively kill microbial cells. It is thought that AMPs work by forming pores in the membrane of prokaryotic cells (Brogden, 2005). AMPs have been recombinantly expressed in a number of organisms, including <em>E. coli</em> (for a review, see Li, 2011) and <em>B. Subtilis</em> (Chen et al., 2009, Yu et al., 2013).</p><br />
<p>Our concept was to design a star peptide with antimicrobial peptide arms. Wiradharma et al. (2012) first showed that placing linear antimicrobial peptides in a star configuration could lead to enhanced antimicrobial activity and decreased hemolytic activity. Although it is unclear why this is the case, it may be due to the ability of neighbouring antimicrobial peptide arms to interact with each other to synergistically rupture the membrane.<br><br />
While Wiradharma used a synthetic peptide sequence, we designed a peptide using the naturally occurring AMP, Magainin 1 (Zasloff, 1987). Magainin 1 peptides will be placed to the ends of each star arm.</p><br />
<br />
<p>Magainin 1 was chosen because the Magainins are one of the major classes of antimicrobial peptides, being well studied and characterised. In addition, we were concerned that tethering the antimicrobial peptide to the star peptide might interfere with its antimicrobial activity. Magainin 1, however, has previously been tethered to surfaces, where it has imparted the surfaces with microbicidal properties (Glinel et al., 2008; Humblot et al., 2009). We surmised that if Magainin 1 maintained its activity while anchored to surfaces, it may also maintain its activity while anchored to a star peptide.</p><br />
<p>The sequence for Magainin 1 is: GIGKFLHSAGKFGKAFVGEIMKS.</p><br />
<p>When attached to a star peptide it will have the following structure:</p><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/MAGAININ_1_peptide.png" width="720" height="300" alt=""/><br />
<p>There are several design elements to note:</p><br />
<ul><br />
<li><br />
The antimicrobial peptide star will be expressed with a SUMO fusion protein. This is because without the fusion, it is likely that the peptide would be toxic to the host cell.</li><br />
<li>The peptide includes a Factor X cutting site between the two cysteines for eventual proteolytic cleavage and formation of the star peptide.</li><br />
</ul><br />
<p>In addition to the star peptide Magainin 1, we synthesised a gene for a linear Magainin 1 peptide as well. This is identical to the construct above, except that there is only one Magainin 1 peptide attached to the SUMO fusion.</p><br />
<br />
<h3>Unstructured Peptide (USP) Construct</h3><br />
<p>In the second approach, we designed a peptide which can be functionalised using chemical approaches. This peptide was designed to have flexible, unstructured arms and was termed the USP construct. Unlike the Magainin 1 star, the arms of this peptide serve not as active peptides themselves, but as inert structural linkers.</p><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/USPI_Peptide.png" width="720" height="216" alt=""/><br />
<p>The arms were designed with the following elements in mind:</p><br />
<ul><br />
<li><br />
<b>Lack of structure.</b> The arms were designed with a bioinspired approach, using the FxFG motif of nucleoporins (where x is a variable amino acid residue). Such segments naturally repeat in nucleoporins and are thought to lead to disorder/lack of stable secondary structure. Nucleoporins are found in mammalian cells, serving as flexible brushes around nuclear pores (Ader et al., 2010).<br />
</li><br />
<li><b>Water-soluble.</b> The arms were designed with several charged amino acids to improve solubility.<br />
</li><br />
<li><b>Designed to form a disulfide bond.</b> Although it is difficult to rationally ensure that the disulfide bond will form between two cysteines in our peptide, we incorporated a beta turn between the two cysteines which may encourage the peptide to fold at the apex of the hairpin loop. This may bring the cysteines into closer proximity, providing more probable bond formation.</li></ul><br />
<br />
<p>The ultimate utility of this peptide lies in its ability to be functionalised with other biomacromolecules. For example, the technique of Native Chemical Ligation(NCL) can be used to join peptides, proteins, and other ligands to the arms (Dawson et al., 1994). The idea of attaching enzymes to the star peptide was explored by the University of Oxford iGEM 2014 team in a collaborative effort between our two teams (See Supplementary Project Work at the end of this page). </p><br />
<br />
<p>&nbsp;</p><br />
<h2>Expression system</h2><br />
<p>In order to successfully express our constructs, we designed our protein expression vectors to include a fusion protein. The fusion protein was necessary for two reasons. First, some of our constructs are very small (e.g. the non-star Magainin 1), and expression levels of very small peptides can be difficult without a fusion partner. Second, two of our constructs code for antimicrobial peptides. Without a fusion partner, it is likely that these genes would be toxic to their hosts upon induction.</p><br />
<p>To find a suitable expression system, we looked towards the Registry of Standard Parts. We used the SUMO protein expression system designed by TU Delft 2014 (for example, see<a target="_blank" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1022101">http://parts.igem.org/wiki/index.php?title=Part:BBa_K1022101</a>). This system essentially consists of a N-terminal HIS-tag followed by the SUMO protein (also known as UlpI).</p><br />
<p><br />
The strategy of expressing toxic AMPs using SUMO has been successfully reported in the literature (Bommarius et al., 2010). We surmise that the SUMO protein could inhibit the antimicrobial activity of single, linear peptides, and that it may also inhibit the activity of our star antimicrobial peptide.</p><br />
<p><br />
SUMO as a fusion protein also has the benefit of leaving no residues at the C-terminal end of the cleavage site. This means that upon cleavage with the SUMO protease, the native protein can be recovered. In our case, this means that one of the arms of the star can be designed without the need to take into account the addition of any amino acid residues left behind by the protease.<br><br />
We used the SUMO peptide sequence reported by TU Delft. However, our construct contained the following unique features:</p><br />
<ol><br />
<li> Standardisation of the biobrick by substituting the T7 promoter and RBS (the origins of which are both not specified in the Delft documentation) with the standard T7 promoter and RBS BioBrick<a target="_blank" href="http://parts.igem.org/Part:BBa_K525998">BBa_K525998</a>. In addition to supporting the principle of standardization, using the well-characterized promoter BioBrick should help assure expression levels.<br><br />
</li><br />
<li>Biobrick BBa_K1022101 lacks a terminator sequence (this was presumably because the part was meant to be integrated into a larger genetic construct with a terminator). A terminator from the registry of standard parts was added (specifically, the wild type terminator from T7 bacteriophage,<a target="_blank" href="http://parts.igem.org/Part:BBa_K731721">BBa_K731721</a>).<br />
</li><br />
<li>The original biobrick BBa_K1022101 codes for three amino acid residues before the HIS-tag (ASM), which appeared to be redundant. Correspondence with the 2013 TU Delft team suggested that these residues were unnecessary and appear to be cleaved within the cell as part of the cells post-translational modifications. However, their presence complicates the addition of additional tags at the N-terminus of the protein (e.g. periplasmic export tags), and therefore were not included.<br><br />
</li><br />
<li>The SUMO sequence was codon optimised for E. coli during synthesis. As the Delft documentation did not specify whether the gene was codon optimal, codon optomisation was undertaken to potentially improve expression levels. The linear Magainin 1 construct, however, was not codon optimised in the SUMO region in order to provide a control condition.</li><br />
</ol><br />
<p>To summarise, the protein expression devices used in our project to the following form:</p><br />
<img src="https://static.igem.org/mediawiki/2014/d/d0/Gene_circuit_export.png" width="720" height="91" alt=""/></td><br />
<p>The protein coding region consisted of a 6x-HIS tag followed by the SUMO protein and the relevant star or linear peptide.</p><br />
<p>&nbsp;</p><br />
<h2>Plasmid preparation: Cloning and acquisition of the genetic constructs</h2><br />
<h3>Magainin 1 Star Peptide</h3><br />
<p>This construct was synthesised in the standard shipping plasmid from Life Technologies- plasmid pMK. We had originally planned to express our protein to the periplasm of E. coli, and therefore had included in the synthesis a periplasmic export tag, the TorTss signal sequence (Steiner et al., 2006).</p><br />
<p><br />
Cytoplasmic and periplasmic expression may both allow for disulfide bond formation in the cell. We decided, however, to focus on cytoplasmic expression. There are distinct advantages to cytoplasmic expression (e.g. the absence of several periplasmic proteases and potentially higher expression levels (Baneyx, 1999)).</p><br />
<p><br />
Another reason the cytoplasm was chosen was to allow us to test the effects of an oxidising versus reducing intracellular environment on disulfide bond formation. We planned to express the construct in both SHuffle T7 cells (oxidising cytoplasm) and BL21(DE3) (reducing cytoplasm) to probe whether there was a difference in disulfide bond formation. Therefore, we needed to remove the periplasmic export tag from the gene.</p><br />
<p><br />
To do this, we use the following cloning strategy (noting that the top row represents the gene initially synthesised in plasmid pMK):</p><br />
<img src="https://static.igem.org/mediawiki/2014/2/2c/Melbourne_cloning_strategy_diagram.jpg" width="900" height="437" alt=""/><br />
<p>The gene was inserted into plasmid pSB1C3 containing the T7 promoter and ribosome binding site, BBa_K525998. It was inserted after the promoter and before the BioBrick suffix. This was accomplished by digesting the destination vector with SpeI and PstI. At the same time, PCR was used to amplify the segment of the gene containing the SUMO fusion and the Magainin 1 Star peptide, adding XbaI and keeping the PstI site existing in the gene.</p><br />
<p><br />
Digestion of the PCR product then allowed for ligation of the insert and destination vector using the PstI sites and XbaI and SpeI (XbaI and SpeI have compatible sticky ends). Note that after the ligation, there will be a scar in the gene where the XbaI and the SpeI sites were ligated.</p><br />
<p><br />
The ligation appeared to be successful. The ligation mixture was transformed to DH5α competent cells and plated onto chloramphenicol-containing agar plates.</p><br />
<p><br />
Plasmid from 5 colonies (C1, C2, C3, C4, and C5) was cultured, extracted, and then digested with EcoRI and PstI.</p><br />
<p><br />
As evident in the figure below, at least 4 of the colonies appeared to contain the insert. The empty, linearised pSB1C3 backbone ran at approximately the correct size (2070 bps), as did the insert (740 bps).</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/9/95/Magainin_1_subcloning.png" width="353" height="400" alt=""/></center><br />
<p>N.B. MW marker is the 100 bp Ladder from Axygen. * indicates the colony picked for sequencing and eventual transformation to the expression cell lines.</p><br />
<p>The DNA from Colony C2 was confirmed using Sanger sequencing at the Australian Genome Research Facility. This DNA appears in the registry of standard parts as BioBrick <a target="_blank" href="http://parts.igem.org/Part:BBa_K1394000">BBa_K1394000</a>.</p><br />
<p><br />
For the USP peptide and the linear Magainin 1 peptide, the genes were synthesised by GenScript and delivered in pSB1C3. The expression vectors had identical gene regulatory elements to that used for the Magainin 1 Star peptide. They only differed in the codon optimisation used, and they also lacked the assembly scar described above.</p><br />
<br />
<h2><br />
Protein expression and characterisation<br />
</h2><br />
<p><br />
After acquiring our genes, the project moved to protein expression and purification. We expressed both star peptides (Magainin 1 Star Peptide and the USP peptide) in both the SHuffle T7 and BL21(DE3) cell lines, both sourced from New England Biolabs. As the Linear Magainin 1 peptide does not have any special disulfide bonding<br />
requirements, we expressed it in BL21(DE3).<br />
</p><br />
<p><br />
The plasmids were transformed to the expression cells, and a single colony was cultured and induced overnight at 17 °C. A whole cell sample both before<br />
IPTG induction (-IPTG) and after the induction period (+IPTG) were boiled in SDS-PAGE sample buffer and loaded on a 15% tris-glycine gel. The whole cell<br />
Coomassie stained gel is shown below alongside the NEB P7712S molecular weight marker.<br />
</p><br />
<br><br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/2/24/Whole_cell_CS.png" height="330" width="439"/><br />
</p><br />
<br><br />
<p><br />
From theoretical prediction of the peptide masses, we would expect the following distribution of molecular weights:<br />
</p><br />
<br><br />
<table border="1" cellpadding="0" cellspacing="0" align="center"><br />
<tbody><br />
<tr><br />
<td width="156"><br />
<p align="center"><br />
Magainin 1 Star Peptide (Mag1 Star)<br />
</p><br />
</td><br />
<td width="145"><br />
<p align="center"><br />
USP<br />
</p><br />
</td><br />
<td width="156"><br />
<p align="center"><br />
Linear Magainin 1 (Linear Mag1)<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="156"><br />
<p align="center"><br />
22.15 kDa<br />
</p><br />
</td><br />
<td width="145"><br />
<p align="center"><br />
29.3 kDa<br />
</p><br />
</td><br />
<td width="156"><br />
<p align="center"><br />
14.32 kDa<br />
</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<br><br />
<p><br />
We looked for bands corresponding to overexpression of the proteins. We observed a thick band, post IPTG around 32 kDa in the BL21(DE3) cells carrying the<br />
USP plasmid. However, we saw this same band appearing in the Linear Magainin 1 lane but the Linear Magainin 1 protein should have a much lower molecular<br />
weight than what was observed. Therefore, we did not assume that we had overexpression overexpression of the USP 1 protein.<br />
</p><br />
<p><br />
We also noted bands in all post-IPTG lanes around 17 kDa. This would be roughly consistent with both the Linear Magainin 1 and Magainin 1 Star Peptide,<br />
noting that small proteins may not run at their expected molecular weight. Again, the analysis is complicated by the fact that the band also appears in the<br />
lane corresponding to the larger molecular weight protein, USP I. Given this ambiguity, we decided to purify all the protein in the sample using Ni-NTA<br />
purification.<br />
</p><br />
<h3><br />
Purification and further characterisation<br />
</h3><br />
<p><br />
A small-scale purification was carried out according to our <a href="https://static.igem.org/mediawiki/2014/e/e1/MELBOURNE_D1V1_Column_Purification_of_His-Tagged_Proteins.pdf">protocol</a>. The cells were lysed with iodoacetamide, an alkylating agent, added to the lysis buffer. Iodoacetamide blocks all free cysteines on proteins with a short alkyl group. This was added<br />
because we wanted to determine if a disulphide bond had formed inside the cell. If we did not block free cysteines, then any bond that had formed could be<br />
attributed to spontaneous/air oxidation of disulfides outside the cell.<br />
</p><br />
<p><br />
After purification, we ran a concurrent Western Blot and Coomassie stain on both the pre-induction samples from above and the purified protein (namely, the<br />
first elution from the batch purification).<br />
</p><br />
<p><br />
The Western Blot used mouse monoclonal antibodies against the N-terminal HIS-tag as primary antibodies and anti-mouse secondary antibodies:<br />
</p><br />
<br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/5/54/Western_Blot_Melb.png" height="400" width="460" border="0"/><br />
</p><br />
<p><br />
The corresponding Coomassie stain is show below:<br />
</p><br />
<br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f0/Purified_CS.png" height="365" width="486" border="0"/><br />
</p><br />
<p><br />
There are several interesting aspects of the Western Blot. Firstly, there was a strong band between 25 and 32 kDa in most lanes. However, it<br />
appears in all of the pre-induction controls, suggesting non-specific binding of the anti-HIS antibodies.<br />
</p><br />
<p><br />
Secondly, we observed a band in most of the lanes around 17 kDa (with the exception being the USP protein in SHuffle cells). As these bands were not<br />
present in the pre-induction controls, we suspected they were a result of the induction.<br />
</p><br />
<h3><br />
Mass spectroscopy<br />
</h3><br />
<p><br />
In order to assess the identity of the bands, we performed an in-gel tryptic digestion on select bands. We digested bands in the Coomassie stain which<br />
corresponded to the HIS-tagged bands in the Western Blot. This was followed by mass spec analysis (LC MS/MS). We focused our analysis on the Magainin 1<br />
Star Peptide bands and the Linear Magainin 1 band. Our USP peptide was, by design, highly rich in basic residues, greatly reducing the likelihood that<br />
tryptic fragments would be detected by the mass spec.<br />
</p><br />
<p><br />
We found that the Linear Magainin 1 peptide appeared to be present in the approximately 17 kDa band, with the following detected tryptic fragments in the<br />
sequence below (bold; basic residues underlined):<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/1/1b/Tryptic_digest_linear_mag1.PNG" height="63" width="601" border="0"/><br />
</p><br />
<p><br />
Note that spaces have been added in the sequence to emphasise distinct domains in the protein (in this case, the fusion protein versus the Magainin 1<br />
peptide). Together with the fact that the protein runs close to the expected molecular weight, this seems to provide good evidence that the protein is<br />
being expressed.<br />
</p><br />
<p><br />
We then examined the bands corresponding to the Magainin 1 Star Peptide.<br />
</p><br />
<p><br />
Again, we digested bands in the Coomassie stained gel corresponding to the prominent HIS-tagged bands on the <em>Western Blot</em> (near 17 kDa).<br />
</p><br />
<p><br />
The mass spec coverage for the Magainin 1 Star Peptide expressed in SHuffle cells was as follows:<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/6/63/Tryptic_digest_Sample_A_Star_Mag_Shuffle.PNG" height="90" width="601" border="0"/><br />
</p><br />
<p><br />
The coverage for the same peptide expressed in BL21(DE3) was found to be:<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/d/db/Tryptic_digest_Sample_A_Star_Mag_BL21%28DE3%29.PNG" height="85" width="602" border="0"/><br />
</p><br />
<p><br />
Again, we can see that tryptic peptides from the expressed protein appear to present in the gel band under analysis.<br />
</p><br />
<p><br />
There is a relatively lower sequence coverage. This could be a limitation of our procedure: for example, improper destaining during the in gel digestion<br />
procedure can inhibit tryptic digestion.<br />
</p><br />
<p><br />
However, even if the digestion was complete, the mass spec will only detect fragments which ionize well. The Magainin 1 Star peptide consists of four<br />
repeats of the Magainin 1 sequence (corresponding to the arms of the star). If the two tryptic fragments within Magainin 1 do not ionize well, then indeed<br />
the entire peptide would not be read.<br />
</p><br />
<p><br />
Finally, it is possible that the protein has been cleaved or degraded. This would account for the slightly lower-than-expected mass on the SDS page gel.<br />
</p><br />
<p><br />
Nonetheless, there is the distinct possibility that the peptide fragments are there but not detected. Replication of the in-gel mass spec would be useful<br />
in clarifying this point, or provided the purity of the sample is acceptable, it may be possible to run an intact mass spec.<br />
</p><br />
<h2><br />
Conclusions<br />
</h2><br />
<p><br />
We designed several novel star peptides based on several rational design criteria. Further, we began the optimisation process required to express these peptides in <em>E. coli</em>.<br />
</p><br />
<p><br />
To this end, we constructed an expression system based on the SUMO protein and standard BioBrick parts. The function of this system was confirmed, as it<br />
appears it does produce HIS-tagged SUMO fusion protein in <em>E. coli</em>, as expected.<br />
</p><br />
<p><br />
Although it appears the expression system works, there was some uncertainty about particular peptides, namely the Magainin 1 Star Peptide and USP Peptide.<br />
A Westerm Blot suggested that at least part of the peptides are present. However, additional sequence coverage in a mass spec analysis and intact mass spec<br />
would help us determine the precise identity of our products.<br />
</p><br />
<p><br />
We have shown that the SUMO fusion can be expressed in our BioBrick. Given the success of SUMO in the general scientific community, we hope our BioBrick<br />
will encourage further use of the fusion domain within the iGEM community. In addition, we hope that our ideas and concepts will inspire future<br />
iGEM efforts to explore the applications of synthetic biology for material science. We continue to believe that bacteria have great promise for the <em>in vivo</em><br />
construction of unique biomaterials, and we look forward to seeing how the synthetic biology community will develop in this area.<br />
</p><br />
<br />
<br />
<br />
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<p><br />
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SULISTIO, A., GURR, P. A., BLENCOWE, A. &amp; QIAO, G. G. 2012. Peptide-Based Star Polymers: The Rising Star in Functional Polymers. <em>Australian Journal of Chemistry,</em> 65<strong>,</strong> 978-984.</p><br />
<p><br />
SULISTIO, A., LOWENTHAL, J., BLENCOWE, A., BONGIOVANNI, M. N., ONG, L., GRAS, S. L., ZHANG, X. &amp; QIAO, G. G. 2011. Folic Acid Conjugated Amino Acid-Based Star Polymers for Active Targeting of Cancer Cells. <em>Biomacromolecules,</em> 12<strong>,</strong> 3469-3477.</p><br />
<p><br />
TANAKA, N., KUSAKABE, Y., ITO, K., YOSHIMOTO, T. & NAKAMURA, K. T. 2002. Crystal Structure of Formaldehyde Dehydrogenase from< i> Pseudomonas putida</i>: the Structural Origin of the Tightly Bound Cofactor in Nicotinoprotein Dehydrogenases. Journal of molecular biology, 324, 519-533.</p><br />
<p><br />
WIRADHARMA, N., LIU, S. Q. &amp; YANG, Y. Y. 2012. Branched and 4-Arm Starlike α-Helical Peptide Structures with Enhanced Antimicrobial Potency and Selectivity. <em>Small,</em> 8<strong>,</strong> 362-366.</p><br />
<p><br />
YU, Z., WANG, Q., MA, Q. &amp; ZHANG, R. 2013. Secretory expression of lacticin Q fused with SUMO in Bacillus subtilis. <em>Protein Expression and Purification,</em> 89<strong>,</strong> 51-55.</p><br />
<p><br />
ZASLOFF, M. 1987. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. <em>Proceedings of the National Academy of Sciences,</em> 84<strong>,</strong> 5449-5453.</p><br />
<p>&nbsp;</p><br />
<br />
<A NAME="Oxford"></A><br />
<h1>Collaboration with Oxford</h1><br />
<p>To strengthen the sense of iGEM community, we contacted <a target="_blank" href="https://2014.igem.org/Team:Oxford">University of Oxford iGEM team</a> to discuss the possibility of collaboration between our teams. The Oxford project aims to develop a system that can dispose of the carcinogenic, hazardous solvent dichloromethane (DCM). To do this, Oxford team has proposed the use of the DcmA enzyme. This enzyme degrades DCM to form a toxic intermediate which in turn is converted by another enzyme, FdhA, into a neutral molecule. Schematically this can be presented in the following way:</p><br />
<br />
<p><br />
Reaction 1: DCM +&nbsp;DcmA <strong>toxic intermediate</strong><br><br />
Reaction 2: <strong>toxic intermediate</strong> + FdhA neutral product.</p><br />
<p>The problem for their system lies in bringing the two enzymes together to ensure efficient reaction kinetics. An idea that we discussed with Oxford was to use the star peptide platform to link the two enzymes together, in a structure similar to the following: </p><br />
<img src="https://static.igem.org/mediawiki/2014/2/2e/Melbourne_Collab_1.png" width="500" height="325" alt=""/><br />
<p>We worked with Oxford to study this system from a theoretical standpoint. Our team studied the 3D structure of both enzymes involved to confirm that the enzyme could in theory be attached to a linker in this manner, while Oxford team did stochastic modelling to determine how reaction rate changes when the linker length, labeled D on the diagram, changes. </p><br />
<p><br />
As a first check, it was necessary to determine whether anchoring these enzymes to our star would be sterically possible. We studied the structures of FdhA and DcmA determined by crystallography using data from the protein data bank. As no crystallographic data was available in the databank for DcmA, GST was examined as a proxy, as GST is structurally homologous to DcmA. </p><br />
<p><br />
The crystalographically resolved structure of FdhA enzyme (Tanaka et al., 2002) from the protein data bank revealed the following image:</p><br />
<img src="https://static.igem.org/mediawiki/2014/d/dc/Melbourne_Collab_2.png" width="700" height="355" alt=""/><br />
<p>The crystalographically resolved structure of GST was as follows (Chang et al., 2014):</p><br />
<img src="https://static.igem.org/mediawiki/2014/e/e9/Melbourne_Collab_3.png" width="800" height="361" alt=""/><br />
<p>It can be seen that the amino- and carboxy-terminals are located away from the active site. This suggests that both enzymes, DcmA and FdhA, might be linked together via linkers that are used in our star peptide. That is, the active site will not be sterically hindered by attachment to the star peptide. That said, it is difficult to predict how anchoring the protein to the star</div>Slowehttp://2014.igem.org/Team:Melbourne/Human_PracticesTeam:Melbourne/Human Practices2014-10-18T02:40:37Z<p>Slowe: </p>
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<br />
<h1>Public Outreach</h1><br />
<h2>&lsquo;Synthetic biology for kids and adults and everyone in between&rsquo; </h2><br />
<p>The University of Melbourne took up the challenge of teaching synthetic biology to all ages, embracing a &lsquo;science for everyone&rsquo; approach. In so doing, we engaged in education outreach activities with kindergarten students from three childcare centres as well as both university and high school students from across Australia.</p><br />
<p>&nbsp; </p><br />
<h3>For kindergarten students </h3><br />
<table width="100%"><br />
<tr><br />
<td width="195" ><img src="https://static.igem.org/mediawiki/2014/4/4b/Melbourne_Adventure_of_E_coli.jpg" width="176" height="264" alt="Adventure of E coli"/></td><br />
<td width="594"><p>We decided to write the first in a series of children&rsquo;s books for pre-schoolers aged 4-6 themed in the spirit of iGEM entitled &lsquo;The Adventures of E. coli&rsquo; for a number of reasons:</p><br />
<ul><br />
<li> Children aged 4-6 are in a key learning stage, during which their interest in science can significantly influence their long-term science education outcomes (Bowman, Donovan, &amp; Burns, 2001, pp. 8-9). </li><br />
<li>There are lots of resources about science for older kids but not so many for kids at such a young age. </li><br />
<li>Picture books provide a springboard for further classroom activities </li><br />
</ul><br />
<p>With the assistance of IndieMosh (Publishers link is here: <A href="http://www.indiemosh.com.au/author-42-iGEM-Melbourne-childrens-biology-science" rel="nofollow">http://www.indiemosh.com.au/author-42-iGEM-Melbourne-childrens-biology-science</A>) – a self-publishing facilitator – we managed to get our book in print and on Amazon, please see <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/</A></p><br />
<p>After we successfully published our book, it was time to take it to the audience. We approached childcare centres across Melbourne and negotiated for our book to be read to pre-schoolers and for our team members to come along and teach the kids about science.</p></td><br />
</tr><br />
</table><p>The book reading was well received and was followed by playing some science-related games, and having a chat about science in groups. Please check out the video we made about the day: </p><br />
<br />
<iframe class="centervid" width="560" height="315" src="//www.youtube.com/embed/RNV_lPKcrSc" frameborder="0" allowfullscreen></iframe><br />
<br />
<br />
<p>We believe that through this book, young children can learn about basic concepts and the language of biology, forming a scientific foundation to be built on in future years. Moreover, it is our hope that this book will inspire children to continue to learn about science and to appreciate all the wonders that it holds. </p><br />
<p>The Kindle book is available from here: <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon-ebook/dp/B00OJ691DI/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon-ebook/dp/B00OJ691DI/</A></p><br />
<p>Paperback available on Amazon here: <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/</A></p><br />
<p>Fixed layout epub (for iPad and Android tablets, phones etc) available on Smashwords here: <A href="http://www.smashwords.com/books/view/484305" rel="nofollow">http://www.smashwords.com/books/view/484305</A></p><br />
<h3>&nbsp;</h3><br />
<h3>For high school students </h3><br />
<p>In an effort to broaden the accessibility of the iGEM competition to those who appeared to be currently underrepresented in the competition (e.g. disadvantaged students, females and Australian high school students), we approached a private high school in Victoria with the following aim: </p><br />
<p>To find a well-resourced, private high school to partner with an iGEM team (e.g. UoM) to field a team consisting of half private high school students/half underrepresented students – who would have their flights to Boston funded by fundraising done primarily by the private high school. </p><br />
<p>After extensive email &amp; phone correspondence spanning several months, the school decided it was not currently in the position to go ahead with the iGEM team proposal due to upcoming infrastructure developments, though would be interested in fielding a team after these are completed (scheduled for 2016). Nonetheless, the school did encourage us that other APS schools would likely be interested in hearing about iGEM and our team concept. </p><br />
<p>Whilst we didn&rsquo;t achieve our ultimate goal, this activity did raise awareness of the iGEM competition with all the key science staff members at the school which requested not to be named on this wiki (for further details please contact our team leader Sean). </p><br />
<p>Finally, we would encourage all Australian teams to work on the following goals: </p><br />
<ul><br />
<li>improve iGEM's accessibility to those of disadvantaged backgrounds </li><br />
<li>increase female representation in science &amp; iGEM </li><br />
<li>increase the participation of Australian/international high school students in the iGEM competition. </li><br />
</ul><br />
<p><BR><br />
In addition to our negotiations with the private high school, we also aimed at widening the participation of high school students in the iGEM competition by collaborating with the University of Sydney on The Strange Nature Writing competition. </p><br />
<p>We spruiked the competition not only on our project flyers which described the iGEM competition, our project and the Strange Nature Writing Competition, but also through our development of a promotional video designed to be shown in high school assemblies as a way of promoting both iGEM and the Strange Nature Writing competition. Please see the video below: </p><br />
<br />
<iframe class="centervid" width="560" height="315" src="//www.youtube.com/embed/ZuSoO3HVMjo" frameborder="0" allowfullscreen></iframe><br />
<br />
<p>Furthermore, we wrote and published articles on Comet.is which is aimed at high school students, university students and recent university graduates, please see 'For university students' for a list of the articles we wrote. </p><br />
<h3>&nbsp;</h3><br />
<h3>For university students </h3><br />
<p>Our main form of outreach toward university students was in writing and publishing articles on a burgeoning and government sponsored website Comet.is which is aimed at high school students, university students and recent graduates. Our articles covered a range of topics from our iGEM experience to considerations of ethical issues of synthetic biology and the potential of synthetic biology. </p><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/d/d5/Comet.jpg" width="640" height="400" alt="Adventure of E coli"/><br />
<br />
<br />
</p> <strong>Here are some excerpts:</strong> </p><br />
<br />
<blockquote></p> "All this leads me to refer to synthetic biology as a ménage à trois of a discipline, utilizing engineering, biology and computer science to produce a fantastically creative activity." </p></blockquote><br />
<br />
</p> "Given assistance in the form of grants or human resources, entrepreneurial forums along with student competitions like InnovationACT and iGEM have the chance to inspire entrepreneurialism in students. It is in these opportunities for students to go on entrepreneurial adventures, replete with late nights, red bulls and a shot at winning a grand prize – that the next generation of entrepreneurs is born." </p><br />
<br />
</p> "A prime example of this is in the International Genetically Engineered Machine (iGEM) competition, which I am a team member of for the University of Melbourne this year. Our team stretches across many disciplines; from telecommunications and business to chemical engineering and science communication, even information technology - we are practically the UN of fields and backgrounds." </p><br />
<br />
</p> "Participating in IGEM can definitely be categorized as having an experience and not just joining a team. The competition brings together all the components of completing a project for undergraduate students to be part of, a little like a trial run of a Masters or Doctorate course." </p><br />
<br />
<p>Here are the links to our articles: </p><br />
<p><A href="https://comet.is/article/2960" rel="nofollow">https://comet.is/article/2960</A> <A href="https://comet.is/article/3499/" rel="nofollow">https://comet.is/article/3499/</A> <A href="https://comet.is/article/3381/" rel="nofollow">https://comet.is/article/3381/</A> <A href="https://comet.is/article/3561/" rel="nofollow">https://comet.is/article/3561/</A> <A href="https://comet.is/article/3562/" rel="nofollow">https://comet.is/article/3562/</A> <A href="https://comet.is/article/3694/" rel="nofollow">https://comet.is/article/3694/</A> <A href="http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/" rel="nofollow">http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/</A> <A href="http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/" rel="nofollow">http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/</A></p><br />
<p>Furthermore, as part of our outreach directed at university students, we combined our recruitment campaign for an IT whiz to help with our wiki, with a presentation on what iGEM was all about. We lecture bashed over several weeks, presenting over 11 times. </p><br />
<br />
<h3><BR><br />
</h3><br />
<h3>For general public </h3><br />
<p>After promoting the iGEM competition to the Victorian State Chair of National Science Week (NSW) - Nick Besley, our team decided to field a stall in &lsquo;Market of the Mind&rsquo; – a NSW event aimed at the general public located in a high foot traffic area alongside the Yarra River in Southbank across the river from Flinders St Station. Luckily, our stall was located next to the bar, which meant there was a lot of foot traffic directed to our happy team members wearing our iGEM team t-shirts. Using a true/false quiz, armed with Ferrero Rochers as rewards for getting all the answers correct, we had over 30 quiz contestants engage with us on the first night and 35+ contestants on the second day. Please see the accompanying video made by the City of Melbourne, which shows our stall/team members from 0:45 </p><br />
<br />
<p><iframe class="centervid" width="560" height="315" src="//www.youtube.com/embed/ahFI3aFBdNc" frameborder="0" allowfullscreen></iframe><p><br />
<br />
<p><BR><br />
<A href="http://www.scienceweek.net.au/market-of-the-mind/" rel="nofollow">http://www.scienceweek.net.au/market-of-the-mind/</A> <A href="http://re-science.org.au/science-event/market-mind-2649" rel="nofollow">http://re-science.org.au/science-event/market-mind-2649</A></p></html><br />
<br />
<br />
<h1>Intellectual Property </h1><br />
<br />
<p>'''Our team considered the question of ‘how should we proceed if we want to retain IP rights on our project’?''' </p><br />
<br />
<p> Before attempting to answer this pertinent IP question, our team leader decided to recruit two more team members who had some intellectual property experience to complement the learning on patent law that a few early team members had acquired in their biotechnology undergraduate degrees. Both the new team members had previously filed provisional patents; one had filed some in Russia and the other in Australia, offering two perspectives on this question of IP. </p><br />
<br />
<p> In answering this question, we approached our universities’ tech transfer office to gain clarity over our team’s discussions and brainstorm sessions on whether our project was patentable. </p><br />
<br />
<p> The tech transfer office advised us that our project may have commercial applications, however it was probably too soon to act on the idea and convert it into a provisional patent, so we decided to hold off and wait till we had a more substantiated grasp on our commercial applications. We were also advised that if at any point we wanted to file for IP protection on the project, it would be really important to not have disclosed the details of our idea to anyone else, as this could result in us losing the right to our IP. </p><br />
<br />
<p> After doing some research on the various law firms who could offer us a provisional patent, we discovered the estimated cost would be in the vicinity of $3500-$6500 depending on the level of detail we wanted to cover e.g. the number and extent of the claims, the full description of the idea, diagrams and experimental protocols as well as the filing & service fees associated. </p><br />
<br />
<p> Due to the relatively high estimates we received, we looked into other options for filing a provisional, including recruiting a law student to join the team and contribute their knowledge to our IP protection. This avenue did not eventuate as our ideas on what we would like to patent continuously evolved over the course of the project and we could not reach a sufficient degree of clarity to bring on a lawyer for the filing of a provisional patent. </p><br />
<br />
<p> Nonetheless, we proceeded with caution, ensuring to follow the advice of the University of Melbourne’s tech transfer office and not disclose our ideas without ensuring our disclosure was protected. An instance when we had to implement this protection was during our scientific collaboration with Oxford, were we ensured Non Disclosure Agreements were signed prior to the disclosure of our project to them. They indicated their interest in using our project as a case study in their ‘iGEM intellectual property guide’, however we felt that this might put our IP rights in jeopardy, so we decided not to proceed as a case study. </p><br />
<br />
<p> At this time, our team is looking to produce at least one provisional patent following the iGEM 2014 competition on at least one of the various techniques we designed during the project. </p><br />
<br />
<br />
<p> '''Our team consulted:''' </p><br />
<p> University of Melbourne Technology Transfer Office </p><br />
<p> University of Melbourne Technology Commercialisation Team </p><br />
<p> UoM Commercial </p><br />
<p> Stanislauv Yatskevich (iGEM team member with Russian patent experience) </p><br />
<p> Peter Collins (iGEM team member with Australian patent experience) </p><br />
<br />
<br />
<p> '''References:''' </p><br />
<br />
<p> http://www.ipaustralia.gov.au/.../factsheet-experimental.../ </p><br />
<br />
<p>http://www.google.com.au/url?sa=t&rct=j&q=&esrc=s...</p></div>Slowehttp://2014.igem.org/Team:Melbourne/Human_PracticesTeam:Melbourne/Human Practices2014-10-18T02:38:49Z<p>Slowe: </p>
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Notebook</a></td><br />
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<a href="https://2014.igem.org/Team:Melbourne/Protocols"style=" color:#000000"> <br />
Protocols</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Safety"style="color:#000000"><br />
Safety</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Sponsors"style="color:#000000"><br />
Sponsors</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Attributions"style="color:#000000"><br />
Attributions</a></td><br />
<br />
<br />
<td align="right"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="46px"></a> </td><br />
</tr><br />
</table><br />
<br />
<br />
<h1>Public Outreach</h1><br />
<h2>&lsquo;Synthetic biology for kids and adults and everyone in between&rsquo; </h2><br />
<p>The University of Melbourne took up the challenge of teaching synthetic biology to all ages, embracing a &lsquo;science for everyone&rsquo; approach. In so doing, we engaged in education outreach activities with kindergarten students from three childcare centres as well as both university and high school students from across Australia.</p><br />
<p>&nbsp; </p><br />
<h3>For kindergarten students </h3><br />
<table width="100%"><br />
<tr><br />
<td width="195" ><img src="https://static.igem.org/mediawiki/2014/4/4b/Melbourne_Adventure_of_E_coli.jpg" width="176" height="264" alt="Adventure of E coli"/></td><br />
<td width="594"><p>We decided to write the first in a series of children&rsquo;s books for pre-schoolers aged 4-6 themed in the spirit of iGEM entitled &lsquo;The Adventures of E. coli&rsquo; for a number of reasons:</p><br />
<ul><br />
<li> Children aged 4-6 are in a key learning stage, during which their interest in science can significantly influence their long-term science education outcomes (Bowman, Donovan, &amp; Burns, 2001, pp. 8-9). </li><br />
<li>There are lots of resources about science for older kids but not so many for kids at such a young age. </li><br />
<li>Picture books provide a springboard for further classroom activities </li><br />
</ul><br />
<p>With the assistance of IndieMosh (Publishers link is here: <A href="http://www.indiemosh.com.au/author-42-iGEM-Melbourne-childrens-biology-science" rel="nofollow">http://www.indiemosh.com.au/author-42-iGEM-Melbourne-childrens-biology-science</A>) – a self-publishing facilitator – we managed to get our book in print and on Amazon, please see <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/</A></p><br />
<p>After we successfully published our book, it was time to take it to the audience. We approached childcare centres across Melbourne and negotiated for our book to be read to pre-schoolers and for our team members to come along and teach the kids about science.</p></td><br />
</tr><br />
</table><p>The book reading was well received and was followed by playing some science-related games, and having a chat about science in groups. Please check out the video we made about the day: </p><br />
<br />
<iframe class="centervid" width="560" height="315" src="//www.youtube.com/embed/RNV_lPKcrSc" frameborder="0" allowfullscreen></iframe><br />
<br />
<br />
<p>We believe that through this book, young children can learn about basic concepts and the language of biology, forming a scientific foundation to be built on in future years. Moreover, it is our hope that this book will inspire children to continue to learn about science and to appreciate all the wonders that it holds. </p><br />
<p>The Kindle book is available from here: <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon-ebook/dp/B00OJ691DI/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon-ebook/dp/B00OJ691DI/</A></p><br />
<p>Paperback available on Amazon here: <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/</A></p><br />
<p>Fixed layout epub (for iPad and Android tablets, phones etc) available on Smashwords here: <A href="http://www.smashwords.com/books/view/484305" rel="nofollow">http://www.smashwords.com/books/view/484305</A></p><br />
<h3>&nbsp;</h3><br />
<h3>For high school students </h3><br />
<p>In an effort to broaden the accessibility of the iGEM competition to those who appeared to be currently underrepresented in the competition (e.g. disadvantaged students, females and Australian high school students), we approached a private high school in Victoria with the following aim: </p><br />
<p>To find a well-resourced, private high school to partner with an iGEM team (e.g. UoM) to field a team consisting of half private high school students/half underrepresented students – who would have their flights to Boston funded by fundraising done primarily by the private high school. </p><br />
<p>After extensive email &amp; phone correspondence spanning several months, the school decided it was not currently in the position to go ahead with the iGEM team proposal due to upcoming infrastructure developments, though would be interested in fielding a team after these are completed (scheduled for 2016). Nonetheless, the school did encourage us that other APS schools would likely be interested in hearing about iGEM and our team concept. </p><br />
<p>Whilst we didn&rsquo;t achieve our ultimate goal, this activity did raise awareness of the iGEM competition with all the key science staff members at the school which requested not to be named on this wiki (for further details please contact our team leader Sean). </p><br />
<p>Finally, we would encourage all Australian teams to work on the following goals: </p><br />
<ul><br />
<li>improve iGEM's accessibility to those of disadvantaged backgrounds </li><br />
<li>increase female representation in science &amp; iGEM </li><br />
<li>increase the participation of Australian/international high school students in the iGEM competition. </li><br />
</ul><br />
<p><BR><br />
In addition to our negotiations with the private high school, we also aimed at widening the participation of high school students in the iGEM competition by collaborating with the University of Sydney on The Strange Nature Writing competition. </p><br />
<p>We spruiked the competition not only on our project flyers which described the iGEM competition, our project and the Strange Nature Writing Competition, but also through our development of a promotional video designed to be shown in high school assemblies as a way of promoting both iGEM and the Strange Nature Writing competition. Please see the video below: </p><br />
<br />
<iframe class="centervid" width="560" height="315" src="//www.youtube.com/embed/ZuSoO3HVMjo" frameborder="0" allowfullscreen></iframe><br />
<br />
<p>Furthermore, we wrote and published articles on Comet.is which is aimed at high school students, university students and recent university graduates, please see 'For university students' for a list of the articles we wrote. </p><br />
<h3>&nbsp;</h3><br />
<h3>For university students </h3><br />
<p>Our main form of outreach toward university students was in writing and publishing articles on a burgeoning and government sponsored website Comet.is which is aimed at high school students, university students and recent graduates. Our articles covered a range of topics from our iGEM experience to considerations of ethical issues of synthetic biology and the potential of synthetic biology. </p><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/d/d5/Comet.jpg" width="640" height="400" alt="Adventure of E coli"/><br />
<br />
<br />
</p> <strong>Here are some excerpts:</strong> </p><br />
<br />
</p> "All this leads me to refer to synthetic biology as a ménage à trois of a discipline, utilizing engineering, biology and computer science to produce a fantastically creative activity." </p><br />
<br />
</p> "Given assistance in the form of grants or human resources, entrepreneurial forums along with student competitions like InnovationACT and iGEM have the chance to inspire entrepreneurialism in students. It is in these opportunities for students to go on entrepreneurial adventures, replete with late nights, red bulls and a shot at winning a grand prize – that the next generation of entrepreneurs is born." </p><br />
<br />
</p> "A prime example of this is in the International Genetically Engineered Machine (iGEM) competition, which I am a team member of for the University of Melbourne this year. Our team stretches across many disciplines; from telecommunications and business to chemical engineering and science communication, even information technology - we are practically the UN of fields and backgrounds." </p><br />
<br />
</p> "Participating in IGEM can definitely be categorized as having an experience and not just joining a team. The competition brings together all the components of completing a project for undergraduate students to be part of, a little like a trial run of a Masters or Doctorate course." </p><br />
<br />
<p>Here are the links to our articles: </p><br />
<p><A href="https://comet.is/article/2960" rel="nofollow">https://comet.is/article/2960</A> <A href="https://comet.is/article/3499/" rel="nofollow">https://comet.is/article/3499/</A> <A href="https://comet.is/article/3381/" rel="nofollow">https://comet.is/article/3381/</A> <A href="https://comet.is/article/3561/" rel="nofollow">https://comet.is/article/3561/</A> <A href="https://comet.is/article/3562/" rel="nofollow">https://comet.is/article/3562/</A> <A href="https://comet.is/article/3694/" rel="nofollow">https://comet.is/article/3694/</A> <A href="http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/" rel="nofollow">http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/</A> <A href="http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/" rel="nofollow">http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/</A></p><br />
<p>Furthermore, as part of our outreach directed at university students, we combined our recruitment campaign for an IT whiz to help with our wiki, with a presentation on what iGEM was all about. We lecture bashed over several weeks, presenting over 11 times. </p><br />
<br />
<h3><BR><br />
</h3><br />
<h3>For general public </h3><br />
<p>After promoting the iGEM competition to the Victorian State Chair of National Science Week (NSW) - Nick Besley, our team decided to field a stall in &lsquo;Market of the Mind&rsquo; – a NSW event aimed at the general public located in a high foot traffic area alongside the Yarra River in Southbank across the river from Flinders St Station. Luckily, our stall was located next to the bar, which meant there was a lot of foot traffic directed to our happy team members wearing our iGEM team t-shirts. Using a true/false quiz, armed with Ferrero Rochers as rewards for getting all the answers correct, we had over 30 quiz contestants engage with us on the first night and 35+ contestants on the second day. Please see the accompanying video made by the City of Melbourne, which shows our stall/team members from 0:45 </p><br />
<br />
<p><iframe class="centervid" width="560" height="315" src="//www.youtube.com/embed/ahFI3aFBdNc" frameborder="0" allowfullscreen></iframe><p><br />
<br />
<p><BR><br />
<A href="http://www.scienceweek.net.au/market-of-the-mind/" rel="nofollow">http://www.scienceweek.net.au/market-of-the-mind/</A> <A href="http://re-science.org.au/science-event/market-mind-2649" rel="nofollow">http://re-science.org.au/science-event/market-mind-2649</A></p></html><br />
<br />
<br />
<h1>Intellectual Property </h1><br />
<br />
<p>'''Our team considered the question of ‘how should we proceed if we want to retain IP rights on our project’?''' </p><br />
<br />
<p> Before attempting to answer this pertinent IP question, our team leader decided to recruit two more team members who had some intellectual property experience to complement the learning on patent law that a few early team members had acquired in their biotechnology undergraduate degrees. Both the new team members had previously filed provisional patents; one had filed some in Russia and the other in Australia, offering two perspectives on this question of IP. </p><br />
<br />
<p> In answering this question, we approached our universities’ tech transfer office to gain clarity over our team’s discussions and brainstorm sessions on whether our project was patentable. </p><br />
<br />
<p> The tech transfer office advised us that our project may have commercial applications, however it was probably too soon to act on the idea and convert it into a provisional patent, so we decided to hold off and wait till we had a more substantiated grasp on our commercial applications. We were also advised that if at any point we wanted to file for IP protection on the project, it would be really important to not have disclosed the details of our idea to anyone else, as this could result in us losing the right to our IP. </p><br />
<br />
<p> After doing some research on the various law firms who could offer us a provisional patent, we discovered the estimated cost would be in the vicinity of $3500-$6500 depending on the level of detail we wanted to cover e.g. the number and extent of the claims, the full description of the idea, diagrams and experimental protocols as well as the filing & service fees associated. </p><br />
<br />
<p> Due to the relatively high estimates we received, we looked into other options for filing a provisional, including recruiting a law student to join the team and contribute their knowledge to our IP protection. This avenue did not eventuate as our ideas on what we would like to patent continuously evolved over the course of the project and we could not reach a sufficient degree of clarity to bring on a lawyer for the filing of a provisional patent. </p><br />
<br />
<p> Nonetheless, we proceeded with caution, ensuring to follow the advice of the University of Melbourne’s tech transfer office and not disclose our ideas without ensuring our disclosure was protected. An instance when we had to implement this protection was during our scientific collaboration with Oxford, were we ensured Non Disclosure Agreements were signed prior to the disclosure of our project to them. They indicated their interest in using our project as a case study in their ‘iGEM intellectual property guide’, however we felt that this might put our IP rights in jeopardy, so we decided not to proceed as a case study. </p><br />
<br />
<p> At this time, our team is looking to produce at least one provisional patent following the iGEM 2014 competition on at least one of the various techniques we designed during the project. </p><br />
<br />
<br />
<p> '''Our team consulted:''' </p><br />
<p> University of Melbourne Technology Transfer Office </p><br />
<p> University of Melbourne Technology Commercialisation Team </p><br />
<p> UoM Commercial </p><br />
<p> Stanislauv Yatskevich (iGEM team member with Russian patent experience) </p><br />
<p> Peter Collins (iGEM team member with Australian patent experience) </p><br />
<br />
<br />
<p> '''References:''' </p><br />
<br />
<p> http://www.ipaustralia.gov.au/.../factsheet-experimental.../ </p><br />
<br />
<p>http://www.google.com.au/url?sa=t&rct=j&q=&esrc=s...</p></div>Slowehttp://2014.igem.org/Team:Melbourne/Human_PracticesTeam:Melbourne/Human Practices2014-10-18T02:38:00Z<p>Slowe: </p>
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Project</a> </td><br />
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Protocols</a></td><br />
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Sponsors</a></td><br />
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<br />
<h1>Public Outreach</h1><br />
<h2>&lsquo;Synthetic biology for kids and adults and everyone in between&rsquo; </h2><br />
<p>The University of Melbourne took up the challenge of teaching synthetic biology to all ages, embracing a &lsquo;science for everyone&rsquo; approach. In so doing, we engaged in education outreach activities with kindergarten students from three childcare centres as well as both university and high school students from across Australia.</p><br />
<p>&nbsp; </p><br />
<h3>For kindergarten students </h3><br />
<table width="100%"><br />
<tr><br />
<td width="195" ><img src="https://static.igem.org/mediawiki/2014/4/4b/Melbourne_Adventure_of_E_coli.jpg" width="176" height="264" alt="Adventure of E coli"/></td><br />
<td width="594"><p>We decided to write the first in a series of children&rsquo;s books for pre-schoolers aged 4-6 themed in the spirit of iGEM entitled &lsquo;The Adventures of E. coli&rsquo; for a number of reasons:</p><br />
<ul><br />
<li> Children aged 4-6 are in a key learning stage, during which their interest in science can significantly influence their long-term science education outcomes (Bowman, Donovan, &amp; Burns, 2001, pp. 8-9). </li><br />
<li>There are lots of resources about science for older kids but not so many for kids at such a young age. </li><br />
<li>Picture books provide a springboard for further classroom activities </li><br />
</ul><br />
<p>With the assistance of IndieMosh (Publishers link is here: <A href="http://www.indiemosh.com.au/author-42-iGEM-Melbourne-childrens-biology-science" rel="nofollow">http://www.indiemosh.com.au/author-42-iGEM-Melbourne-childrens-biology-science</A>) – a self-publishing facilitator – we managed to get our book in print and on Amazon, please see <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/</A></p><br />
<p>After we successfully published our book, it was time to take it to the audience. We approached childcare centres across Melbourne and negotiated for our book to be read to pre-schoolers and for our team members to come along and teach the kids about science.</p></td><br />
</tr><br />
</table><p>The book reading was well received and was followed by playing some science-related games, and having a chat about science in groups. Please check out the video we made about the day: </p><br />
<br />
<iframe class="centervid" width="560" height="315" src="//www.youtube.com/embed/RNV_lPKcrSc" frameborder="0" allowfullscreen></iframe><br />
<br />
<br />
<p>We believe that through this book, young children can learn about basic concepts and the language of biology, forming a scientific foundation to be built on in future years. Moreover, it is our hope that this book will inspire children to continue to learn about science and to appreciate all the wonders that it holds. </p><br />
<p>The Kindle book is available from here: <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon-ebook/dp/B00OJ691DI/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon-ebook/dp/B00OJ691DI/</A></p><br />
<p>Paperback available on Amazon here: <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/</A></p><br />
<p>Fixed layout epub (for iPad and Android tablets, phones etc) available on Smashwords here: <A href="http://www.smashwords.com/books/view/484305" rel="nofollow">http://www.smashwords.com/books/view/484305</A></p><br />
<h3>&nbsp;</h3><br />
<h3>For high school students </h3><br />
<p>In an effort to broaden the accessibility of the iGEM competition to those who appeared to be currently underrepresented in the competition (e.g. disadvantaged students, females and Australian high school students), we approached a private high school in Victoria with the following aim: </p><br />
<p>To find a well-resourced, private high school to partner with an iGEM team (e.g. UoM) to field a team consisting of half private high school students/half underrepresented students – who would have their flights to Boston funded by fundraising done primarily by the private high school. </p><br />
<p>After extensive email &amp; phone correspondence spanning several months, the school decided it was not currently in the position to go ahead with the iGEM team proposal due to upcoming infrastructure developments, though would be interested in fielding a team after these are completed (scheduled for 2016). Nonetheless, the school did encourage us that other APS schools would likely be interested in hearing about iGEM and our team concept. </p><br />
<p>Whilst we didn&rsquo;t achieve our ultimate goal, this activity did raise awareness of the iGEM competition with all the key science staff members at the school which requested not to be named on this wiki (for further details please contact our team leader Sean). </p><br />
<p>Finally, we would encourage all Australian teams to work on the following goals: </p><br />
<ul><br />
<li>improve iGEM's accessibility to those of disadvantaged backgrounds </li><br />
<li>increase female representation in science &amp; iGEM </li><br />
<li>increase the participation of Australian/international high school students in the iGEM competition. </li><br />
</ul><br />
<p><BR><br />
In addition to our negotiations with the private high school, we also aimed at widening the participation of high school students in the iGEM competition by collaborating with the University of Sydney on The Strange Nature Writing competition. </p><br />
<p>We spruiked the competition not only on our project flyers which described the iGEM competition, our project and the Strange Nature Writing Competition, but also through our development of a promotional video designed to be shown in high school assemblies as a way of promoting both iGEM and the Strange Nature Writing competition. Please see the video below: </p><br />
<br />
<iframe class="centervid" width="560" height="315" src="//www.youtube.com/embed/ZuSoO3HVMjo" frameborder="0" allowfullscreen></iframe><br />
<br />
<p>Furthermore, we wrote and published articles on Comet.is which is aimed at high school students, university students and recent university graduates, please see 'For university students' for a list of the articles we wrote. </p><br />
<h3>&nbsp;</h3><br />
<h3>For university students </h3><br />
<p>Our main form of outreach toward university students was in writing and publishing articles on a burgeoning and government sponsored website Comet.is which is aimed at high school students, university students and recent graduates. Our articles covered a range of topics from our iGEM experience to considerations of ethical issues of synthetic biology and the potential of synthetic biology. </p><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/d/d5/Comet.jpg" width="640" height="400" alt="Adventure of E coli"/><br />
<br />
<br />
</p> Here are some excerpts: </p><br />
<br />
</p> "All this leads me to refer to synthetic biology as a ménage à trois of a discipline, utilizing engineering, biology and computer science to produce a fantastically creative activity." </p><br />
<br />
</p> "Given assistance in the form of grants or human resources, entrepreneurial forums along with student competitions like InnovationACT and iGEM have the chance to inspire entrepreneurialism in students. It is in these opportunities for students to go on entrepreneurial adventures, replete with late nights, red bulls and a shot at winning a grand prize – that the next generation of entrepreneurs is born." </p><br />
<br />
</p> "A prime example of this is in the International Genetically Engineered Machine (iGEM) competition, which I am a team member of for the University of Melbourne this year. Our team stretches across many disciplines; from telecommunications and business to chemical engineering and science communication, even information technology - we are practically the UN of fields and backgrounds." </p><br />
<br />
</p> "Participating in IGEM can definitely be categorized as having an experience and not just joining a team. The competition brings together all the components of completing a project for undergraduate students to be part of, a little like a trial run of a Masters or Doctorate course." </p><br />
<br />
<p>Here are the links to our articles: </p><br />
<p><A href="https://comet.is/article/2960" rel="nofollow">https://comet.is/article/2960</A> <A href="https://comet.is/article/3499/" rel="nofollow">https://comet.is/article/3499/</A> <A href="https://comet.is/article/3381/" rel="nofollow">https://comet.is/article/3381/</A> <A href="https://comet.is/article/3561/" rel="nofollow">https://comet.is/article/3561/</A> <A href="https://comet.is/article/3562/" rel="nofollow">https://comet.is/article/3562/</A> <A href="https://comet.is/article/3694/" rel="nofollow">https://comet.is/article/3694/</A> <A href="http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/" rel="nofollow">http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/</A> <A href="http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/" rel="nofollow">http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/</A></p><br />
<p>Furthermore, as part of our outreach directed at university students, we combined our recruitment campaign for an IT whiz to help with our wiki, with a presentation on what iGEM was all about. We lecture bashed over several weeks, presenting over 11 times. </p><br />
<br />
<h3><BR><br />
</h3><br />
<h3>For general public </h3><br />
<p>After promoting the iGEM competition to the Victorian State Chair of National Science Week (NSW) - Nick Besley, our team decided to field a stall in &lsquo;Market of the Mind&rsquo; – a NSW event aimed at the general public located in a high foot traffic area alongside the Yarra River in Southbank across the river from Flinders St Station. Luckily, our stall was located next to the bar, which meant there was a lot of foot traffic directed to our happy team members wearing our iGEM team t-shirts. Using a true/false quiz, armed with Ferrero Rochers as rewards for getting all the answers correct, we had over 30 quiz contestants engage with us on the first night and 35+ contestants on the second day. Please see the accompanying video made by the City of Melbourne, which shows our stall/team members from 0:45 </p><br />
<br />
<p><iframe class="centervid" width="560" height="315" src="//www.youtube.com/embed/ahFI3aFBdNc" frameborder="0" allowfullscreen></iframe><p><br />
<br />
<p><BR><br />
<A href="http://www.scienceweek.net.au/market-of-the-mind/" rel="nofollow">http://www.scienceweek.net.au/market-of-the-mind/</A> <A href="http://re-science.org.au/science-event/market-mind-2649" rel="nofollow">http://re-science.org.au/science-event/market-mind-2649</A></p></html><br />
<br />
<br />
<h1>Intellectual Property </h1><br />
<br />
<p>'''Our team considered the question of ‘how should we proceed if we want to retain IP rights on our project’?''' </p><br />
<br />
<p> Before attempting to answer this pertinent IP question, our team leader decided to recruit two more team members who had some intellectual property experience to complement the learning on patent law that a few early team members had acquired in their biotechnology undergraduate degrees. Both the new team members had previously filed provisional patents; one had filed some in Russia and the other in Australia, offering two perspectives on this question of IP. </p><br />
<br />
<p> In answering this question, we approached our universities’ tech transfer office to gain clarity over our team’s discussions and brainstorm sessions on whether our project was patentable. </p><br />
<br />
<p> The tech transfer office advised us that our project may have commercial applications, however it was probably too soon to act on the idea and convert it into a provisional patent, so we decided to hold off and wait till we had a more substantiated grasp on our commercial applications. We were also advised that if at any point we wanted to file for IP protection on the project, it would be really important to not have disclosed the details of our idea to anyone else, as this could result in us losing the right to our IP. </p><br />
<br />
<p> After doing some research on the various law firms who could offer us a provisional patent, we discovered the estimated cost would be in the vicinity of $3500-$6500 depending on the level of detail we wanted to cover e.g. the number and extent of the claims, the full description of the idea, diagrams and experimental protocols as well as the filing & service fees associated. </p><br />
<br />
<p> Due to the relatively high estimates we received, we looked into other options for filing a provisional, including recruiting a law student to join the team and contribute their knowledge to our IP protection. This avenue did not eventuate as our ideas on what we would like to patent continuously evolved over the course of the project and we could not reach a sufficient degree of clarity to bring on a lawyer for the filing of a provisional patent. </p><br />
<br />
<p> Nonetheless, we proceeded with caution, ensuring to follow the advice of the University of Melbourne’s tech transfer office and not disclose our ideas without ensuring our disclosure was protected. An instance when we had to implement this protection was during our scientific collaboration with Oxford, were we ensured Non Disclosure Agreements were signed prior to the disclosure of our project to them. They indicated their interest in using our project as a case study in their ‘iGEM intellectual property guide’, however we felt that this might put our IP rights in jeopardy, so we decided not to proceed as a case study. </p><br />
<br />
<p> At this time, our team is looking to produce at least one provisional patent following the iGEM 2014 competition on at least one of the various techniques we designed during the project. </p><br />
<br />
<br />
<p> '''Our team consulted:''' </p><br />
<p> University of Melbourne Technology Transfer Office </p><br />
<p> University of Melbourne Technology Commercialisation Team </p><br />
<p> UoM Commercial </p><br />
<p> Stanislauv Yatskevich (iGEM team member with Russian patent experience) </p><br />
<p> Peter Collins (iGEM team member with Australian patent experience) </p><br />
<br />
<br />
<p> '''References:''' </p><br />
<br />
<p> http://www.ipaustralia.gov.au/.../factsheet-experimental.../ </p><br />
<br />
<p>http://www.google.com.au/url?sa=t&rct=j&q=&esrc=s...</p></div>Slowehttp://2014.igem.org/Team:Melbourne/Human_PracticesTeam:Melbourne/Human Practices2014-10-18T02:35:03Z<p>Slowe: </p>
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Home</a> </td><br />
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Team</a> </td><br />
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Project</a> </td><br />
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Human Practices</a></td><br />
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Achievements</a></td><br />
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Notebook</a></td><br />
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Protocols</a></td><br />
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Safety</a></td><br />
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<a href="https://2014.igem.org/Team:Melbourne/Sponsors"style="color:#000000"><br />
Sponsors</a></td><br />
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Attributions</a></td><br />
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<td align="right"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="46px"></a> </td><br />
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<br />
<br />
<h1>Public Outreach</h1><br />
<h2>&lsquo;Synthetic biology for kids and adults and everyone in between&rsquo; </h2><br />
<p>The University of Melbourne took up the challenge of teaching synthetic biology to all ages, embracing a &lsquo;science for everyone&rsquo; approach. In so doing, we engaged in education outreach activities with kindergarten students from three childcare centres as well as both university and high school students from across Australia.</p><br />
<p>&nbsp; </p><br />
<h3>For kindergarten students </h3><br />
<table width="100%"><br />
<tr><br />
<td width="195" ><img src="https://static.igem.org/mediawiki/2014/4/4b/Melbourne_Adventure_of_E_coli.jpg" width="176" height="264" alt="Adventure of E coli"/></td><br />
<td width="594"><p>We decided to write the first in a series of children&rsquo;s books for pre-schoolers aged 4-6 themed in the spirit of iGEM entitled &lsquo;The Adventures of E. coli&rsquo; for a number of reasons:</p><br />
<ul><br />
<li> Children aged 4-6 are in a key learning stage, during which their interest in science can significantly influence their long-term science education outcomes (Bowman, Donovan, &amp; Burns, 2001, pp. 8-9). </li><br />
<li>There are lots of resources about science for older kids but not so many for kids at such a young age. </li><br />
<li>Picture books provide a springboard for further classroom activities </li><br />
</ul><br />
<p>With the assistance of IndieMosh (Publishers link is here: <A href="http://www.indiemosh.com.au/author-42-iGEM-Melbourne-childrens-biology-science" rel="nofollow">http://www.indiemosh.com.au/author-42-iGEM-Melbourne-childrens-biology-science</A>) – a self-publishing facilitator – we managed to get our book in print and on Amazon, please see <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/</A></p><br />
<p>After we successfully published our book, it was time to take it to the audience. We approached childcare centres across Melbourne and negotiated for our book to be read to pre-schoolers and for our team members to come along and teach the kids about science.</p></td><br />
</tr><br />
</table><p>The book reading was well received and was followed by playing some science-related games, and having a chat about science in groups. Please check out the video we made about the day: </p><br />
<br />
<iframe class="centervid" width="560" height="315" src="//www.youtube.com/embed/RNV_lPKcrSc" frameborder="0" allowfullscreen></iframe><br />
<br />
<br />
<p>We believe that through this book, young children can learn about basic concepts and the language of biology, forming a scientific foundation to be built on in future years. Moreover, it is our hope that this book will inspire children to continue to learn about science and to appreciate all the wonders that it holds. </p><br />
<p>The Kindle book is available from here: <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon-ebook/dp/B00OJ691DI/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon-ebook/dp/B00OJ691DI/</A></p><br />
<p>Paperback available on Amazon here: <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/</A></p><br />
<p>Fixed layout epub (for iPad and Android tablets, phones etc) available on Smashwords here: <A href="http://www.smashwords.com/books/view/484305" rel="nofollow">http://www.smashwords.com/books/view/484305</A></p><br />
<h3>&nbsp;</h3><br />
<h3>For high school students </h3><br />
<p>In an effort to broaden the accessibility of the iGEM competition to those who appeared to be currently underrepresented in the competition (e.g. disadvantaged students, females and Australian high school students), we approached a private high school in Victoria with the following aim: </p><br />
<p>To find a well-resourced, private high school to partner with an iGEM team (e.g. UoM) to field a team consisting of half private high school students/half underrepresented students – who would have their flights to Boston funded by fundraising done primarily by the private high school. </p><br />
<p>After extensive email &amp; phone correspondence spanning several months, the school decided it was not currently in the position to go ahead with the iGEM team proposal due to upcoming infrastructure developments, though would be interested in fielding a team after these are completed (scheduled for 2016). Nonetheless, the school did encourage us that other APS schools would likely be interested in hearing about iGEM and our team concept. </p><br />
<p>Whilst we didn&rsquo;t achieve our ultimate goal, this activity did raise awareness of the iGEM competition with all the key science staff members at the school which requested not to be named on this wiki (for further details please contact our team leader Sean). </p><br />
<p>Finally, we would encourage all Australian teams to work on the following goals: </p><br />
<ul><br />
<li>improve iGEM's accessibility to those of disadvantaged backgrounds </li><br />
<li>increase female representation in science &amp; iGEM </li><br />
<li>increase the participation of Australian/international high school students in the iGEM competition. </li><br />
</ul><br />
<p><BR><br />
In addition to our negotiations with the private high school, we also aimed at widening the participation of high school students in the iGEM competition by collaborating with the University of Sydney on The Strange Nature Writing competition. </p><br />
<p>We spruiked the competition not only on our project flyers which described the iGEM competition, our project and the Strange Nature Writing Competition, but also through our development of a promotional video designed to be shown in high school assemblies as a way of promoting both iGEM and the Strange Nature Writing competition. Please see the video below: </p><br />
<br />
<iframe class="centervid" width="560" height="315" src="//www.youtube.com/embed/ZuSoO3HVMjo" frameborder="0" allowfullscreen></iframe><br />
<br />
<p>Furthermore, we wrote and published articles on Comet.is which is aimed at high school students, university students and recent university graduates, please see 'For university students' for a list of the articles we wrote. </p><br />
<h3>&nbsp;</h3><br />
<h3>For university students </h3><br />
<p>Our main form of outreach toward university students was in writing and publishing articles on a burgeoning and government sponsored website Comet.is which is aimed at high school students, university students and recent graduates. Our articles covered a range of topics from our iGEM experience to considerations of ethical issues of synthetic biology and the potential of synthetic biology. </p><br />
<br />
<td width="195" ><img src="https://static.igem.org/mediawiki/2014/d/d5/Comet.jpg" width="375" height="400" alt="Adventure of E coli"/></td><br />
<br />
<br />
</p> Here are some excerpts: </p><br />
<br />
</p> "All this leads me to refer to synthetic biology as a ménage à trois of a discipline, utilizing engineering, biology and computer science to produce a fantastically creative activity." </p><br />
<br />
</p> "Given assistance in the form of grants or human resources, entrepreneurial forums along with student competitions like InnovationACT and iGEM have the chance to inspire entrepreneurialism in students. It is in these opportunities for students to go on entrepreneurial adventures, replete with late nights, red bulls and a shot at winning a grand prize – that the next generation of entrepreneurs is born." </p><br />
<br />
</p> "A prime example of this is in the International Genetically Engineered Machine (iGEM) competition, which I am a team member of for the University of Melbourne this year. Our team stretches across many disciplines; from telecommunications and business to chemical engineering and science communication, even information technology - we are practically the UN of fields and backgrounds." </p><br />
<br />
</p> "Participating in IGEM can definitely be categorized as having an experience and not just joining a team. The competition brings together all the components of completing a project for undergraduate students to be part of, a little like a trial run of a Masters or Doctorate course." </p><br />
<br />
<p>Here are the links to our articles: </p><br />
<p><A href="https://comet.is/article/2960" rel="nofollow">https://comet.is/article/2960</A> <A href="https://comet.is/article/3499/" rel="nofollow">https://comet.is/article/3499/</A> <A href="https://comet.is/article/3381/" rel="nofollow">https://comet.is/article/3381/</A> <A href="https://comet.is/article/3561/" rel="nofollow">https://comet.is/article/3561/</A> <A href="https://comet.is/article/3562/" rel="nofollow">https://comet.is/article/3562/</A> <A href="https://comet.is/article/3694/" rel="nofollow">https://comet.is/article/3694/</A> <A href="http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/" rel="nofollow">http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/</A> <A href="http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/" rel="nofollow">http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/</A></p><br />
<p>Furthermore, as part of our outreach directed at university students, we combined our recruitment campaign for an IT whiz to help with our wiki, with a presentation on what iGEM was all about. We lecture bashed over several weeks, presenting over 11 times. </p><br />
<br />
<h3><BR><br />
</h3><br />
<h3>For general public </h3><br />
<p>After promoting the iGEM competition to the Victorian State Chair of National Science Week (NSW) - Nick Besley, our team decided to field a stall in &lsquo;Market of the Mind&rsquo; – a NSW event aimed at the general public located in a high foot traffic area alongside the Yarra River in Southbank across the river from Flinders St Station. Luckily, our stall was located next to the bar, which meant there was a lot of foot traffic directed to our happy team members wearing our iGEM team t-shirts. Using a true/false quiz, armed with Ferrero Rochers as rewards for getting all the answers correct, we had over 30 quiz contestants engage with us on the first night and 35+ contestants on the second day. Please see the accompanying video made by the City of Melbourne, which shows our stall/team members from 0:45 </p><br />
<br />
<p><iframe class="centervid" width="560" height="315" src="//www.youtube.com/embed/ahFI3aFBdNc" frameborder="0" allowfullscreen></iframe><p><br />
<br />
<p><BR><br />
<A href="http://www.scienceweek.net.au/market-of-the-mind/" rel="nofollow">http://www.scienceweek.net.au/market-of-the-mind/</A> <A href="http://re-science.org.au/science-event/market-mind-2649" rel="nofollow">http://re-science.org.au/science-event/market-mind-2649</A></p></html><br />
<br />
<br />
<h1>Intellectual Property </h1><br />
<br />
<p>'''Our team considered the question of ‘how should we proceed if we want to retain IP rights on our project’?''' </p><br />
<br />
<p> Before attempting to answer this pertinent IP question, our team leader decided to recruit two more team members who had some intellectual property experience to complement the learning on patent law that a few early team members had acquired in their biotechnology undergraduate degrees. Both the new team members had previously filed provisional patents; one had filed some in Russia and the other in Australia, offering two perspectives on this question of IP. </p><br />
<br />
<p> In answering this question, we approached our universities’ tech transfer office to gain clarity over our team’s discussions and brainstorm sessions on whether our project was patentable. </p><br />
<br />
<p> The tech transfer office advised us that our project may have commercial applications, however it was probably too soon to act on the idea and convert it into a provisional patent, so we decided to hold off and wait till we had a more substantiated grasp on our commercial applications. We were also advised that if at any point we wanted to file for IP protection on the project, it would be really important to not have disclosed the details of our idea to anyone else, as this could result in us losing the right to our IP. </p><br />
<br />
<p> After doing some research on the various law firms who could offer us a provisional patent, we discovered the estimated cost would be in the vicinity of $3500-$6500 depending on the level of detail we wanted to cover e.g. the number and extent of the claims, the full description of the idea, diagrams and experimental protocols as well as the filing & service fees associated. </p><br />
<br />
<p> Due to the relatively high estimates we received, we looked into other options for filing a provisional, including recruiting a law student to join the team and contribute their knowledge to our IP protection. This avenue did not eventuate as our ideas on what we would like to patent continuously evolved over the course of the project and we could not reach a sufficient degree of clarity to bring on a lawyer for the filing of a provisional patent. </p><br />
<br />
<p> Nonetheless, we proceeded with caution, ensuring to follow the advice of the University of Melbourne’s tech transfer office and not disclose our ideas without ensuring our disclosure was protected. An instance when we had to implement this protection was during our scientific collaboration with Oxford, were we ensured Non Disclosure Agreements were signed prior to the disclosure of our project to them. They indicated their interest in using our project as a case study in their ‘iGEM intellectual property guide’, however we felt that this might put our IP rights in jeopardy, so we decided not to proceed as a case study. </p><br />
<br />
<p> At this time, our team is looking to produce at least one provisional patent following the iGEM 2014 competition on at least one of the various techniques we designed during the project. </p><br />
<br />
<br />
<p> '''Our team consulted:''' </p><br />
<p> University of Melbourne Technology Transfer Office </p><br />
<p> University of Melbourne Technology Commercialisation Team </p><br />
<p> UoM Commercial </p><br />
<p> Stanislauv Yatskevich (iGEM team member with Russian patent experience) </p><br />
<p> Peter Collins (iGEM team member with Australian patent experience) </p><br />
<br />
<br />
<p> '''References:''' </p><br />
<br />
<p> http://www.ipaustralia.gov.au/.../factsheet-experimental.../ </p><br />
<br />
<p>http://www.google.com.au/url?sa=t&rct=j&q=&esrc=s...</p></div>Slowehttp://2014.igem.org/Team:Melbourne/Human_PracticesTeam:Melbourne/Human Practices2014-10-18T02:34:35Z<p>Slowe: </p>
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<img src="https://static.igem.org/mediawiki/2014/9/9b/Melbourne_Banner.png" alt="Banner" height="225" width="1000"><br />
<br />
<br />
<table width="100%" style="text-transform: uppercase; font-family: 'Lato', sans-serif;"><br />
<br />
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<br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne"style="color:#000000"><br />
Home</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Team"style="color:#000000"><br />
Team</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Project"style="color:#000000"><br />
Project</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Human_Practices"style="color:#000000"> <br />
Human Practices</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Achievements"style="color:#000000"><br />
Achievements</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Notebook"style="color:#000000"><br />
Notebook</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Protocols"style=" color:#000000"> <br />
Protocols</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Safety"style="color:#000000"><br />
Safety</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Sponsors"style="color:#000000"><br />
Sponsors</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Attributions"style="color:#000000"><br />
Attributions</a></td><br />
<br />
<br />
<td align="right"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="46px"></a> </td><br />
</tr><br />
</table><br />
<br />
<br />
<h1>Public Outreach</h1><br />
<h2>&lsquo;Synthetic biology for kids and adults and everyone in between&rsquo; </h2><br />
<p>The University of Melbourne took up the challenge of teaching synthetic biology to all ages, embracing a &lsquo;science for everyone&rsquo; approach. In so doing, we engaged in education outreach activities with kindergarten students from three childcare centres as well as both university and high school students from across Australia.</p><br />
<p>&nbsp; </p><br />
<h3>For kindergarten students </h3><br />
<table width="100%"><br />
<tr><br />
<td width="195" ><img src="https://static.igem.org/mediawiki/2014/4/4b/Melbourne_Adventure_of_E_coli.jpg" width="176" height="264" alt="Adventure of E coli"/></td><br />
<td width="594"><p>We decided to write the first in a series of children&rsquo;s books for pre-schoolers aged 4-6 themed in the spirit of iGEM entitled &lsquo;The Adventures of E. coli&rsquo; for a number of reasons:</p><br />
<ul><br />
<li> Children aged 4-6 are in a key learning stage, during which their interest in science can significantly influence their long-term science education outcomes (Bowman, Donovan, &amp; Burns, 2001, pp. 8-9). </li><br />
<li>There are lots of resources about science for older kids but not so many for kids at such a young age. </li><br />
<li>Picture books provide a springboard for further classroom activities </li><br />
</ul><br />
<p>With the assistance of IndieMosh (Publishers link is here: <A href="http://www.indiemosh.com.au/author-42-iGEM-Melbourne-childrens-biology-science" rel="nofollow">http://www.indiemosh.com.au/author-42-iGEM-Melbourne-childrens-biology-science</A>) – a self-publishing facilitator – we managed to get our book in print and on Amazon, please see <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/</A></p><br />
<p>After we successfully published our book, it was time to take it to the audience. We approached childcare centres across Melbourne and negotiated for our book to be read to pre-schoolers and for our team members to come along and teach the kids about science.</p></td><br />
</tr><br />
</table><p>The book reading was well received and was followed by playing some science-related games, and having a chat about science in groups. Please check out the video we made about the day: </p><br />
<br />
<iframe class="centervid" width="560" height="315" src="//www.youtube.com/embed/RNV_lPKcrSc" frameborder="0" allowfullscreen></iframe><br />
<br />
<br />
<p>We believe that through this book, young children can learn about basic concepts and the language of biology, forming a scientific foundation to be built on in future years. Moreover, it is our hope that this book will inspire children to continue to learn about science and to appreciate all the wonders that it holds. </p><br />
<p>The Kindle book is available from here: <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon-ebook/dp/B00OJ691DI/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon-ebook/dp/B00OJ691DI/</A></p><br />
<p>Paperback available on Amazon here: <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/</A></p><br />
<p>Fixed layout epub (for iPad and Android tablets, phones etc) available on Smashwords here: <A href="http://www.smashwords.com/books/view/484305" rel="nofollow">http://www.smashwords.com/books/view/484305</A></p><br />
<h3>&nbsp;</h3><br />
<h3>For high school students </h3><br />
<p>In an effort to broaden the accessibility of the iGEM competition to those who appeared to be currently underrepresented in the competition (e.g. disadvantaged students, females and Australian high school students), we approached a private high school in Victoria with the following aim: </p><br />
<p>To find a well-resourced, private high school to partner with an iGEM team (e.g. UoM) to field a team consisting of half private high school students/half underrepresented students – who would have their flights to Boston funded by fundraising done primarily by the private high school. </p><br />
<p>After extensive email &amp; phone correspondence spanning several months, the school decided it was not currently in the position to go ahead with the iGEM team proposal due to upcoming infrastructure developments, though would be interested in fielding a team after these are completed (scheduled for 2016). Nonetheless, the school did encourage us that other APS schools would likely be interested in hearing about iGEM and our team concept. </p><br />
<p>Whilst we didn&rsquo;t achieve our ultimate goal, this activity did raise awareness of the iGEM competition with all the key science staff members at the school which requested not to be named on this wiki (for further details please contact our team leader Sean). </p><br />
<p>Finally, we would encourage all Australian teams to work on the following goals: </p><br />
<ul><br />
<li>improve iGEM's accessibility to those of disadvantaged backgrounds </li><br />
<li>increase female representation in science &amp; iGEM </li><br />
<li>increase the participation of Australian/international high school students in the iGEM competition. </li><br />
</ul><br />
<p><BR><br />
In addition to our negotiations with the private high school, we also aimed at widening the participation of high school students in the iGEM competition by collaborating with the University of Sydney on The Strange Nature Writing competition. </p><br />
<p>We spruiked the competition not only on our project flyers which described the iGEM competition, our project and the Strange Nature Writing Competition, but also through our development of a promotional video designed to be shown in high school assemblies as a way of promoting both iGEM and the Strange Nature Writing competition. Please see the video below: </p><br />
<br />
<iframe width="560" height="315" src="//www.youtube.com/embed/ZuSoO3HVMjo" frameborder="0" allowfullscreen></iframe><br />
<br />
<p>Furthermore, we wrote and published articles on Comet.is which is aimed at high school students, university students and recent university graduates, please see 'For university students' for a list of the articles we wrote. </p><br />
<h3>&nbsp;</h3><br />
<h3>For university students </h3><br />
<p>Our main form of outreach toward university students was in writing and publishing articles on a burgeoning and government sponsored website Comet.is which is aimed at high school students, university students and recent graduates. Our articles covered a range of topics from our iGEM experience to considerations of ethical issues of synthetic biology and the potential of synthetic biology. </p><br />
<br />
<td width="195" ><img src="https://static.igem.org/mediawiki/2014/d/d5/Comet.jpg" width="375" height="400" alt="Adventure of E coli"/></td><br />
<br />
<br />
</p> Here are some excerpts: </p><br />
<br />
</p> "All this leads me to refer to synthetic biology as a ménage à trois of a discipline, utilizing engineering, biology and computer science to produce a fantastically creative activity." </p><br />
<br />
</p> "Given assistance in the form of grants or human resources, entrepreneurial forums along with student competitions like InnovationACT and iGEM have the chance to inspire entrepreneurialism in students. It is in these opportunities for students to go on entrepreneurial adventures, replete with late nights, red bulls and a shot at winning a grand prize – that the next generation of entrepreneurs is born." </p><br />
<br />
</p> "A prime example of this is in the International Genetically Engineered Machine (iGEM) competition, which I am a team member of for the University of Melbourne this year. Our team stretches across many disciplines; from telecommunications and business to chemical engineering and science communication, even information technology - we are practically the UN of fields and backgrounds." </p><br />
<br />
</p> "Participating in IGEM can definitely be categorized as having an experience and not just joining a team. The competition brings together all the components of completing a project for undergraduate students to be part of, a little like a trial run of a Masters or Doctorate course." </p><br />
<br />
<p>Here are the links to our articles: </p><br />
<p><A href="https://comet.is/article/2960" rel="nofollow">https://comet.is/article/2960</A> <A href="https://comet.is/article/3499/" rel="nofollow">https://comet.is/article/3499/</A> <A href="https://comet.is/article/3381/" rel="nofollow">https://comet.is/article/3381/</A> <A href="https://comet.is/article/3561/" rel="nofollow">https://comet.is/article/3561/</A> <A href="https://comet.is/article/3562/" rel="nofollow">https://comet.is/article/3562/</A> <A href="https://comet.is/article/3694/" rel="nofollow">https://comet.is/article/3694/</A> <A href="http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/" rel="nofollow">http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/</A> <A href="http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/" rel="nofollow">http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/</A></p><br />
<p>Furthermore, as part of our outreach directed at university students, we combined our recruitment campaign for an IT whiz to help with our wiki, with a presentation on what iGEM was all about. We lecture bashed over several weeks, presenting over 11 times. </p><br />
<br />
<h3><BR><br />
</h3><br />
<h3>For general public </h3><br />
<p>After promoting the iGEM competition to the Victorian State Chair of National Science Week (NSW) - Nick Besley, our team decided to field a stall in &lsquo;Market of the Mind&rsquo; – a NSW event aimed at the general public located in a high foot traffic area alongside the Yarra River in Southbank across the river from Flinders St Station. Luckily, our stall was located next to the bar, which meant there was a lot of foot traffic directed to our happy team members wearing our iGEM team t-shirts. Using a true/false quiz, armed with Ferrero Rochers as rewards for getting all the answers correct, we had over 30 quiz contestants engage with us on the first night and 35+ contestants on the second day. Please see the accompanying video made by the City of Melbourne, which shows our stall/team members from 0:45 </p><br />
<br />
<p><iframe width="560" height="315" src="//www.youtube.com/embed/ahFI3aFBdNc" frameborder="0" allowfullscreen></iframe><p><br />
<br />
<p><BR><br />
<A href="http://www.scienceweek.net.au/market-of-the-mind/" rel="nofollow">http://www.scienceweek.net.au/market-of-the-mind/</A> <A href="http://re-science.org.au/science-event/market-mind-2649" rel="nofollow">http://re-science.org.au/science-event/market-mind-2649</A></p></html><br />
<br />
<br />
<h1>Intellectual Property </h1><br />
<br />
<p>'''Our team considered the question of ‘how should we proceed if we want to retain IP rights on our project’?''' </p><br />
<br />
<p> Before attempting to answer this pertinent IP question, our team leader decided to recruit two more team members who had some intellectual property experience to complement the learning on patent law that a few early team members had acquired in their biotechnology undergraduate degrees. Both the new team members had previously filed provisional patents; one had filed some in Russia and the other in Australia, offering two perspectives on this question of IP. </p><br />
<br />
<p> In answering this question, we approached our universities’ tech transfer office to gain clarity over our team’s discussions and brainstorm sessions on whether our project was patentable. </p><br />
<br />
<p> The tech transfer office advised us that our project may have commercial applications, however it was probably too soon to act on the idea and convert it into a provisional patent, so we decided to hold off and wait till we had a more substantiated grasp on our commercial applications. We were also advised that if at any point we wanted to file for IP protection on the project, it would be really important to not have disclosed the details of our idea to anyone else, as this could result in us losing the right to our IP. </p><br />
<br />
<p> After doing some research on the various law firms who could offer us a provisional patent, we discovered the estimated cost would be in the vicinity of $3500-$6500 depending on the level of detail we wanted to cover e.g. the number and extent of the claims, the full description of the idea, diagrams and experimental protocols as well as the filing & service fees associated. </p><br />
<br />
<p> Due to the relatively high estimates we received, we looked into other options for filing a provisional, including recruiting a law student to join the team and contribute their knowledge to our IP protection. This avenue did not eventuate as our ideas on what we would like to patent continuously evolved over the course of the project and we could not reach a sufficient degree of clarity to bring on a lawyer for the filing of a provisional patent. </p><br />
<br />
<p> Nonetheless, we proceeded with caution, ensuring to follow the advice of the University of Melbourne’s tech transfer office and not disclose our ideas without ensuring our disclosure was protected. An instance when we had to implement this protection was during our scientific collaboration with Oxford, were we ensured Non Disclosure Agreements were signed prior to the disclosure of our project to them. They indicated their interest in using our project as a case study in their ‘iGEM intellectual property guide’, however we felt that this might put our IP rights in jeopardy, so we decided not to proceed as a case study. </p><br />
<br />
<p> At this time, our team is looking to produce at least one provisional patent following the iGEM 2014 competition on at least one of the various techniques we designed during the project. </p><br />
<br />
<br />
<p> '''Our team consulted:''' </p><br />
<p> University of Melbourne Technology Transfer Office </p><br />
<p> University of Melbourne Technology Commercialisation Team </p><br />
<p> UoM Commercial </p><br />
<p> Stanislauv Yatskevich (iGEM team member with Russian patent experience) </p><br />
<p> Peter Collins (iGEM team member with Australian patent experience) </p><br />
<br />
<br />
<p> '''References:''' </p><br />
<br />
<p> http://www.ipaustralia.gov.au/.../factsheet-experimental.../ </p><br />
<br />
<p>http://www.google.com.au/url?sa=t&rct=j&q=&esrc=s...</p></div>Slowehttp://2014.igem.org/Team:Melbourne/Human_PracticesTeam:Melbourne/Human Practices2014-10-18T02:31:13Z<p>Slowe: </p>
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Home</a> </td><br />
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Team</a> </td><br />
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Human Practices</a></td><br />
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Achievements</a></td><br />
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Notebook</a></td><br />
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Protocols</a></td><br />
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Safety</a></td><br />
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<br />
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<td align="right"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="46px"></a> </td><br />
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<br />
<br />
<h1>Public Outreach</h1><br />
<h2>&lsquo;Synthetic biology for kids and adults and everyone in between&rsquo; </h2><br />
<p>The University of Melbourne took up the challenge of teaching synthetic biology to all ages, embracing a &lsquo;science for everyone&rsquo; approach. In so doing, we engaged in education outreach activities with kindergarten students from three childcare centres as well as both university and high school students from across Australia.</p><br />
<p>&nbsp; </p><br />
<h3>For kindergarten students </h3><br />
<table width="100%"><br />
<tr><br />
<td width="195" ><img src="https://static.igem.org/mediawiki/2014/4/4b/Melbourne_Adventure_of_E_coli.jpg" width="176" height="264" alt="Adventure of E coli"/></td><br />
<td width="594"><p>We decided to write the first in a series of children&rsquo;s books for pre-schoolers aged 4-6 themed in the spirit of iGEM entitled &lsquo;The Adventures of E. coli&rsquo; for a number of reasons:</p><br />
<ul><br />
<li> Children aged 4-6 are in a key learning stage, during which their interest in science can significantly influence their long-term science education outcomes (Bowman, Donovan, &amp; Burns, 2001, pp. 8-9). </li><br />
<li>There are lots of resources about science for older kids but not so many for kids at such a young age. </li><br />
<li>Picture books provide a springboard for further classroom activities </li><br />
</ul><br />
<p>With the assistance of IndieMosh (Publishers link is here: <A href="http://www.indiemosh.com.au/author-42-iGEM-Melbourne-childrens-biology-science" rel="nofollow">http://www.indiemosh.com.au/author-42-iGEM-Melbourne-childrens-biology-science</A>) – a self-publishing facilitator – we managed to get our book in print and on Amazon, please see <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/</A></p><br />
<p>After we successfully published our book, it was time to take it to the audience. We approached childcare centres across Melbourne and negotiated for our book to be read to pre-schoolers and for our team members to come along and teach the kids about science.</p></td><br />
</tr><br />
</table><p>The book reading was well received and was followed by playing some science-related games, and having a chat about science in groups. Please check out the video we made about the day: </p><br />
<br />
<iframe class="centervid" width="560" height="315" src="//www.youtube.com/embed/RNV_lPKcrSc" frameborder="0" allowfullscreen></iframe><br />
<br />
<br />
<p>We believe that through this book, young children can learn about basic concepts and the language of biology, forming a scientific foundation to be built on in future years. Moreover, it is our hope that this book will inspire children to continue to learn about science and to appreciate all the wonders that it holds. </p><br />
<p>The Kindle book is available from here: <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon-ebook/dp/B00OJ691DI/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon-ebook/dp/B00OJ691DI/</A></p><br />
<p>Paperback available on Amazon here: <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/</A></p><br />
<p>Fixed layout epub (for iPad and Android tablets, phones etc) available on Smashwords here: <A href="http://www.smashwords.com/books/view/484305" rel="nofollow">http://www.smashwords.com/books/view/484305</A></p><br />
<h3>&nbsp;</h3><br />
<h3>For high school students </h3><br />
<p>In an effort to broaden the accessibility of the iGEM competition to those who appeared to be currently underrepresented in the competition (e.g. disadvantaged students, females and Australian high school students), we approached a private high school in Victoria with the following aim: </p><br />
<p>To find a well-resourced, private high school to partner with an iGEM team (e.g. UoM) to field a team consisting of half private high school students/half underrepresented students – who would have their flights to Boston funded by fundraising done primarily by the private high school. </p><br />
<p>After extensive email &amp; phone correspondence spanning several months, the school decided it was not currently in the position to go ahead with the iGEM team proposal due to upcoming infrastructure developments, though would be interested in fielding a team after these are completed (scheduled for 2016). Nonetheless, the school did encourage us that other APS schools would likely be interested in hearing about iGEM and our team concept. </p><br />
<p>Whilst we didn&rsquo;t achieve our ultimate goal, this activity did raise awareness of the iGEM competition with all the key science staff members at the school which requested not to be named on this wiki (for further details please contact our team leader Sean). </p><br />
<p>Finally, we would encourage all Australian teams to work on the following goals: </p><br />
<ul><br />
<li>improve iGEM's accessibility to those of disadvantaged backgrounds </li><br />
<li>increase female representation in science &amp; iGEM </li><br />
<li>increase the participation of Australian/international high school students in the iGEM competition. </li><br />
</ul><br />
<p><BR><br />
In addition to our negotiations with the private high school, we also aimed at widening the participation of high school students in the iGEM competition by collaborating with the University of Sydney on The Strange Nature Writing competition. </p><br />
<p>We spruiked the competition not only on our project flyers which described the iGEM competition, our project and the Strange Nature Writing Competition, but also through our development of a promotional video designed to be shown in high school assemblies as a way of promoting both iGEM and the Strange Nature Writing competition. Please see the video below: </p><br />
<br />
<iframe width="560" height="315" src="//www.youtube.com/embed/ZuSoO3HVMjo" frameborder="0" allowfullscreen></iframe><br />
<br />
<p>Furthermore, we wrote and published articles on Comet.is which is aimed at high school students, university students and recent university graduates, please see 'For university students' for a list of the articles we wrote. </p><br />
<h3>&nbsp;</h3><br />
<h3>For university students </h3><br />
<p>Our main form of outreach toward university students was in writing and publishing articles on a burgeoning and government sponsored website Comet.is which is aimed at high school students, university students and recent graduates. Our articles covered a range of topics from our iGEM experience to considerations of ethical issues of synthetic biology and the potential of synthetic biology. </p><br />
<br />
<td width="195" ><img src="https://static.igem.org/mediawiki/2014/d/d5/Comet.jpg" width="375" height="400" alt="Adventure of E coli"/></td><br />
<br />
<br />
</p> Here are some excerpts: </p><br />
<br />
</p> "All this leads me to refer to synthetic biology as a ménage à trois of a discipline, utilizing engineering, biology and computer science to produce a fantastically creative activity." </p><br />
<br />
</p> "Given assistance in the form of grants or human resources, entrepreneurial forums along with student competitions like InnovationACT and iGEM have the chance to inspire entrepreneurialism in students. It is in these opportunities for students to go on entrepreneurial adventures, replete with late nights, red bulls and a shot at winning a grand prize – that the next generation of entrepreneurs is born." </p><br />
<br />
</p> "A prime example of this is in the International Genetically Engineered Machine (iGEM) competition, which I am a team member of for the University of Melbourne this year. Our team stretches across many disciplines; from telecommunications and business to chemical engineering and science communication, even information technology - we are practically the UN of fields and backgrounds." </p><br />
<br />
</p> "Participating in IGEM can definitely be categorized as having an experience and not just joining a team. The competition brings together all the components of completing a project for undergraduate students to be part of, a little like a trial run of a Masters or Doctorate course." </p><br />
<br />
<p>Here are the links to our articles: </p><br />
<p><A href="https://comet.is/article/2960" rel="nofollow">https://comet.is/article/2960</A> <A href="https://comet.is/article/3499/" rel="nofollow">https://comet.is/article/3499/</A> <A href="https://comet.is/article/3381/" rel="nofollow">https://comet.is/article/3381/</A> <A href="https://comet.is/article/3561/" rel="nofollow">https://comet.is/article/3561/</A> <A href="https://comet.is/article/3562/" rel="nofollow">https://comet.is/article/3562/</A> <A href="https://comet.is/article/3694/" rel="nofollow">https://comet.is/article/3694/</A> <A href="http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/" rel="nofollow">http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/</A> <A href="http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/" rel="nofollow">http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/</A></p><br />
<p>Furthermore, as part of our outreach directed at university students, we combined our recruitment campaign for an IT whiz to help with our wiki, with a presentation on what iGEM was all about. We lecture bashed over several weeks, presenting over 11 times. </p><br />
<br />
<h3><BR><br />
</h3><br />
<h3>For general public </h3><br />
<p>After promoting the iGEM competition to the Victorian State Chair of National Science Week (NSW) - Nick Besley, our team decided to field a stall in &lsquo;Market of the Mind&rsquo; – a NSW event aimed at the general public located in a high foot traffic area alongside the Yarra River in Southbank across the river from Flinders St Station. Luckily, our stall was located next to the bar, which meant there was a lot of foot traffic directed to our happy team members wearing our iGEM team t-shirts. Using a true/false quiz, armed with Ferrero Rochers as rewards for getting all the answers correct, we had over 30 quiz contestants engage with us on the first night and 35+ contestants on the second day. Please see the accompanying video made by the City of Melbourne, which shows our stall/team members from 0:45 </p><br />
<br />
<p><iframe width="560" height="315" src="//www.youtube.com/embed/ahFI3aFBdNc" frameborder="0" allowfullscreen></iframe><p><br />
<br />
<p><BR><br />
<A href="http://www.scienceweek.net.au/market-of-the-mind/" rel="nofollow">http://www.scienceweek.net.au/market-of-the-mind/</A> <A href="http://re-science.org.au/science-event/market-mind-2649" rel="nofollow">http://re-science.org.au/science-event/market-mind-2649</A></p></html><br />
<br />
<br />
<h1>Intellectual Property </h1><br />
<br />
<p>'''Our team considered the question of ‘how should we proceed if we want to retain IP rights on our project’?''' </p><br />
<br />
<p> Before attempting to answer this pertinent IP question, our team leader decided to recruit two more team members who had some intellectual property experience to complement the learning on patent law that a few early team members had acquired in their biotechnology undergraduate degrees. Both the new team members had previously filed provisional patents; one had filed some in Russia and the other in Australia, offering two perspectives on this question of IP. </p><br />
<br />
<p> In answering this question, we approached our universities’ tech transfer office to gain clarity over our team’s discussions and brainstorm sessions on whether our project was patentable. </p><br />
<br />
<p> The tech transfer office advised us that our project may have commercial applications, however it was probably too soon to act on the idea and convert it into a provisional patent, so we decided to hold off and wait till we had a more substantiated grasp on our commercial applications. We were also advised that if at any point we wanted to file for IP protection on the project, it would be really important to not have disclosed the details of our idea to anyone else, as this could result in us losing the right to our IP. </p><br />
<br />
<p> After doing some research on the various law firms who could offer us a provisional patent, we discovered the estimated cost would be in the vicinity of $3500-$6500 depending on the level of detail we wanted to cover e.g. the number and extent of the claims, the full description of the idea, diagrams and experimental protocols as well as the filing & service fees associated. </p><br />
<br />
<p> Due to the relatively high estimates we received, we looked into other options for filing a provisional, including recruiting a law student to join the team and contribute their knowledge to our IP protection. This avenue did not eventuate as our ideas on what we would like to patent continuously evolved over the course of the project and we could not reach a sufficient degree of clarity to bring on a lawyer for the filing of a provisional patent. </p><br />
<br />
<p> Nonetheless, we proceeded with caution, ensuring to follow the advice of the University of Melbourne’s tech transfer office and not disclose our ideas without ensuring our disclosure was protected. An instance when we had to implement this protection was during our scientific collaboration with Oxford, were we ensured Non Disclosure Agreements were signed prior to the disclosure of our project to them. They indicated their interest in using our project as a case study in their ‘iGEM intellectual property guide’, however we felt that this might put our IP rights in jeopardy, so we decided not to proceed as a case study. </p><br />
<br />
<p> At this time, our team is looking to produce at least one provisional patent following the iGEM 2014 competition on at least one of the various techniques we designed during the project. </p><br />
<br />
<br />
<p> '''Our team consulted:''' </p><br />
<p> University of Melbourne Technology Transfer Office </p><br />
<p> University of Melbourne Technology Commercialisation Team </p><br />
<p> UoM Commercial </p><br />
<p> Stanislauv Yatskevich (iGEM team member with Russian patent experience) </p><br />
<p> Peter Collins (iGEM team member with Australian patent experience) </p><br />
<br />
<br />
<p> '''References:''' </p><br />
<br />
<p> http://www.ipaustralia.gov.au/.../factsheet-experimental.../ </p><br />
<br />
<p>http://www.google.com.au/url?sa=t&rct=j&q=&esrc=s...</p></div>Slowehttp://2014.igem.org/Team:Melbourne/AchievementsTeam:Melbourne/Achievements2014-10-18T02:23:19Z<p>Slowe: </p>
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<a href="https://2014.igem.org/Team:Melbourne/Project"style="color:#000000"><br />
Project</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Human_Practices"style="color:#000000"> <br />
Human Practices</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Achievements"style="color:#000000"><br />
Achievements</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Notebook"style="color:#000000"><br />
Notebook</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Protocols"style=" color:#000000"> <br />
Protocols</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Safety"style="color:#000000"><br />
Safety</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Sponsors"style="color:#000000"><br />
Sponsors</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Attributions"style="color:#000000"><br />
Attributions</a></td><br />
<br />
<br />
<td align="right"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="46px"></a> </td><br />
</tr><br />
</table><br />
<br />
<br />
<h1>Achievements</h1><br />
<h2>Bronze medal requirements</h2><br />
<ul><br />
<li>Register the team, have a great summer, and plan to have<br />
fun at the Giant Jamboree.</li><br />
<ul><br />
<li>We have met this criterion</li><br />
</ul><br />
<li>Successfully complete and submit this iGEM 2014 Judging<br />
form.</li><br />
<ul><br />
<li>We have met this criterion</li><br />
</ul><br />
<li>Create and share a Description of the team's project using<br />
the iGEM wiki and the team's parts using the Registry of Standard<br />
Biological Parts.</li><br />
<ul><br />
<li>We have met this criterion</li><br />
</ul><br />
<li>Plan to present a Poster and Talk at the iGEM Jamboree.</li><br />
<ul><br />
<li>We meet this criterion</li><br />
</ul><br />
<li>The description of each project must clearly attribute work<br />
done by the students and distinguish it from work done by others,<br />
including host labs, advisors, instructors, sponsors, professional<br />
website designers, artists, and commercial services.</li><br />
<ul><br />
<li>This is documented on the <a<br />
href="https://2014.igem.org/Team:Melbourne/Attributions">Attributions<br />
Page</a>.</li><br />
</ul><br />
<li>Document at least one new standard BioBrick Part or Device<br />
used in your project/central to your project and submit this part to<br />
the iGEM Registry</li><br />
<ul><br />
<li>Submitted &nbsp;<a<br />
href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1394000">BBa_K1394000</a>,<br />
<a<br />
href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1394001">BBa_K1394001</a>,<br />
and <a<br />
href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1394002">BBa_K1394002</a>,<br />
the parts central to our project.</li><br />
</ul><br />
</ul><br />
<h2>Silver medal requirements</h2><br />
<ul><br />
<li>Experimentally validate that at least one new BioBrick Part<br />
or Device of your own design and construction works as expected.</li><br />
<ul><br />
<li>As described on our <a<br />
href="https://2014.igem.org/Team:Melbourne/Project">project<br />
page</a>, we validated that our protein expression BioBricks<br />
worked and led to protein expression (e.g. <a<br />
href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1394001">BBa_K1394001</a>).</li><br />
</ul><br />
<li>Document the characterization of this part in the Main Page<br />
section of that Part's/Device's Registry entry.</li><br />
<ul><br />
<li>These characterization efforts have been documented.</li><br />
</ul><br />
<li>Submit this new part to the iGEM Parts Registry<br />
(submissions must adhere to the iGEM Registry guidelines)</li><br />
<ul><br />
<li>These parts have been submitted.</li><br />
</ul><br />
<li>iGEM projects involve important questions beyond the bench,<br />
for example relating to (but not limited to) ethics, sustainability,<br />
social justice, safety, security, or intellectual property rights.<br />
Articulate at least one question encountered by your team, and describe<br />
how your team considered the(se) question(s) within your project.<br />
Include attributions to all experts and stakeholders consulted.</li><br />
<ul><br />
<li>We asked the general question "how can we improve access<br />
to science education across a broad age range, including early<br />
childhood education." With this effort came additional questions,<br />
including "How can we make biology more accessible to young children,"<br />
and (for high school aged students) "How can we improve access to<br />
science education for disadvantaged youth in Australia?" We consulted<br />
with a variety of stakeholders within the science education community<br />
in order to answer these questions.</li><br />
<li>Our project concept could potentially have commercial<br />
value and therefore we had to address intellectual property issues. We<br />
consulted with a number of experts and stakeholders in the intellectual<br />
property space, as described in our <a<br />
href="https://2014.igem.org/Team:Melbourne/Public_Outreach">Human<br />
Practices</a> page.</li><br />
</ul><br />
</ul><br />
<h2>Gold medal requirements</h2><br />
<ul><br />
<li>Improve the function OR characterization of an existing<br />
BioBrick Part or Device (created by another team or your own<br />
institution in a previous year), enter this information in the<br />
Registry. Please see the Registry help page on how to document a<br />
contribution to an existing part.</li><br />
<ul><br />
<li>Our BioBrick protein expression device improves the 2013<br />
TU Delft SUMO fusion <a<br />
href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1022101">BioBrick</a>.<br />
This discussed in detail on our <a<br />
href="https://2014.igem.org/Team:Melbourne/Project">project<br />
page</a>.</li><br />
</ul><br />
<li>Help any registered iGEM team from another school or<br />
institution by, for example, characterizing a part, debugging a<br />
construct, or modeling or simulating their system.</li><br />
<ul><br />
<li>Our team formed a collaborative relationship with the<br />
University of Oxford. We assisted Oxford by providing them with a<br />
design concept for attaching enzymes to star peptides, discussing how<br />
we could improve their enzymatic system with star peptides, and<br />
providing research on linking their particular enzymes to a peptide<br />
linker. See our <a<br />
href="https://2014.igem.org/Team:Melbourne/Project#Oxford">project<br />
page</a> for details.</li><br />
<li>Our team help significantly with the promotion of the<br />
University of Sydney's outreach program, the Strange Nature writing<br />
competition. We created a promotional video for the competition which<br />
was distributed to high school teachers and students across Australia.<br />
Further, we contacted high school teachers directly to help distribute<br />
video and word of the competition. See our <a<br />
href="https://2014.igem.org/Team:Melbourne/Public_Outreach">Human<br />
Practices</a><br />
page.</li><br />
</ul><br />
</ul><br />
<br />
<br />
<br />
</html></div>Slowehttp://2014.igem.org/Team:Melbourne/ProjectTeam:Melbourne/Project2014-10-18T02:12:44Z<p>Slowe: </p>
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Safety</a></td><br />
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Sponsors</a></td><br />
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Attributions</a></td><br />
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<td align="right"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="46px"></a> </td><br />
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<h1>Project and results</h1><br />
<br />
<p>Read about our experimental work below, or jump to our theoretical collaboration with the <A HREF="#Oxford">University of Oxford</A><br />
<br />
<h2>Introduction and theory</h2><br />
<p>Synthetic peptide chemists have long produced peptide-based materials <em>in vitro</em>. Star-shaped peptides are a promising type of biomaterial being explored in the field of nanomedicine (Sulistio et al., 2012). Star peptides can have several biomedical uses, such as acting as drug delivery vehicles (Sulistio et al., 2011) or linkers for other biomacromolecules. Star peptides generally take the form of several linear peptide arms linked together in a central core. One way of linking these linear peptide arms together is to used covalent bonds such as those in disulfides. Typically, disulfide bonds are formed synthetically by taking several linear arms and treating them with an oxidant <em>in vitro</em>. Here, we introduce a new approach to forming star peptides using <em>E. coli</em> and synthetic biology. Thus, we aimed to show how the peptides synthesis and disulfide bond forming machinery of <em>E. coli</em> can be used to form disulfide linked star peptides and key star peptide precursors.</p><br />
<p>&nbsp;</p><br />
<h2>Synthesis approach</h2><br />
<p><em>E. coli</em> naturally possesses the capacity to form disulfide bonds. In native strains, disulfide bonds are naturally formed by an array of enzymes which are part of the Dsb family (e.g. DsbA and DsbC) (Kadokura et al., 2003, Kadokura and Beckwith, 2009). Normally, these enzymes are found in the oxidizing periplasm of the cell. However, several new strains of <em>E. coli</em> have recently been engineered which contain an oxidizing cytoplasm conducive to disulfide bond formation. One example of this is the SHuffle cell line (Lobstein et al., 2012). The cell line contains mutations to key enzymes responsible for the reducing nature of the cytoplasm, namely thioredoxin reductase (<em>trxB</em>) and glutathione reductase (<em>gor</em>). Furthermore, the Shuffle cell line over expresses the disulfide bond isomerase DsbC to the cytoplasm. Together, these mutations allow SHuffle to fold disulfide-bonded proteins in the cytoplasm at a higher success rate compared to non-mutants. We aimed to take advantage of the disulfide bond forming capabilities of this strain of <em>E. coli</em> to synthesize star peptides in cells. As shown in the figure below, the synthesis steps may proceed as follows:</p><br />
<p>&nbsp;</p><br />
<ol><br />
<li> Express a short peptide containing two cysteine residues either to the <em>E. coli</em> periplasm or the cytoplasm of a <em>trxB gor </em>mutant.</li><br />
<li><em>E. coli</em> disulfide bond forming enzymes fold the peptide into a hairpin loop structure.<br />
</li><br />
<li>Cut the loop at a protease recognition site engineered into the peptide. This may be done by extracting the folded peptide from the cell and treating it with the protease in vitro. Alternatively, the protease may be co-expressed in the cell to allow for in vivo cleavage.<br />
</li><br />
</ol><br />
<p>&nbsp;</p><br />
<table align="center"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9d/Project_concept-v2.png" width="480" height="600" alt=""/></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<p>This synthesis approach has several benefits over purely <em>in vitro</em> approaches. Firstly, the exact peptide sequence can be precisely programmed into <em>E. coli</em> using recombinant DNA synthesis. Secondly, by performing the disulfide bond formation in cells and optionally the proteolytic cleavage, several synthesis steps which would need to be performed <em>in vitro</em> are eliminated. From a scale up perspective, this would eliminate entire unit operations which would otherwise be required to produce this product. Given these benefits, in the current study, we aimed to express a star peptide precursor to the cytoplasm of SHuffle cells, which would later be extracted and externally digested with the star-forming protease. In order to achieve this, we first designed several star peptides which might be amenable to this synthetic strategy. These peptides are described below.</p><br />
<p>&nbsp;</p><br />
<h2>Rationally designed peptides</h2><br />
<p>There are two approaches to functionalising star peptides. In the first, the identity of the arms can be chosen to be bioactive peptide molecules. This is the simplest approach to producing a biologically-relevant star. In the second, the arms can be functionalised by ligating molecules to them at any point. We used both of these approaches to design two separate peptides.</p><br />
<h3>Magainin 1 star and linear peptides</h3><br />
<p>Our first strategy was to make a star peptide using antimicrobial peptides(AMPs) as building blocks. AMPs are small, approximately 50 residue peptides secreted by some bacterial and eukaryotic cells which selectively kill microbial cells. It is thought that AMPs work by forming pores in the membrane of prokaryotic cells (Brogden, 2005). AMPs have been recombinantly expressed in a number of organisms, including <em>E. coli</em> (for a review, see Li, 2011) and <em>B. Subtilis</em> (Chen et al., 2009, Yu et al., 2013).</p><br />
<p>Our concept was to design a star peptide with antimicrobial peptide arms. Wiradharma et al. (2012) first showed that placing linear antimicrobial peptides in a star configuration could lead to enhanced antimicrobial activity and decreased hemolytic activity. Although it is unclear why this is the case, it may be due to the ability of neighbouring antimicrobial peptide arms to interact with each other to synergistically rupture the membrane.<br><br />
While Wiradharma used a synthetic peptide sequence, we designed a peptide using the naturally occurring AMP, Magainin 1 (Zasloff, 1987). Magainin 1 peptides will be placed to the ends of each star arm.</p><br />
<br />
<p>Magainin 1 was chosen because the Magainins are one of the major classes of antimicrobial peptides, being well studied and characterised. In addition, we were concerned that tethering the antimicrobial peptide to the star peptide might interfere with its antimicrobial activity. Magainin 1, however, has previously been tethered to surfaces, where it has imparted the surfaces with microbicidal properties (Glinel et al., 2008; Humblot et al., 2009). We surmised that if Magainin 1 maintained its activity while anchored to surfaces, it may also maintain its activity while anchored to a star peptide.</p><br />
<p>The sequence for Magainin 1 is: GIGKFLHSAGKFGKAFVGEIMKS.</p><br />
<p>When attached to a star peptide it will have the following structure:</p><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/MAGAININ_1_peptide.png" width="720" height="300" alt=""/><br />
<p>There are several design elements to note:</p><br />
<ul><br />
<li><br />
The antimicrobial peptide star will be expressed with a SUMO fusion protein. This is because without the fusion, it is likely that the peptide would be toxic to the host cell.</li><br />
<li>The peptide includes a Factor X cutting site between the two cysteines for eventual proteolytic cleavage and formation of the star peptide.</li><br />
</ul><br />
<p>In addition to the star peptide Magainin 1, we synthesised a gene for a linear Magainin 1 peptide as well. This is identical to the construct above, except that there is only one Magainin 1 peptide attached to the SUMO fusion.</p><br />
<br />
<h3>Unstructured Peptide (USP) Construct</h3><br />
<p>In the second approach, we designed a peptide which can be functionalised using chemical approaches. This peptide was designed to have flexible, unstructured arms and was termed the USP construct. Unlike the Magainin 1 star, the arms of this peptide serve not as active peptides themselves, but as inert structural linkers.</p><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/USPI_Peptide.png" width="720" height="216" alt=""/><br />
<p>The arms were designed with the following elements in mind:</p><br />
<ul><br />
<li><br />
<b>Lack of structure.</b> The arms were designed with a bioinspired approach, using the FxFG motif of nucleoporins (where x is a variable amino acid residue). Such segments naturally repeat in nucleoporins and are thought to lead to disorder/lack of stable secondary structure. Nucleoporins are found in mammalian cells, serving as flexible brushes around nuclear pores (Ader et al., 2010).<br />
</li><br />
<li><b>Water-soluble.</b> The arms were designed with several charged amino acids to improve solubility.<br />
</li><br />
<li><b>Designed to form a disulfide bond.</b> Although it is difficult to rationally ensure that the disulfide bond will form between two cysteines in our peptide, we incorporated a beta turn between the two cysteines which may encourage the peptide to fold at the apex of the hairpin loop. This may bring the cysteines into closer proximity, providing more probable bond formation.</li></ul><br />
<br />
<p>The ultimate utility of this peptide lies in its ability to be functionalised with other biomacromolecules. For example, the technique of Native Chemical Ligation(NCL) can be used to join peptides, proteins, and other ligands to the arms (Dawson et al., 1994). The idea of attaching enzymes to the star peptide was explored by the University of Oxford iGEM 2014 team in a collaborative effort between our two teams (See Supplementary Project Work at the end of this page). </p><br />
<br />
<p>&nbsp;</p><br />
<h2>Expression system</h2><br />
<p>In order to successfully express our constructs, we designed our protein expression vectors to include a fusion protein. The fusion protein was necessary for two reasons. First, some of our constructs are very small (e.g. the non-star Magainin 1), and expression levels of very small peptides can be difficult without a fusion partner. Second, two of our constructs code for antimicrobial peptides. Without a fusion partner, it is likely that these genes would be toxic to their hosts upon induction.</p><br />
<p>To find a suitable expression system, we looked towards the Registry of Standard Parts. We used the SUMO protein expression system designed by TU Delft 2014 (for example, see<a target="_blank" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1022101">http://parts.igem.org/wiki/index.php?title=Part:BBa_K1022101</a>). This system essentially consists of a N-terminal HIS-tag followed by the SUMO protein (also known as UlpI).</p><br />
<p><br />
The strategy of expressing toxic AMPs using SUMO has been successfully reported in the literature (Bommarius et al., 2010). We surmise that the SUMO protein could inhibit the antimicrobial activity of single, linear peptides, and that it may also inhibit the activity of our star antimicrobial peptide.</p><br />
<p><br />
SUMO as a fusion protein also has the benefit of leaving no residues at the C-terminal end of the cleavage site. This means that upon cleavage with the SUMO protease, the native protein can be recovered. In our case, this means that one of the arms of the star can be designed without the need to take into account the addition of any amino acid residues left behind by the protease.<br><br />
We used the SUMO peptide sequence reported by TU Delft. However, our construct contained the following unique features:</p><br />
<ol><br />
<li> Standardisation of the biobrick by substituting the T7 promoter and RBS (the origins of which are both not specified in the Delft documentation) with the standard T7 promoter and RBS BioBrick<a target="_blank" href="http://parts.igem.org/Part:BBa_K525998">BBa_K525998</a>. In addition to supporting the principle of standardization, using the well-characterized promoter BioBrick should help assure expression levels.<br><br />
</li><br />
<li>Biobrick BBa_K1022101 lacks a terminator sequence (this was presumably because the part was meant to be integrated into a larger genetic construct with a terminator). A terminator from the registry of standard parts was added (specifically, the wild type terminator from T7 bacteriophage,<a target="_blank" href="http://parts.igem.org/Part:BBa_K731721">BBa_K731721</a>).<br />
</li><br />
<li>The original biobrick BBa_K1022101 codes for three amino acid residues before the HIS-tag (ASM), which appeared to be redundant. Correspondence with the 2013 TU Delft team suggested that these residues were unnecessary and appear to be cleaved within the cell as part of the cells post-translational modifications. However, their presence complicates the addition of additional tags at the N-terminus of the protein (e.g. periplasmic export tags), and therefore were not included.<br><br />
</li><br />
<li>The SUMO sequence was codon optimised for E. coli during synthesis. As the Delft documentation did not specify whether the gene was codon optimal, codon optomisation was undertaken to potentially improve expression levels. The linear Magainin 1 construct, however, was not codon optimised in the SUMO region in order to provide a control condition.</li><br />
</ol><br />
<p>To summarise, the protein expression devices used in our project to the following form:</p><br />
<img src="https://static.igem.org/mediawiki/2014/d/d0/Gene_circuit_export.png" width="720" height="91" alt=""/></td><br />
<p>The protein coding region consisted of a 6x-HIS tag followed by the SUMO protein and the relevant star or linear peptide.</p><br />
<p>&nbsp;</p><br />
<h2>Plasmid preparation: Cloning and acquisition of the genetic constructs</h2><br />
<h3>Magainin 1 Star Peptide</h3><br />
<p>This construct was synthesised in the standard shipping plasmid from Life Technologies- plasmid pMK. We had originally planned to express our protein to the periplasm of E. coli, and therefore had included in the synthesis a periplasmic export tag, the TorTss signal sequence (Steiner et al., 2006).</p><br />
<p><br />
Cytoplasmic and periplasmic expression may both allow for disulfide bond formation in the cell. We decided, however, to focus on cytoplasmic expression. There are distinct advantages to cytoplasmic expression (e.g. the absence of several periplasmic proteases and potentially higher expression levels (Baneyx, 1999)).</p><br />
<p><br />
Another reason the cytoplasm was chosen was to allow us to test the effects of an oxidising versus reducing intracellular environment on disulfide bond formation. We planned to express the construct in both SHuffle T7 cells (oxidising cytoplasm) and BL21(DE3) (reducing cytoplasm) to probe whether there was a difference in disulfide bond formation. Therefore, we needed to remove the periplasmic export tag from the gene.</p><br />
<p><br />
To do this, we use the following cloning strategy (noting that the top row represents the gene initially synthesised in plasmid pMK):</p><br />
<img src="https://static.igem.org/mediawiki/2014/2/2c/Melbourne_cloning_strategy_diagram.jpg" width="900" height="437" alt=""/><br />
<p>The gene was inserted into plasmid pSB1C3 containing the T7 promoter and ribosome binding site, BBa_K525998. It was inserted after the promoter and before the BioBrick suffix. This was accomplished by digesting the destination vector with SpeI and PstI. At the same time, PCR was used to amplify the segment of the gene containing the SUMO fusion and the Magainin 1 Star peptide, adding XbaI and keeping the PstI site existing in the gene.</p><br />
<p><br />
Digestion of the PCR product then allowed for ligation of the insert and destination vector using the PstI sites and XbaI and SpeI (XbaI and SpeI have compatible sticky ends). Note that after the ligation, there will be a scar in the gene where the XbaI and the SpeI sites were ligated.</p><br />
<p><br />
The ligation appeared to be successful. The ligation mixture was transformed to DH5α competent cells and plated onto chloramphenicol-containing agar plates.</p><br />
<p><br />
Plasmid from 5 colonies (C1, C2, C3, C4, and C5) was cultured, extracted, and then digested with EcoRI and PstI.</p><br />
<p><br />
As evident in the figure below, at least 4 of the colonies appeared to contain the insert. The empty, linearised pSB1C3 backbone ran at approximately the correct size (2070 bps), as did the insert (740 bps).</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/9/95/Magainin_1_subcloning.png" width="353" height="400" alt=""/></center><br />
<p>N.B. MW marker is the 100 bp Ladder from Axygen. * indicates the colony picked for sequencing and eventual transformation to the expression cell lines.</p><br />
<p>The DNA from Colony C2 was confirmed using Sanger sequencing at the Australian Genome Research Facility. This DNA appears in the registry of standard parts as BioBrick <a target="_blank" href="http://parts.igem.org/Part:BBa_K1394000">BBa_K1394000</a>.</p><br />
<p><br />
For the USP peptide and the linear Magainin 1 peptide, the genes were synthesised by GenScript and delivered in pSB1C3. The expression vectors had identical gene regulatory elements to that used for the Magainin 1 Star peptide. They only differed in the codon optimisation used, and they also lacked the assembly scar described above.</p><br />
<br />
<h2><br />
Protein expression and characterisation<br />
</h2><br />
<p><br />
After acquiring our genes, the project moved to protein expression and purification. We expressed both star peptides (Magainin 1 Star Peptide and the USP peptide) in both the SHuffle T7 and BL21(DE3) cell lines, both sourced from New England Biolabs. As the Linear Magainin 1 peptide does not have any special disulfide bonding<br />
requirements, we expressed it in BL21(DE3).<br />
</p><br />
<p><br />
The plasmids were transformed to the expression cells, and a single colony was cultured and induced overnight at 17 °C. A whole cell sample both before<br />
IPTG induction (-IPTG) and after the induction period (+IPTG) were boiled in SDS-PAGE sample buffer and loaded on a 15% tris-glycine gel. The whole cell<br />
Coomassie stained gel is shown below alongside the NEB P7712S molecular weight marker.<br />
</p><br />
<br><br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/2/24/Whole_cell_CS.png" height="330" width="439"/><br />
</p><br />
<br><br />
<p><br />
From theoretical prediction of the peptide masses, we would expect the following distribution of molecular weights:<br />
</p><br />
<br><br />
<table border="1" cellpadding="0" cellspacing="0" align="center"><br />
<tbody><br />
<tr><br />
<td width="156"><br />
<p align="center"><br />
Magainin 1 Star Peptide (Mag1 Star)<br />
</p><br />
</td><br />
<td width="145"><br />
<p align="center"><br />
USP<br />
</p><br />
</td><br />
<td width="156"><br />
<p align="center"><br />
Linear Magainin 1 (Linear Mag1)<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="156"><br />
<p align="center"><br />
22.15 kDa<br />
</p><br />
</td><br />
<td width="145"><br />
<p align="center"><br />
29.3 kDa<br />
</p><br />
</td><br />
<td width="156"><br />
<p align="center"><br />
14.32 kDa<br />
</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<br><br />
<p><br />
We looked for bands corresponding to overexpression of the proteins. We observed a thick band, post IPTG around 32 kDa in the BL21(DE3) cells carrying the<br />
USP plasmid. However, we saw this same band appearing in the Linear Magainin 1 lane but the Linear Magainin 1 protein should have a much lower molecular<br />
weight than what was observed. Therefore, we did not assume that we had overexpression overexpression of the USP 1 protein.<br />
</p><br />
<p><br />
We also noted bands in all post-IPTG lanes around 17 kDa. This would be roughly consistent with both the Linear Magainin 1 and Magainin 1 Star Peptide,<br />
noting that small proteins may not run at their expected molecular weight. Again, the analysis is complicated by the fact that the band also appears in the<br />
lane corresponding to the larger molecular weight protein, USP I. Given this ambiguity, we decided to purify all the protein in the sample using Ni-NTA<br />
purification.<br />
</p><br />
<h3><br />
Purification and further characterisation<br />
</h3><br />
<p><br />
A small-scale purification was carried out according to our <a href="https://static.igem.org/mediawiki/2014/e/e1/MELBOURNE_D1V1_Column_Purification_of_His-Tagged_Proteins.pdf">protocol</a>. The cells were lysed with iodoacetamide, an alkylating agent, added to the lysis buffer. Iodoacetamide blocks all free cysteines on proteins with a short alkyl group. This was added<br />
because we wanted to determine if a disulphide bond had formed inside the cell. If we did not block free cysteines, then any bond that had formed could be<br />
attributed to spontaneous/air oxidation of disulfides outside the cell.<br />
</p><br />
<p><br />
After purification, we ran a concurrent Western Blot and Coomassie stain on both the pre-induction samples from above and the purified protein (namely, the<br />
first elution from the batch purification).<br />
</p><br />
<p><br />
The Western Blot used mouse monoclonal antibodies against the N-terminal HIS-tag as primary antibodies and anti-mouse secondary antibodies:<br />
</p><br />
<br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/5/54/Western_Blot_Melb.png" height="400" width="460" border="0"/><br />
</p><br />
<p><br />
The corresponding Coomassie stain is show below:<br />
</p><br />
<br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f0/Purified_CS.png" height="365" width="486" border="0"/><br />
</p><br />
<p><br />
There are several interesting aspects of the Western Blot. Firstly, there was a strong band between 25 and 32 kDa in most lanes. However, it<br />
appears in all of the pre-induction controls, suggesting non-specific binding of the anti-HIS antibodies.<br />
</p><br />
<p><br />
Secondly, we observed a band in most of the lanes around 17 kDa (with the exception being the USP protein in SHuffle cells). As these bands were not<br />
present in the pre-induction controls, we suspected they were a result of the induction.<br />
</p><br />
<h3><br />
Mass spectroscopy<br />
</h3><br />
<p><br />
In order to assess the identity of the bands, we performed an in-gel tryptic digestion on select bands. We digested bands in the Coomassie stain which<br />
corresponded to the HIS-tagged bands in the Western Blot. This was followed by mass spec analysis (LC MS/MS). We focused our analysis on the Magainin 1<br />
Star Peptide bands and the Linear Magainin 1 band. Our USP peptide was, by design, highly rich in basic residues, greatly reducing the likelihood that<br />
tryptic fragments would be detected by the mass spec.<br />
</p><br />
<p><br />
We found that the Linear Magainin 1 peptide appeared to be present in the approximately 17 kDa band, with the following detected tryptic fragments in the<br />
sequence below (bold; basic residues underlined):<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/1/1b/Tryptic_digest_linear_mag1.PNG" height="63" width="601" border="0"/><br />
</p><br />
<p><br />
Note that spaces have been added in the sequence to emphasise distinct domains in the protein (in this case, the fusion protein versus the Magainin 1<br />
peptide). Together with the fact that the protein runs close to the expected molecular weight, this seems to provide good evidence that the protein is<br />
being expressed.<br />
</p><br />
<p><br />
We then examined the bands corresponding to the Magainin 1 Star Peptide.<br />
</p><br />
<p><br />
Again, we digested bands in the Coomassie stained gel corresponding to the prominent HIS-tagged bands on the <em>Western Blot</em> (near 17 kDa).<br />
</p><br />
<p><br />
The mass spec coverage for the Magainin 1 Star Peptide expressed in SHuffle cells was as follows:<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/6/63/Tryptic_digest_Sample_A_Star_Mag_Shuffle.PNG" height="90" width="601" border="0"/><br />
</p><br />
<p><br />
The coverage for the same peptide expressed in BL21(DE3) was found to be:<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/d/db/Tryptic_digest_Sample_A_Star_Mag_BL21%28DE3%29.PNG" height="85" width="602" border="0"/><br />
</p><br />
<p><br />
Again, we can see that tryptic peptides from the expressed protein appear to present in the gel band under analysis.<br />
</p><br />
<p><br />
There is a relatively lower sequence coverage. This could be a limitation of our procedure: for example, improper destaining during the in gel digestion<br />
procedure can inhibit tryptic digestion.<br />
</p><br />
<p><br />
However, even if the digestion was complete, the mass spec will only detect fragments which ionize well. The Magainin 1 Star peptide consists of four<br />
repeats of the Magainin 1 sequence (corresponding to the arms of the star). If the two tryptic fragments within Magainin 1 do not ionize well, then indeed<br />
the entire peptide would not be read.<br />
</p><br />
<p><br />
Finally, it is possible that the protein has been cleaved or degraded. This would account for the slightly lower-than-expected mass on the SDS page gel.<br />
</p><br />
<p><br />
Nonetheless, there is the distinct possibility that the peptide fragments are there but not detected. Replication of the in-gel mass spec would be useful<br />
in clarifying this point, or provided the purity of the sample is acceptable, it may be possible to run an intact mass spec.<br />
</p><br />
<h2><br />
Conclusions<br />
</h2><br />
<p><br />
We designed several novel star peptides based on several rational design criteria. Further, we began the optimisation process required to express these peptides in <em>E. coli</em>.<br />
</p><br />
<p><br />
To this end, we constructed an expression system based on the SUMO protein and standard BioBrick parts. The function of this system was confirmed, as it<br />
appears it does produce HIS-tagged SUMO fusion protein in <em>E. coli</em>, as expected.<br />
</p><br />
<p><br />
Although it appears the expression system works, there was some uncertainty about particular peptides, namely the Magainin 1 Star Peptide and USP Peptide.<br />
A Westerm Blot suggested that at least part of the peptides are present. However, additional sequence coverage in a mass spec analysis and intact mass spec<br />
would help us determine the precise identity of our products.<br />
</p><br />
<p><br />
We have shown that the SUMO fusion can be expressed in our BioBrick. Given the success of SUMO in the general scientific community, we hope our BioBrick<br />
will encourage further use of the fusion domain within the iGEM community. In addition, we hope that our ideas and concepts will inspire future<br />
iGEM efforts to explore the applications of synthetic biology for material science. We continue to believe that bacteria have great promise for the <em>in vivo</em><br />
construction of unique biomaterials, and we look forward to seeing how the synthetic biology community will develop in this area.<br />
</p><br />
<br />
<br />
<br />
<h2>References</h2><br />
<p>ADER, C., FREY, S., MAAS, W., SCHMIDT, H. B., GÖRLICH, D. &amp; BALDUS, M. 2010. Amyloid-like interactions within nucleoporin FG hydrogels. <em>Proceedings of the National Academy of Sciences,</em> 107<strong>,</strong> 6281-6285.</p><br />
<p><br />
BANEYX, F. 1999. Recombinant protein expression in Escherichia coli. <em>Current Opinion in Biotechnology,</em> 10<strong>,</strong> 411-421.</p><br />
<p><br />
BOMMARIUS, B., JENSSEN, H., ELLIOTT, M., KINDRACHUK, J., PASUPULETI, M., GIEREN, H., JAEGER, K. E., HANCOCK, R. E. W. &amp; KALMAN, D. 2010. Cost-effective expression and purification of antimicrobial and host defense peptides in Escherichia coli. <em>Peptides,</em> 31<strong>,</strong> 1957-1965.</p><br />
<p><br />
BROGDEN, K. A. 2005. Antimicrobial peptides: Pore formers or metabolic inhibitors in bacteria? <em>Nature Reviews Microbiology,</em> 3<strong>,</strong> 238-250.</p><br />
<p><br />
CHANG, C., CHHOR, G., CLANCY, S. & JOACHIMIAK, A. 2014. Crystal Structure of Glutathione S-transferase Domain Protein From Haliangium Ochraceum DSM 14365 [Online]. ''NCBI. Available: http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=4w66' [Accessed September 9 2014].</p><br />
<p><br />
CHEN, X., ZHU, F., CAO, Y. &amp; QIAO, S. 2009. Novel expression vector for secretion of cecropin AD in Bacillus subtilis with enhanced antimicrobial activity. <em>Antimicrobial agents and chemotherapy,</em> 53<strong>,</strong> 3683-3689.</p><br />
<p><br />
GLINEL, K., JONAS, A. M., JOUENNE, T., LEPRINCE, J., GALAS, L. & HUCK, W. T. 2008. Antibacterial and antifouling polymer brushes incorporating antimicrobial peptide. <em>Bioconjug Chem,</em> 20<strong>,</strong> 71-77.</p><br />
<p><br />
HUMBLOT, V., YALA, J.-F., THEBAULT, P., BOUKERMA, K., HÉQUET, A., BERJEAUD, J.-M. & PRADIER, C.-M. 2009. The antibacterial activity of Magainin I immobilized onto mixed thiols self-assembled monolayers. <em>Biomaterials,</em> 30<strong>,</strong> 3503-3512.</p><br />
<p><br />
DAWSON, P. E., MUIR, T. W., CLARK-LEWIS, I. &amp; KENT, S. 1994. Synthesis of proteins by native chemical ligation. <em>Science,</em> 266<strong>,</strong> 776-779.</p><br />
<p><br />
KADOKURA, H. &amp; BECKWITH, J. 2009. Detecting folding intermediates of a protein as it passes through the bacterial translocation channel. <em>Cell,</em> 138<strong>,</strong> 1164-1173.</p><br />
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KADOKURA, H., KATZEN, F. &amp; BECKWITH, J. 2003. Protein disulfide bond formation in prokaryotes. <em>Annual review of biochemistry,</em> 72<strong>,</strong> 111-135.</p><br />
<p><br />
LI, Y. 2011. Recombinant production of antimicrobial peptides in&lt; i&gt; Escherichia coli&lt;/i&gt;: A review. <em>Protein Expression and Purification,</em> 80<strong>,</strong> 260-267.</p><br />
<p><br />
LOBSTEIN, J., EMRICH, C. A., JEANS, C., FAULKNER, M., RIGGS, P. &amp; BERKMEN, M. 2012. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. <em>Microb Cell Fact,</em> 11<strong>,</strong> 56-56.</p><br />
<p><br />
PORTAL, P. M. Query result, GST and DcmA [Online]. Available: http://www.proteinmodelportal.org/query/uniprot/P21161 [Accessed September 9 2014].</p><br />
<p><br />
STEINER, D., FORRER, P., STUMPP, M. T. &amp; PLUCKTHUN, A. 2006. Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display. <em>Nat Biotech,</em> 24<strong>,</strong> 823-831.</p><br />
<p><br />
SULISTIO, A., GURR, P. A., BLENCOWE, A. &amp; QIAO, G. G. 2012. Peptide-Based Star Polymers: The Rising Star in Functional Polymers. <em>Australian Journal of Chemistry,</em> 65<strong>,</strong> 978-984.</p><br />
<p><br />
SULISTIO, A., LOWENTHAL, J., BLENCOWE, A., BONGIOVANNI, M. N., ONG, L., GRAS, S. L., ZHANG, X. &amp; QIAO, G. G. 2011. Folic Acid Conjugated Amino Acid-Based Star Polymers for Active Targeting of Cancer Cells. <em>Biomacromolecules,</em> 12<strong>,</strong> 3469-3477.</p><br />
<p><br />
TANAKA, N., KUSAKABE, Y., ITO, K., YOSHIMOTO, T. & NAKAMURA, K. T. 2002. Crystal Structure of Formaldehyde Dehydrogenase from< i> Pseudomonas putida</i>: the Structural Origin of the Tightly Bound Cofactor in Nicotinoprotein Dehydrogenases. Journal of molecular biology, 324, 519-533.</p><br />
<p><br />
WIRADHARMA, N., LIU, S. Q. &amp; YANG, Y. Y. 2012. Branched and 4-Arm Starlike α-Helical Peptide Structures with Enhanced Antimicrobial Potency and Selectivity. <em>Small,</em> 8<strong>,</strong> 362-366.</p><br />
<p><br />
YU, Z., WANG, Q., MA, Q. &amp; ZHANG, R. 2013. Secretory expression of lacticin Q fused with SUMO in Bacillus subtilis. <em>Protein Expression and Purification,</em> 89<strong>,</strong> 51-55.</p><br />
<p><br />
ZASLOFF, M. 1987. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. <em>Proceedings of the National Academy of Sciences,</em> 84<strong>,</strong> 5449-5453.</p><br />
<p>&nbsp;</p><br />
<br />
<A NAME="Oxford"></A><br />
<h1>Collaboration with Oxford</h1><br />
<p>To strengthen the sense of iGEM community, we contacted <a target="_blank" href="https://2014.igem.org/Team:Oxford">University of Oxford iGEM team</a> to discuss the possibility of collaboration between our teams. The Oxford project aims to develop a system that can dispose of the carcinogenic, hazardous solvent dichloromethane (DCM). To do this, Oxford team has proposed the use of the DcmA enzyme. This enzyme degrades DCM to form a toxic intermediate which in turn is converted by another enzyme, FdhA, into a neutral molecule. Schematically this can be presented in the following way:</p><br />
<br />
<p><br />
Reaction 1: DCM +&nbsp;DcmA <strong>toxic intermediate</strong><br><br />
Reaction 2: <strong>toxic intermediate</strong> + FdhA neutral product.</p><br />
<p>The problem for their system lies in bringing the two enzymes together to ensure efficient reaction kinetics. An idea that we discussed with Oxford was to use the star peptide platform to link the two enzymes together, in a structure similar to the following: </p><br />
<img src="https://static.igem.org/mediawiki/2014/2/2e/Melbourne_Collab_1.png" width="500" height="325" alt=""/><br />
<p>We worked with Oxford to study this system from a theoretical standpoint. Our team studied the 3D structure of both enzymes involved to confirm that the enzyme could in theory be attached to a linker in this manner, while Oxford team did stochastic modelling to determine how reaction rate changes when the linker length, labeled D on the diagram, changes. </p><br />
<p><br />
As a first check, it was necessary to determine whether anchoring these enzymes to our star would be sterically possible. We studied the structures of FdhA and DcmA determined by crystallography using data from the protein data bank. As no crystallographic data was available in the databank for DcmA, GST was examined as a proxy, as GST is structurally homologous to DcmA. </p><br />
<p><br />
The crystalographically resolved structure of FdhA enzyme (Tanaka et al., 2002) from the protein data bank revealed the following image:</p><br />
<img src="https://static.igem.org/mediawiki/2014/d/dc/Melbourne_Collab_2.png" width="700" height="355" alt=""/><br />
<p>The crystalographically resolved structure of GST was as follows (Chang et al., 2014):</p><br />
<img src="https://static.igem.org/mediawiki/2014/e/e9/Melbourne_Collab_3.png" width="800" height="361" alt=""/><br />
<p>It can be seen that the amino- and carboxy-terminals are located away from the active site. This suggests that both enzymes, DcmA and FdhA, might be linked together via linkers that are used in our star peptide. That is, the active site will not be sterically hindered by attachment to the star peptide. That said, it is difficult to predict how anchoring the protein to the star</div>Slowehttp://2014.igem.org/Team:Melbourne/ProjectTeam:Melbourne/Project2014-10-18T02:08:05Z<p>Slowe: </p>
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<h1>Project and results</h1><br />
<br />
<p>Read about our experimental work below, or jump to our theoretical collaboration with the <A HREF="#Oxford">University of Oxford</A><br />
<br />
<h2>Introduction and theory</h2><br />
<p>Synthetic peptide chemists have long produced peptide-based materials <em>in vitro</em>. Star-shaped peptides are a promising type of biomaterial being explored in the field of nanomedicine (Sulistio et al., 2012). Star peptides can have several biomedical uses, such as acting as drug delivery vehicles (Sulistio et al., 2011) or linkers for other biomacromolecules. Star peptides generally take the form of several linear peptide arms linked together in a central core. One way of linking these linear peptide arms together is to used covalent bonds such as those in disulfides. Typically, disulfide bonds are formed synthetically by taking several linear arms and treating them with an oxidant <em>in vitro</em>. Here, we introduce a new approach to forming star peptides using <em>E. coli</em> and synthetic biology. Thus, we aimed to show how the peptides synthesis and disulfide bond forming machinery of <em>E. coli</em> can be used to form disulfide linked star peptides and key star peptide precursors.</p><br />
<p>&nbsp;</p><br />
<h2>Synthesis approach</h2><br />
<p><em>E. coli</em> naturally possesses the capacity to form disulfide bonds. In native strains, disulfide bonds are naturally formed by an array of enzymes which are part of the Dsb family (e.g. DsbA and DsbC) (Kadokura et al., 2003, Kadokura and Beckwith, 2009). Normally, these enzymes are found in the oxidizing periplasm of the cell. However, several new strains of <em>E. coli</em> have recently been engineered which contain an oxidizing cytoplasm conducive to disulfide bond formation. One example of this is the SHuffle cell line (Lobstein et al., 2012). The cell line contains mutations to key enzymes responsible for the reducing nature of the cytoplasm, namely thioredoxin reductase (<em>trxB</em>) and glutathione reductase (<em>gor</em>). Furthermore, the Shuffle cell line over expresses the disulfide bond isomerase DsbC to the cytoplasm. Together, these mutations allow SHuffle to fold disulfide-bonded proteins in the cytoplasm at a higher success rate compared to non-mutants. We aimed to take advantage of the disulfide bond forming capabilities of this strain of <em>E. coli</em> to synthesize star peptides in cells. As shown in the figure below, the synthesis steps may proceed as follows:</p><br />
<p>&nbsp;</p><br />
<ol><br />
<li> Express a short peptide containing two cysteine residues either to the <em>E. coli</em> periplasm or the cytoplasm of a <em>trxB gor </em>mutant.</li><br />
<li><em>E. coli</em> disulfide bond forming enzymes fold the peptide into a hairpin loop structure.<br />
</li><br />
<li>Cut the loop at a protease recognition site engineered into the peptide. This may be done by extracting the folded peptide from the cell and treating it with the protease in vitro. Alternatively, the protease may be co-expressed in the cell to allow for in vivo cleavage.<br />
</li><br />
</ol><br />
<p>&nbsp;</p><br />
<table align="center"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9d/Project_concept-v2.png" width="480" height="600" alt=""/></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<p>This synthesis approach has several benefits over purely <em>in vitro</em> approaches. Firstly, the exact peptide sequence can be precisely programmed into <em>E. coli</em> using recombinant DNA synthesis. Secondly, by performing the disulfide bond formation in cells and optionally the proteolytic cleavage, several synthesis steps which would need to be performed <em>in vitro</em> are eliminated. From a scale up perspective, this would eliminate entire unit operations which would otherwise be required to produce this product. Given these benefits, in the current study, we aimed to express a star peptide precursor to the cytoplasm of SHuffle cells, which would later be extracted and externally digested with the star-forming protease. In order to achieve this, we first designed several star peptides which might be amenable to this synthetic strategy. These peptides are described below.</p><br />
<p>&nbsp;</p><br />
<h2>Rationally designed peptides</h2><br />
<p>There are two approaches to functionalising star peptides. In the first, the identity of the arms can be chosen to be bioactive peptide molecules. This is the simplest approach to producing a biologically-relevant star. In the second, the arms can be functionalised by ligating molecules to them at any point. We used both of these approaches to design two separate peptides.</p><br />
<h3>Magainin 1 star and linear peptides</h3><br />
<p>Our first strategy was to make a star peptide using antimicrobial peptides(AMPs) as building blocks. AMPs are small, approximately 50 residue peptides secreted by some bacterial and eukaryotic cells which selectively kill microbial cells. It is thought that AMPs work by forming pores in the membrane of prokaryotic cells (Brogden, 2005). AMPs have been recombinantly expressed in a number of organisms, including <em>E. coli</em> (for a review, see Li, 2011) and <em>B. Subtilis</em> (Chen et al., 2009, Yu et al., 2013).</p><br />
<p>Our concept was to design a star peptide with antimicrobial peptide arms. Wiradharma et al. (2012) first showed that placing linear antimicrobial peptides in a star configuration could lead to enhanced antimicrobial activity and decreased hemolytic activity. Although it is unclear why this is the case, it may be due to the ability of neighbouring antimicrobial peptide arms to interact with each other to synergistically rupture the membrane.<br><br />
While Wiradharma used a synthetic peptide sequence, we designed a peptide using the naturally occurring AMP, Magainin 1 (Zasloff, 1987). Magainin 1 peptides will be placed to the ends of each star arm.</p><br />
<br />
<p>Magainin 1 was chosen because the Magainins are one of the major classes of antimicrobial peptides, being well studied and characterised. In addition, we were concerned that tethering the antimicrobial peptide to the star peptide might interfere with its antimicrobial activity. Magainin 1, however, has previously been tethered to surfaces, where it has imparted the surfaces with microbicidal properties (Glinel et al., 2008; Humblot et al., 2009). We surmised that if Magainin 1 maintained its activity while anchored to surfaces, it may also maintain its activity while anchored to a star peptide.</p><br />
<p>The sequence for Magainin 1 is: GIGKFLHSAGKFGKAFVGEIMKS.</p><br />
<p>When attached to a star peptide it will have the following structure:</p><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/MAGAININ_1_peptide.png" width="720" height="300" alt=""/><br />
<p>There are several design elements to note:</p><br />
<ul><br />
<li><br />
The antimicrobial peptide star will be expressed with a SUMO fusion protein. This is because without the fusion, it is likely that the peptide would be toxic to the host cell.</li><br />
<li>The peptide includes a Factor X cutting site between the two cysteines for eventual proteolytic cleavage and formation of the star peptide.</li><br />
</ul><br />
<p>In addition to the star peptide Magainin 1, we synthesised a gene for a linear Magainin 1 peptide as well. This is identical to the construct above, except that there is only one Magainin 1 peptide attached to the SUMO fusion.</p><br />
<br />
<h3>Unstructured Peptide (USP) Construct</h3><br />
<p>In the second approach, we designed a peptide which can be functionalised using chemical approaches. This peptide was designed to have flexible, unstructured arms and was termed the USP construct. Unlike the Magainin 1 star, the arms of this peptide serve not as active peptides themselves, but as inert structural linkers.</p><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/USPI_Peptide.png" width="720" height="216" alt=""/><br />
<p>The arms were designed with the following elements in mind:</p><br />
<ul><br />
<li><br />
<b>Lack of structure.</b> The arms were designed with a bioinspired approach, using the FxFG motif of nucleoporins (where x is a variable amino acid residue). Such segments naturally repeat in nucleoporins and are thought to lead to disorder/lack of stable secondary structure. Nucleoporins are found in mammalian cells, serving as flexible brushes around nuclear pores (Ader et al., 2010).<br />
</li><br />
<li><b>Water-soluble.</b> The arms were designed with several charged amino acids to improve solubility.<br />
</li><br />
<li><b>Designed to form a disulfide bond.</b> Although it is difficult to rationally ensure that the disulfide bond will form between two cysteines in our peptide, we incorporated a beta turn between the two cysteines which may encourage the peptide to fold at the apex of the hairpin loop. This may bring the cysteines into closer proximity, providing more probable bond formation.</li></ul><br />
<br />
<p>The ultimate utility of this peptide lies in its ability to be functionalised with other biomacromolecules. For example, the technique of Native Chemical Ligation(NCL) can be used to join peptides, proteins, and other ligands to the arms (Dawson et al., 1994). The idea of attaching enzymes to the star peptide was explored by the University of Oxford iGEM 2014 team in a collaborative effort between our two teams (See Supplementary Project Work at the end of this page). </p><br />
<br />
<p>&nbsp;</p><br />
<h2>Expression system</h2><br />
<p>In order to successfully express our constructs, we designed our protein expression vectors to include a fusion protein. The fusion protein was necessary for two reasons. First, some of our constructs are very small (e.g. the non-star Magainin 1), and expression levels of very small peptides can be difficult without a fusion partner. Second, two of our constructs code for antimicrobial peptides. Without a fusion partner, it is likely that these genes would be toxic to their hosts upon induction.</p><br />
<p>To find a suitable expression system, we looked towards the Registry of Standard Parts. We used the SUMO protein expression system designed by TU Delft 2014 (for example, see<a target="_blank" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1022101">http://parts.igem.org/wiki/index.php?title=Part:BBa_K1022101</a>). This system essentially consists of a N-terminal HIS-tag followed by the SUMO protein (also known as UlpI).</p><br />
<p><br />
The strategy of expressing toxic AMPs using SUMO has been successfully reported in the literature (Bommarius et al., 2010). We surmise that the SUMO protein could inhibit the antimicrobial activity of single, linear peptides, and that it may also inhibit the activity of our star antimicrobial peptide.</p><br />
<p><br />
SUMO as a fusion protein also has the benefit of leaving no residues at the C-terminal end of the cleavage site. This means that upon cleavage with the SUMO protease, the native protein can be recovered. In our case, this means that one of the arms of the star can be designed without the need to take into account the addition of any amino acid residues left behind by the protease.<br><br />
We used the SUMO peptide sequence reported by TU Delft. However, our construct contained the following unique features:</p><br />
<ol><br />
<li> Standardisation of the biobrick by substituting the T7 promoter and RBS (the origins of which are both not specified in the Delft documentation) with the standard T7 promoter and RBS BioBrick<a target="_blank" href="http://parts.igem.org/Part:BBa_K525998">BBa_K525998</a>. In addition to supporting the principle of standardization, using the well-characterized promoter BioBrick should help assure expression levels.<br><br />
</li><br />
<li>Biobrick BBa_K1022101 lacks a terminator sequence (this was presumably because the part was meant to be integrated into a larger genetic construct with a terminator). A terminator from the registry of standard parts was added (specifically, the wild type terminator from T7 bacteriophage,<a target="_blank" href="http://parts.igem.org/Part:BBa_K731721">BBa_K731721</a>).<br />
</li><br />
<li>The original biobrick BBa_K1022101 codes for three amino acid residues before the HIS-tag (ASM), which appeared to be redundant. Correspondence with the 2013 TU Delft team suggested that these residues were unnecessary and appear to be cleaved within the cell as part of the cells post-translational modifications. However, their presence complicates the addition of additional tags at the N-terminus of the protein (e.g. periplasmic export tags), and therefore were not included.<br><br />
</li><br />
<li>The SUMO sequence was codon optimised for E. coli during synthesis. As the Delft documentation did not specify whether the gene was codon optimal, codon optomisation was undertaken to potentially improve expression levels. The linear Magainin 1 construct, however, was not codon optimised in the SUMO region in order to provide a control condition.</li><br />
</ol><br />
<p>To summarise, the protein expression devices used in our project to the following form:</p><br />
<img src="https://static.igem.org/mediawiki/2014/d/d0/Gene_circuit_export.png" width="720" height="91" alt=""/></td><br />
<p>The protein coding region consisted of a 6x-HIS tag followed by the SUMO protein and the relevant star or linear peptide.</p><br />
<p>&nbsp;</p><br />
<h2>Plasmid preparation: Cloning and acquisition of the genetic constructs</h2><br />
<h3>Magainin 1 Star Peptide</h3><br />
<p>This construct was synthesised in the standard shipping plasmid from Life Technologies- plasmid pMK. We had originally planned to express our protein to the periplasm of E. coli, and therefore had included in the synthesis a periplasmic export tag, the TorTss signal sequence (Steiner et al., 2006).</p><br />
<p><br />
Cytoplasmic and periplasmic expression may both allow for disulfide bond formation in the cell. We decided, however, to focus on cytoplasmic expression. There are distinct advantages to cytoplasmic expression (e.g. the absence of several periplasmic proteases and potentially higher expression levels (Baneyx, 1999)).</p><br />
<p><br />
Another reason the cytoplasm was chosen was to allow us to test the effects of an oxidising versus reducing intracellular environment on disulfide bond formation. We planned to express the construct in both SHuffle T7 cells (oxidising cytoplasm) and BL21(DE3) (reducing cytoplasm) to probe whether there was a difference in disulfide bond formation. Therefore, we needed to remove the periplasmic export tag from the gene.</p><br />
<p><br />
To do this, we use the following cloning strategy (noting that the top row represents the gene initially synthesised in plasmid pMK):</p><br />
<img src="https://static.igem.org/mediawiki/2014/2/2c/Melbourne_cloning_strategy_diagram.jpg" width="900" height="437" alt=""/><br />
<p>The gene was inserted into plasmid pSB1C3 containing the T7 promoter and ribosome binding site, BBa_K525998. It was inserted after the promoter and before the BioBrick suffix. This was accomplished by digesting the destination vector with SpeI and PstI. At the same time, PCR was used to amplify the segment of the gene containing the SUMO fusion and the Magainin 1 Star peptide, adding XbaI and keeping the PstI site existing in the gene.</p><br />
<p><br />
Digestion of the PCR product then allowed for ligation of the insert and destination vector using the PstI sites and XbaI and SpeI (XbaI and SpeI have compatible sticky ends). Note that after the ligation, there will be a scar in the gene where the XbaI and the SpeI sites were ligated.</p><br />
<p><br />
The ligation appeared to be successful. The ligation mixture was transformed to DH5α competent cells and plated onto chloramphenicol-containing agar plates.</p><br />
<p><br />
Plasmid from 5 colonies (C1, C2, C3, C4, and C5) was cultured, extracted, and then digested with EcoRI and PstI.</p><br />
<p><br />
As evident in the figure below, at least 4 of the colonies appeared to contain the insert. The empty, linearised pSB1C3 backbone ran at approximately the correct size (2070 bps), as did the insert (740 bps).</p><br />
<img src="https://static.igem.org/mediawiki/2014/9/95/Magainin_1_subcloning.png" width="353" height="400" alt=""/><br />
<p>N.B. MW marker is the 100 bp Ladder from Axygen. * indicates the colony picked for sequencing and eventual transformation to the expression cell lines.</p><br />
<p>The DNA from Colony C2 was confirmed using Sanger sequencing at the Australian Genome Research Facility. This DNA appears in the registry of standard parts as BioBrick <a target="_blank" href="http://parts.igem.org/Part:BBa_K1394000">BBa_K1394000</a>.</p><br />
<p><br />
For the USP peptide and the linear Magainin 1 peptide, the genes were synthesised by GenScript and delivered in pSB1C3. The expression vectors had identical gene regulatory elements to that used for the Magainin 1 Star peptide. They only differed in the codon optimisation used, and they also lacked the assembly scar described above.</p><br />
<br />
<h2><br />
Protein expression and characterisation<br />
</h2><br />
<p><br />
After acquiring our genes, the project moved to protein expression and purification. We expressed both star peptides (Magainin 1 Star Peptide and the USP peptide) in both the SHuffle T7 and BL21(DE3) cell lines, both sourced from New England Biolabs. As the Linear Magainin 1 peptide does not have any special disulfide bonding<br />
requirements, we expressed it in BL21(DE3).<br />
</p><br />
<p><br />
The plasmids were transformed to the expression cells, and a single colony was cultured and induced overnight at 17 °C. A whole cell sample both before<br />
IPTG induction (-IPTG) and after the induction period (+IPTG) were boiled in SDS-PAGE sample buffer and loaded on a 15% tris-glycine gel. The whole cell<br />
Coomassie stained gel is shown below alongside the NEB P7712S molecular weight marker.<br />
</p><br />
<br><br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/2/24/Whole_cell_CS.png" height="330" width="439"/><br />
</p><br />
<br><br />
<p><br />
From theoretical prediction of the peptide masses, we would expect the following distribution of molecular weights:<br />
</p><br />
<br><br />
<table border="1" cellpadding="0" cellspacing="0" align="center"><br />
<tbody><br />
<tr><br />
<td width="156"><br />
<p align="center"><br />
Magainin 1 Star Peptide (Mag1 Star)<br />
</p><br />
</td><br />
<td width="145"><br />
<p align="center"><br />
USP<br />
</p><br />
</td><br />
<td width="156"><br />
<p align="center"><br />
Linear Magainin 1 (Linear Mag1)<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="156"><br />
<p align="center"><br />
22.15 kDa<br />
</p><br />
</td><br />
<td width="145"><br />
<p align="center"><br />
29.3 kDa<br />
</p><br />
</td><br />
<td width="156"><br />
<p align="center"><br />
14.32 kDa<br />
</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<br><br />
<p><br />
We looked for bands corresponding to overexpression of the proteins. We observed a thick band, post IPTG around 32 kDa in the BL21(DE3) cells carrying the<br />
USP plasmid. However, we saw this same band appearing in the Linear Magainin 1 lane but the Linear Magainin 1 protein should have a much lower molecular<br />
weight than what was observed. Therefore, we did not assume that we had overexpression overexpression of the USP 1 protein.<br />
</p><br />
<p><br />
We also noted bands in all post-IPTG lanes around 17 kDa. This would be roughly consistent with both the Linear Magainin 1 and Magainin 1 Star Peptide,<br />
noting that small proteins may not run at their expected molecular weight. Again, the analysis is complicated by the fact that the band also appears in the<br />
lane corresponding to the larger molecular weight protein, USP I. Given this ambiguity, we decided to purify all the protein in the sample using Ni-NTA<br />
purification.<br />
</p><br />
<h3><br />
Purification and further characterisation<br />
</h3><br />
<p><br />
A small-scale purification was carried out according to our <a href="https://static.igem.org/mediawiki/2014/e/e1/MELBOURNE_D1V1_Column_Purification_of_His-Tagged_Proteins.pdf">protocol</a>. The cells were lysed with iodoacetamide, an alkylating agent, added to the lysis buffer. Iodoacetamide blocks all free cysteines on proteins with a short alkyl group. This was added<br />
because we wanted to determine if a disulphide bond had formed inside the cell. If we did not block free cysteines, then any bond that had formed could be<br />
attributed to spontaneous/air oxidation of disulfides outside the cell.<br />
</p><br />
<p><br />
After purification, we ran a concurrent Western Blot and Coomassie stain on both the pre-induction samples from above and the purified protein (namely, the<br />
first elution from the batch purification).<br />
</p><br />
<p><br />
The Western Blot used mouse monoclonal antibodies against the N-terminal HIS-tag as primary antibodies and anti-mouse secondary antibodies:<br />
</p><br />
<br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/5/54/Western_Blot_Melb.png" height="400" width="460" border="0"/><br />
</p><br />
<p><br />
The corresponding Coomassie stain is show below:<br />
</p><br />
<br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f0/Purified_CS.png" height="365" width="486" border="0"/><br />
</p><br />
<p><br />
There are several interesting aspects of the Western Blot. Firstly, there was a strong band between 25 and 32 kDa in most lanes. However, it<br />
appears in all of the pre-induction controls, suggesting non-specific binding of the anti-HIS antibodies.<br />
</p><br />
<p><br />
Secondly, we observed a band in most of the lanes around 17 kDa (with the exception being the USP protein in SHuffle cells). As these bands were not<br />
present in the pre-induction controls, we suspected they were a result of the induction.<br />
</p><br />
<h3><br />
Mass spectroscopy<br />
</h3><br />
<p><br />
In order to assess the identity of the bands, we performed an in-gel tryptic digestion on select bands. We digested bands in the Coomassie stain which<br />
corresponded to the HIS-tagged bands in the Western Blot. This was followed by mass spec analysis (LC MS/MS). We focused our analysis on the Magainin 1<br />
Star Peptide bands and the Linear Magainin 1 band. Our USP peptide was, by design, highly rich in basic residues, greatly reducing the likelihood that<br />
tryptic fragments would be detected by the mass spec.<br />
</p><br />
<p><br />
We found that the Linear Magainin 1 peptide appeared to be present in the approximately 17 kDa band, with the following detected tryptic fragments in the<br />
sequence below (bold; basic residues underlined):<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/1/1b/Tryptic_digest_linear_mag1.PNG" height="63" width="601" border="0"/><br />
</p><br />
<p><br />
Note that spaces have been added in the sequence to emphasise distinct domains in the protein (in this case, the fusion protein versus the Magainin 1<br />
peptide). Together with the fact that the protein runs close to the expected molecular weight, this seems to provide good evidence that the protein is<br />
being expressed.<br />
</p><br />
<p><br />
We then examined the bands corresponding to the Magainin 1 Star Peptide.<br />
</p><br />
<p><br />
Again, we digested bands in the Coomassie stained gel corresponding to the prominent HIS-tagged bands on the <em>Western Blot</em> (near 17 kDa).<br />
</p><br />
<p><br />
The mass spec coverage for the Magainin 1 Star Peptide expressed in SHuffle cells was as follows:<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/6/63/Tryptic_digest_Sample_A_Star_Mag_Shuffle.PNG" height="90" width="601" border="0"/><br />
</p><br />
<p><br />
The coverage for the same peptide expressed in BL21(DE3) was found to be:<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/d/db/Tryptic_digest_Sample_A_Star_Mag_BL21%28DE3%29.PNG" height="85" width="602" border="0"/><br />
</p><br />
<p><br />
Again, we can see that tryptic peptides from the expressed protein appear to present in the gel band under analysis.<br />
</p><br />
<p><br />
There is a relatively lower sequence coverage. This could be a limitation of our procedure: for example, improper destaining during the in gel digestion<br />
procedure can inhibit tryptic digestion.<br />
</p><br />
<p><br />
However, even if the digestion was complete, the mass spec will only detect fragments which ionize well. The Magainin 1 Star peptide consists of four<br />
repeats of the Magainin 1 sequence (corresponding to the arms of the star). If the two tryptic fragments within Magainin 1 do not ionize well, then indeed<br />
the entire peptide would not be read.<br />
</p><br />
<p><br />
Finally, it is possible that the protein has been cleaved or degraded. This would account for the slightly lower-than-expected mass on the SDS page gel.<br />
</p><br />
<p><br />
Nonetheless, there is the distinct possibility that the peptide fragments are there but not detected. Replication of the in-gel mass spec would be useful<br />
in clarifying this point, or provided the purity of the sample is acceptable, it may be possible to run an intact mass spec.<br />
</p><br />
<h2><br />
Conclusions<br />
</h2><br />
<p><br />
We designed several novel star peptides based on several rational design criteria. Further, we began the optimisation process required to express these peptides in <em>E. coli</em>.<br />
</p><br />
<p><br />
To this end, we constructed an expression system based on the SUMO protein and standard BioBrick parts. The function of this system was confirmed, as it<br />
appears it does produce HIS-tagged SUMO fusion protein in <em>E. coli</em>, as expected.<br />
</p><br />
<p><br />
Although it appears the expression system works, there was some uncertainty about particular peptides, namely the Magainin 1 Star Peptide and USP Peptide.<br />
A Westerm Blot suggested that at least part of the peptides are present. However, additional sequence coverage in a mass spec analysis and intact mass spec<br />
would help us determine the precise identity of our products.<br />
</p><br />
<p><br />
We have shown that the SUMO fusion can be expressed in our BioBrick. Given the success of SUMO in the general scientific community, we hope our BioBrick<br />
will encourage further use of the fusion domain within the iGEM community. In addition, we hope that our ideas and concepts will inspire future<br />
iGEM efforts to explore the applications of synthetic biology for material science. We continue to believe that bacteria have great promise for the <em>in vivo</em><br />
construction of unique biomaterials, and we look forward to seeing how the synthetic biology community will develop in this area.<br />
</p><br />
<br />
<br />
<br />
<h2>References</h2><br />
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<p><br />
BANEYX, F. 1999. Recombinant protein expression in Escherichia coli. <em>Current Opinion in Biotechnology,</em> 10<strong>,</strong> 411-421.</p><br />
<p><br />
BOMMARIUS, B., JENSSEN, H., ELLIOTT, M., KINDRACHUK, J., PASUPULETI, M., GIEREN, H., JAEGER, K. E., HANCOCK, R. E. W. &amp; KALMAN, D. 2010. Cost-effective expression and purification of antimicrobial and host defense peptides in Escherichia coli. <em>Peptides,</em> 31<strong>,</strong> 1957-1965.</p><br />
<p><br />
BROGDEN, K. A. 2005. Antimicrobial peptides: Pore formers or metabolic inhibitors in bacteria? <em>Nature Reviews Microbiology,</em> 3<strong>,</strong> 238-250.</p><br />
<p><br />
CHANG, C., CHHOR, G., CLANCY, S. & JOACHIMIAK, A. 2014. Crystal Structure of Glutathione S-transferase Domain Protein From Haliangium Ochraceum DSM 14365 [Online]. ''NCBI. Available: http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=4w66' [Accessed September 9 2014].</p><br />
<p><br />
CHEN, X., ZHU, F., CAO, Y. &amp; QIAO, S. 2009. Novel expression vector for secretion of cecropin AD in Bacillus subtilis with enhanced antimicrobial activity. <em>Antimicrobial agents and chemotherapy,</em> 53<strong>,</strong> 3683-3689.</p><br />
<p><br />
GLINEL, K., JONAS, A. M., JOUENNE, T., LEPRINCE, J., GALAS, L. & HUCK, W. T. 2008. Antibacterial and antifouling polymer brushes incorporating antimicrobial peptide. <em>Bioconjug Chem,</em> 20<strong>,</strong> 71-77.</p><br />
<p><br />
HUMBLOT, V., YALA, J.-F., THEBAULT, P., BOUKERMA, K., HÉQUET, A., BERJEAUD, J.-M. & PRADIER, C.-M. 2009. The antibacterial activity of Magainin I immobilized onto mixed thiols self-assembled monolayers. <em>Biomaterials,</em> 30<strong>,</strong> 3503-3512.</p><br />
<p><br />
DAWSON, P. E., MUIR, T. W., CLARK-LEWIS, I. &amp; KENT, S. 1994. Synthesis of proteins by native chemical ligation. <em>Science,</em> 266<strong>,</strong> 776-779.</p><br />
<p><br />
KADOKURA, H. &amp; BECKWITH, J. 2009. Detecting folding intermediates of a protein as it passes through the bacterial translocation channel. <em>Cell,</em> 138<strong>,</strong> 1164-1173.</p><br />
<p><br />
KADOKURA, H., KATZEN, F. &amp; BECKWITH, J. 2003. Protein disulfide bond formation in prokaryotes. <em>Annual review of biochemistry,</em> 72<strong>,</strong> 111-135.</p><br />
<p><br />
LI, Y. 2011. Recombinant production of antimicrobial peptides in&lt; i&gt; Escherichia coli&lt;/i&gt;: A review. <em>Protein Expression and Purification,</em> 80<strong>,</strong> 260-267.</p><br />
<p><br />
LOBSTEIN, J., EMRICH, C. A., JEANS, C., FAULKNER, M., RIGGS, P. &amp; BERKMEN, M. 2012. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. <em>Microb Cell Fact,</em> 11<strong>,</strong> 56-56.</p><br />
<p><br />
PORTAL, P. M. Query result, GST and DcmA [Online]. Available: http://www.proteinmodelportal.org/query/uniprot/P21161 [Accessed September 9 2014].</p><br />
<p><br />
STEINER, D., FORRER, P., STUMPP, M. T. &amp; PLUCKTHUN, A. 2006. Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display. <em>Nat Biotech,</em> 24<strong>,</strong> 823-831.</p><br />
<p><br />
SULISTIO, A., GURR, P. A., BLENCOWE, A. &amp; QIAO, G. G. 2012. Peptide-Based Star Polymers: The Rising Star in Functional Polymers. <em>Australian Journal of Chemistry,</em> 65<strong>,</strong> 978-984.</p><br />
<p><br />
SULISTIO, A., LOWENTHAL, J., BLENCOWE, A., BONGIOVANNI, M. N., ONG, L., GRAS, S. L., ZHANG, X. &amp; QIAO, G. G. 2011. Folic Acid Conjugated Amino Acid-Based Star Polymers for Active Targeting of Cancer Cells. <em>Biomacromolecules,</em> 12<strong>,</strong> 3469-3477.</p><br />
<p><br />
TANAKA, N., KUSAKABE, Y., ITO, K., YOSHIMOTO, T. & NAKAMURA, K. T. 2002. Crystal Structure of Formaldehyde Dehydrogenase from< i> Pseudomonas putida</i>: the Structural Origin of the Tightly Bound Cofactor in Nicotinoprotein Dehydrogenases. Journal of molecular biology, 324, 519-533.</p><br />
<p><br />
WIRADHARMA, N., LIU, S. Q. &amp; YANG, Y. Y. 2012. Branched and 4-Arm Starlike α-Helical Peptide Structures with Enhanced Antimicrobial Potency and Selectivity. <em>Small,</em> 8<strong>,</strong> 362-366.</p><br />
<p><br />
YU, Z., WANG, Q., MA, Q. &amp; ZHANG, R. 2013. Secretory expression of lacticin Q fused with SUMO in Bacillus subtilis. <em>Protein Expression and Purification,</em> 89<strong>,</strong> 51-55.</p><br />
<p><br />
ZASLOFF, M. 1987. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. <em>Proceedings of the National Academy of Sciences,</em> 84<strong>,</strong> 5449-5453.</p><br />
<p>&nbsp;</p><br />
<br />
<A NAME="Oxford"></A><br />
<h1>Collaboration with Oxford</h1><br />
<p>To strengthen the sense of iGEM community, we contacted <a target="_blank" href="https://2014.igem.org/Team:Oxford">University of Oxford iGEM team</a> to discuss the possibility of collaboration between our teams. The Oxford project aims to develop a system that can dispose of the carcinogenic, hazardous solvent dichloromethane (DCM). To do this, Oxford team has proposed the use of the DcmA enzyme. This enzyme degrades DCM to form a toxic intermediate which in turn is converted by another enzyme, FdhA, into a neutral molecule. Schematically this can be presented in the following way:</p><br />
<br />
<p><br />
Reaction 1: DCM +&nbsp;DcmA <strong>toxic intermediate</strong><br><br />
Reaction 2: <strong>toxic intermediate</strong> + FdhA neutral product.</p><br />
<p>The problem for their system lies in bringing the two enzymes together to ensure efficient reaction kinetics. An idea that we discussed with Oxford was to use the star peptide platform to link the two enzymes together, in a structure similar to the following: </p><br />
<img src="https://static.igem.org/mediawiki/2014/2/2e/Melbourne_Collab_1.png" width="500" height="325" alt=""/><br />
<p>We worked with Oxford to study this system from a theoretical standpoint. Our team studied the 3D structure of both enzymes involved to confirm that the enzyme could in theory be attached to a linker in this manner, while Oxford team did stochastic modelling to determine how reaction rate changes when the linker length, labeled D on the diagram, changes. </p><br />
<p><br />
As a first check, it was necessary to determine whether anchoring these enzymes to our star would be sterically possible. We studied the structures of FdhA and DcmA determined by crystallography using data from the protein data bank. As no crystallographic data was available in the databank for DcmA, GST was examined as a proxy, as GST is structurally homologous to DcmA. </p><br />
<p><br />
The crystalographically resolved structure of FdhA enzyme (Tanaka et al., 2002) from the protein data bank revealed the following image:</p><br />
<img src="https://static.igem.org/mediawiki/2014/d/dc/Melbourne_Collab_2.png" width="700" height="355" alt=""/><br />
<p>The crystalographically resolved structure of GST was as follows (Chang et al., 2014):</p><br />
<img src="https://static.igem.org/mediawiki/2014/e/e9/Melbourne_Collab_3.png" width="800" height="361" alt=""/><br />
<p>It can be seen that the amino- and carboxy-terminals are located away from the active site. This suggests that both enzymes, DcmA and FdhA, might be linked together via linkers that are used in our star peptide. That is, the active site will not be sterically hindered by attachment to the star peptide. That said, it is difficult to predict how anchoring the protein to the star</div>Slowehttp://2014.igem.org/Team:Melbourne/TeamTeam:Melbourne/Team2014-10-18T01:35:40Z<p>Slowe: </p>
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<h1 >Team</h1><br />
<br />
<h3>About The University of Melbourne</h3><br />
<p>Located in Melbourne, Victoria, The University of Melbourne was founded in 1853 and is Australia’s second oldest university. The university is consistently ranked among the leading universities in the world, with international rankings of world universities placing it at number 1 in Australia and number 34 in the world (Times Higher Education World University Ranking 2013-2014).</p><br />
<br />
<h3>About The 2014 Melbourne iGEM Team</h3><br />
<p>The 2014 Melbourne iGEM team consists of 19 highly-motivated undergraduate students and 1 postgraduate student from a range of disciplines, including biochemistry and molecular biology, chemistry, chemical engineering and bioengineering. The team’s academic and research credentials are well-tested. This year, the team comprises three of The University of Melbourne’s Chancellor’s Scholars (top 0.1% of all Australian high school students) and several team members with research experience at Melbourne’s high-profile research institutes, such as the prestigious Walter and Eliza Hall Institute, The Peter MacCallum Cancer Centre, and The Baker IDI Heart and Diabetes Institute. The team is supervised by Dr Heung-Chin Cheng, Professor Paul Gooley, Dr Angus Johnson and Dr Neil O’Brien-Simpson.<br />
The Melbourne iGEM team was founded by a group of enthusiastic and motivated students in 2013. The 2013 team played a key role in establishing the foundations of this year’s team and generating this year’s project. Unfortunately many of the 2013 team members could not continue their work for iGEM in 2014, so credit must also be given to Pedro Avellar Costa, Michelle Tie, Georgina Panshem, Morgana Cerqueira, Jeong Yoon Esther Kim, Winnie Tan, Hannah Nguyen, Mary Teo, Cathy Pitt and Joyce Kant.</p><br />
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<h3>Supervisors and Advisors</h3><br />
<table width="100%"><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/d/d4/Melbourne_Cheng.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Heung-Chin Cheng (Supervisor)</strong></p><br />
An Associate Professor with the Department of Biochemistry and Molecular Biology, Heung-Chin has been studying the biochemical basis of the regulation of protein kinases and phosphatases since he was a graduate student at the University of California in 1982. His PhD project unravelled the active site structure of c-AMP-dependent protein kinase, how the kinase recognises its protein substrates and how the kinase is selectively inhibited by its endogenous inhibitor. Heung-Chin was the Melbourne iGEM team&rsquo;s supervisor and he kindly offered us space to work in his lab and assisted us when we had questions about practical procedures, troubleshooting or theory.</td><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/7/74/Melbourne_Gooley.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Paul Gooley (Advisor)</strong></p><br />
Associate Professor Paul Gooley directs a structural biology research group that focuses on the application of NMR spectroscopy to elucidate structure and protein interactions. He obtained his degrees at The University of New South Wales and spent 10 years in the USA, including 5 years at the pharmaceutical company Merk and Co. Over the last 10 years his group has conducted and published NMR structural and dynamical analyses on a number of protein domains and systems that have biological functions in stress and infection, in lipid transport, in protein and membrane trafficking, and in receptor signalling. Paul assisted the 2014 Melbourne iGEM team by providing advice, answering questions and brainstorming.</td><br />
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<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/9/97/Melbourne_Angus.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Angus Johnston (Advisor)</strong></p><br />
Dr Angus Johnston is a researcher at the Monash Institute of Pharmaceutical Science. Angus is an ARC Future Fellow who’s work focuses on developing better ways to deliver drugs, making them more therapeutically active and limiting side effects. He has extensive knowledge and expertise in nanomaterials assembly, material characterisation, cellular interactions and advanced imaging techniques. Angus’ research interests include targeted vaccine therapy, sensors for cellular imaging, understanding cellular processing of nanoparticles, self assembling peptides as drug carrier and the toxicology of nanomaterials. Angus assisted the 2014 Melbourne iGEM team by providing advice, answering questions and brainstorming.</td><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/f/fd/Melbourne_Neil_O%E2%80%99Brien-Simpson.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Neil O’Brien-Simpson (Advisor)</strong></p><br />
Associate Professor Neil O’Brien-Simpson is a researcher at the Royal Dental Hospital in Melbourne and has an interdisciplinary background, combining organic and peptide chemistry with immunology to develop novel vaccines and therapeutics and investigating the immune response to pathogens. His research into vaccine design and periodontitis has resulted in Neil being awarded the Colgate Prize for Dental Research (1999), The IADR Hatton Award (2000) and the Oral Biology Award (2003). In 2004 he became program co-leader for the Novel Diagnostics, Vaccines and Pharmaceuticals Research Program in the Oral Health CRC. Neil assisted the 2014 Melbourne iGEM team by providing advice, answering questions and brainstorming.</td><br />
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</table><br />
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<h3>Team Members</h3><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/2/25/Melbourne_Sean.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Sean Lowe (Team Leader)</strong></p><br />
Sean started the 2014 Melbourne iGEM team while completing his Master of Engineering in chemical engineering. As the team leader, Sean was responsible for communicating with supervisors and sponsors, setting up the lab, and making sure the team ran smoothly. Sean is passionate about science and saw iGEM as an amazing opportunity to combine his interests in chemical engineering and biotechnology. He found that the best part of iGEM was the creative process, and thoroughly enjoyed discussing many great ideas with his bright and capable teammates. He looks forward to passing on the torch to the next generation of Melbourne Uni iGEMers.</td><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/a/a6/Melbourne_Lizzy.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Elizabeth Brookes</strong></p><br />
Elizabeth is in her second year of the Bachelor of Biomedicine degree and is majoring in Immunology. In the future Elizabeth hopes to continue to develop her passion for research while studying postgraduate medicine. Elizabeth was new to iGEM in 2014 and contributed to Melbourne’s iGEM project by working in the laboratory, developing protocols and performing various administrative duties. For Elizabeth, the most enjoyable aspect of iGEM was getting hands-on experience while meeting like-minded people. In her free time Elizabeth enjoys playing netball and discovering new restaurants with friends.</td><br />
</tr><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/f/f6/Melbourne_Peter.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Peter Collins</strong></p><br />
Peter is in his final year of the Bachelor of Science at The Australian National University in Canberra and is majoring in Science Communications, as well as completing his first year of the Bachelor of Environments at The University of Melbourne. In the future, Peter aims to work on The Human Variome Project, develop the iGEM competition in Australia and work for UNESCO. Peter joined the Melbourne iGEM team in 2014 and was in charge of human practices and outreach, working to establish relations with high schools in Melbourne for on-going public outreach efforts in the future. As such, Peter has enjoyed getting an insight into managing science innovation teams and the tremendous array of challenges that accompany this. In his spare time, Peter enjoys learning about esoteric topics that may have unrealised commercial applications.</td><br />
</tr><br />
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<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/1/1a/Melbourne_Sheryl.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Sheryl Ding</strong></p><br />
Sheryl is in the first year of a Bachelor of Biomedicine degree and is hoping to major in Bioengineering Systems. In the future, Sheryl would like to work in biomedical research, preferably specialising in engineering or molecular and cellular biology. After joining the iGEM team in early 2014, Sheryl contributed to the laboratory work and some administrative tasks for the project. Sheryl enjoyed all aspects of this experience, but particularly the opportunity to gain practical insight into laboratory research and meet the people on the iGEM team. Outside of science, Sheryl enjoys visual art and watching TV shows.</td><br />
</tr><br />
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<td width="180" ><img src="https://static.igem.org/mediawiki/2014/6/66/Melbourne_Robyn.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Robyn Esterbauer</strong></p><br />
As a second year student of the Bachelor of Science with a major in Microbiology and Immunology, Robyn hopes to undertake a masters and PhD, specialising in infectious disease research. After joining the team in March 2014, Robyn contributed to the team with her work in the laboratory, research into cysteine bond formation and computational protein design analysis, and public outreach activities. Robyn has enjoyed experiencing a group research environment and learning some very useful practical laboratory skills. In her free time, Robyn loves to read cutting edge infectious disease research and playing soccer.</td><br />
</tr><br />
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<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/d/d4/Melbourne_Tobias.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Tobias Gustavsson</strong></p><br />
Tobias is in his second year of the Bachelor of Biomedicine degree and is majoring in Immunology. In the future Tobias hopes to study medicine and continue to pursue research in immunology. Tobias was new to iGEM in 2014 and contributed to the theoretical design and research of the team’s project. iGEM presented Tobias with the opportunity to finally partake in the laboratory work that he had heard so much about in lectures.</td><br />
</tr><br />
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<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/e/e8/Melbourne_Henry.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Henry Howard</strong></p><br />
Henry has just completed his Honours year for the Bachelor of Biomedicine and in the future he hopes to undertake a PhD and work in Melbourne’s bustling medical research sector. After joining the iGEM team in February 2013, Henry predominantly contributed to the theoretical design of the project. He most enoyed having access to resources that have allowed us to undertake serious scientific research of our own design. Outside of iGEM, Henry is a black belt in Tang Soo Tao and enjoys planting trees and completing jigsaw puzzles.</td><br />
</tr><br />
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<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/8/8f/Melbourne_Aishwarya.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Aishwarya Kulkarni</strong></p><br />
Aishwarya is in her second year of the Bachelor of Science and is majoring in Biochemistry and Molecular Biology. Her future plans include completing a PhD and research in the area of translational bioinformatics and personalised medicine. As part of the Melbourne iGEM 2014 team, Aishwarya contributed to laboratory and budgeting tasks. She enjoyed both the atmosphere of the team and the challenges with which we were confronted when trying to work independently to solve problems. Outside of iGEM, Aishwarya also enjoys playing tennis.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/2/2a/Melbourne_Ranit.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Ranit Lahmy</strong></p><br />
Ranit is in her final year of the Bachelor of Science degree and is completing her major in Chemistry. Ranit joined the Melbourne iGEM team in early 2013 and has contributed to many facets of the project, including laboratory work, administration and human outreach. As a consequence, Ranit has really enjoyed the chance to meet new people while learning research and laboratory skills. When not working on iGEM, Ranit enjoys reading books, playing tennis and hanging out with friends.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/b/bc/Melbourne_Jeffrey.JPG" width="150" height="196" alt=""/></td><br />
<td><p><strong>Jeffrey Lai</strong></p><br />
Jeffrey is in his first year of the Bachelor of Biomedicine at The University of Melbourne and in the future he would like to become a medical practitioner or researcher. After joining the Melbourne team in July 2014, Jeffrey helped out in the laboratory work and the iGEM website. Jeffrey found it incredibly rewarding when we finally saw some promising results after putting so much time and effort into the project. In his spare time, Jeffrey likes to play the piano and compose symphonic music.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/1/10/Melbourne_Melissa.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Melissa Leckie</strong></p><br />
Melissa is in her final year of the Bachelor of Science and is completing a major in biochemistry and molecular biology. Competing in her first iGEM jamboree in 2008, Melissa joined the current team in 2013 and has contributed to project design, administration and human outreach. Through this experience Melissa has enjoyed making life long friends and facing the challenges of being part of a student run team. Melissa wishes to pursue a career in Medical Technology and in her spare time she enjoys reading and travelling.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/3/3c/Melbourne_Keit.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Keit Loi</strong></p><br />
Keit is in his final year of the Bachelor of Biomedicine and is majoring in infection and immunity. Since joining the Melbourne iGEM team in early 2014, Keit has mainly contributed to the laboratory work for the project and he has enjoyed that this has given him a good understanding of what working in research would be like. In the future, Keit would like to pursue this passion and work towards a PhD and university lecturing. Outside of iGEM, Keit works on projects with high school students and enjoys chilling with friends and kicking back with some video games.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/0/07/Melbourne_Darcy.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Darcy Marum</strong></p><br />
Darcy is in his second year of the Bachelor of Science degree and is majoring in Microbiology and Immunology. Darcy hopes to pursue research in these exciting fields after completing an honours year. After joining the team in early 2014, Darcy predominantly worked on laboratory work for the project and enjoyed the practical experience that he gained.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/2/2e/Melbourne_Gayle.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Gayle Pereira</strong></p><br />
Gayle is in her final year of the Bachelor of Science and is majoring in Pathology. In the future Gayle hopes to pursue a career either as a clinical pathologist or secondary school teacher. Gayle joined the iGEM team in 2013 and was the senior laboratory leader for the group, tirelessly working and teaching others in the laboratory. Working on the project, Gayle appreciated gaining the laboratory experience that she requires for her future ambitions. In her free time Gayle enjoys supporting her team in the Australian Football League and doing handicraft-based hobbies.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/b/b1/Melbourne_Lumi.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Lumi Tomren</strong></p><br />
Lumi is in her first year of the Bachelor of Biomedicine at The University of Melbourne and is planning on majoring in Microbiology and Immunology. With a desire to learn more about synthetic biology and the microscopic world around us, Lumi joined the team in Spring 2014. Lumi has contributed to the team by performing lab work and she has really enjoyed learning a lot of new practical techniques. She has also enjoyed discovering many of the possibilities of synthetic biology by researching past projects undertaken by iGEM teams. Outside of iGEM and science, Lumi enjoys visual art.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/d/d6/Melbourne_Michael.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Michael Wei</strong></p><br />
Michael is in his first year of the Bachelor of Biomedicine and plans on majoring in neuroscience. Michael was new to iGEM in 2014 and worked primarily in the laboratory and designing primers. Michael is interested in working towards a career in neurosurgery or cancer tumour research, looking specifically at the epigenetic mechanisms underlying cancer regulation. As part of the iGEM team, Michael has enjoyed meeting like-minded individuals who share similar interests. Michael is fascinated by marine life and enjoys reading in his leisure time.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/0/07/Melbourne_Stan.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Stanislav Yatskevich</strong></p><br />
Although Stanislav started studying his first year of a Bachelor of Biomedicine at The University of Melbourne, he has now commenced study at The University of Oxford reading Biochemistry. Stanislav joined the Melbourne iGEM team in March 2014 and contributed greatly to the theoretical project design and laboratory work. After moving to Oxford, Stanislav also facilitated communication between our team and the Oxford iGEM team. In the future, Stanislav would like to pursue research in Biochemistry and he has appreciated the amount of experience he was able to gain in such a short time while taking part in iGEM. Outside of Biochemistry, Stanislav enjoys playing football and procrastinating his study.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/d/dc/Melbourne_Nicholas.JPG" width="150" height="196" alt=""/></td><br />
<td><p><strong>Nicholas Yee</strong></p><br />
Nicholas has just completed the Honours year of his Bachelor of Biomedicine degree, studying novel pathway inhibitors to elucidate mechanisms of brain tumours. He plans to continue his research into brain tumours by undertaking a PhD and potentially studying medicine. After joining the team in 2014, Nicholas enjoyed helping the younger students in the team by teaching them new laboratory techniques and troubleshooting their experiments. When not in the laboratory, Nicholas enjoys hanging out with friends and playing badminton.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/1/1b/Melbourne_Michelle.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Michelle Zheng</strong></p><br />
Michelle is in her second year of the Bachelor of Science and is undertaking the Biochemistry major. At the completion of her degree, Michelle seeks to pursue further research through the honours or masters programmes. Michelle joined the Melbourne iGEM team in 2014 and completed many hours of laboratory work contributing to the project. Being part of a team dedicated to science, the laboratory work and the challenge of overcoming troubleshooting problems were the aspects of iGEM that excited Michelle most. In her free time she enjoys playing the violin.</td><br />
</tr><br />
<br />
<tr><br />
<td width="180" ><img src="https://static.igem.org/mediawiki/2014/0/0f/Melbourne_Wayne.jpg" width="150" height="196" alt=""/></td><br />
<td><p><strong>Wayne Zheng</strong></p><br />
Wayne is in his first year of the Bachelor of Biomedicine and hopes to eventually major in Human Structure and Function. In the future, Wayne would like to continue his involvement in biomedical research while also studying and practising medicine. In contributing to the 2014 Melbourne iGEM team Wayne performed laboratory work as well as some theoretical research. Wayne loved learning new techniques in the laboratory and reading into a wide range of research topics.</td><br />
</tr><br />
<br />
</table><br />
<br />
<br />
<h3>Advisors</h3><br />
<p>A special mention must also be made to our Honours, Masters and PhD student advisors in the Cheng Lab who tirelessly and patiently assisted with our troubleshooting and planning of experimental procedures. These students were: <br />
<br />
Ashfaqul Hoque (PhD student), Gahana Advani (PhD student), George Cao (Masters student), Ken Ang (Honours student) and David Zula (Honours student).</p><br />
<br />
<table><br />
<tr><br />
<td width="152" ><img src="https://static.igem.org/mediawiki/2014/5/58/Melbourne_Ashfaqul.jpg" width="150" height="196" alt=""/></td><br />
<td width="10" ></td><br />
<td width="152" ><img src="https://static.igem.org/mediawiki/2014/d/da/Melbourne_Gahana.jpg" width="150" height="196" alt=""/></td><br />
<td width="10" ></td><br />
<td width="152" ><img src="https://static.igem.org/mediawiki/2014/f/f8/Melbourne_George.jpg" width="150" height="196" alt=""/></td><br />
<td width="10" ></td><br />
<td width="152" ><img src="https://static.igem.org/mediawiki/2014/a/aa/Melbourne_Ken.jpg" width="150" height="196" alt=""/></td><br />
<td width="10" ></td><br />
<td width="152" ><img src="https://static.igem.org/mediawiki/2014/9/9c/Melbourne_Dave.jpg" width="150" height="196" alt=""/></td><br />
</tr><br />
<br />
<tr><br />
<td align="center"><strong>Ashfaqul Hoque</strong></td><br />
<td></td><br />
<td align="center"> <strong>Gahana Advani</strong></td><br />
<td></td><br />
<td align="center"> <strong>George Cao</strong></td><br />
<td></td><br />
<td align="center"><strong>Ken Ang</strong></td><br />
<td></td><br />
<td align="center"><strong>David Zula</strong></td><br />
</tr><br />
<br />
</table><br />
<br />
<p>&nbsp;</p><br />
<p>Many thanks also go to <strong>Sze-Ting Bong</strong> and <strong>Anna Gakusurudoi</strong>, Masters students in the Cheng lab whose assistance with practical methods was extremely helpful.<br />
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* 1. Correct font family not being inherited in all browsers.<br />
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* 3. Address margins set differently in Firefox 4+, Safari 5, and Chrome.<br />
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<br />
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input {<br />
line-height: normal;<br />
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<br />
/**<br />
* Address inconsistent `text-transform` inheritance for `button` and `select`.<br />
* All other form control elements do not inherit `text-transform` values.<br />
* Correct `button` style inheritance in Chrome, Safari 5+, and IE 8+.<br />
* Correct `select` style inheritance in Firefox 4+ and Opera.<br />
*/<br />
<br />
button,<br />
select {<br />
text-transform: none;<br />
}<br />
<br />
/**<br />
* 1. Avoid the WebKit bug in Android 4.0.* where (2) destroys native `audio`<br />
* and `video` controls.<br />
* 2. Correct inability to style clickable `input` types in iOS.<br />
* 3. Improve usability and consistency of cursor style between image-type<br />
* `input` and others.<br />
*/<br />
<br />
button,<br />
html input[type="button"], /* 1 */<br />
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*/<br />
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button[disabled],<br />
html input[disabled] {<br />
cursor: default;<br />
}<br />
<br />
/**<br />
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}<br />
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* 2. Address `box-sizing` set to `border-box` in Safari 5 and Chrome<br />
* (include `-moz` to future-proof).<br />
*/<br />
<br />
input[type="search"] {<br />
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<br />
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* 1. Remove default vertical scrollbar in IE 8/9.<br />
* 2. Improve readability and alignment in all browsers.<br />
*/<br />
<br />
textarea {<br />
overflow: auto; /* 1 */<br />
vertical-align: top; /* 2 */<br />
}<br />
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/* ==========================================================================<br />
Tables<br />
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<br />
/**<br />
* Remove most spacing between table cells.<br />
*/<br />
<br />
table {<br />
border-collapse: collapse;<br />
border-spacing: 0;<br />
}<br />
<br />
html, body {<br />
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<br />
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</style><br />
<img src="https://static.igem.org/mediawiki/2014/9/9b/Melbourne_Banner.png" alt="Banner" height="225" width="1000"><br />
<br />
<br />
<table width="100%" style="text-transform: uppercase; font-family: 'Lato', sans-serif;"><br />
<br />
<tr height="10px"><br />
<br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne"style="color:#000000"><br />
Home</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Team"style="color:#000000"><br />
Team</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Project"style="color:#000000"><br />
Project</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Public_Outreach"style="color:#000000"> <br />
Outreach</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Achievements"style="color:#000000"><br />
Achievements</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Notebook"style="color:#000000"><br />
Notebook</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Protocols"style=" color:#000000"> <br />
Protocols</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Safety"style="color:#000000"><br />
Safety</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Sponsors"style="color:#000000"><br />
Sponsors</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Attributions"style="color:#000000"><br />
Attributions</a></td><br />
<br />
<br />
<td align="right"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="46px"></a> </td><br />
</tr><br />
</table><br />
<br />
<br />
<h1>Sponsors</h1><br />
<br />
The Melbourne University iGEM team, and the incredible learning experience and science outreach that we participated in, would not be possible without the support of our sponsors. We would like to sincerely thank them for their generosity and support of young scientists in Australia.<br />
<br />
<br><br><br />
<h2>Founding sponsors of the iGEM team</2><br />
<table width="889"><br />
<tr><br />
<td width="364" ><p><img src="https://static.igem.org/mediawiki/2014/4/4c/Melbourne_DSI.jpg" width="363" height="128" alt=""/></p></td><br />
<td width="167"><p><img src="https://static.igem.org/mediawiki/2014/e/e8/Melbourne_Bio21_Institute.jpg" width="115" height="196" alt=""/></p></td><br />
<td width="342" ><p><img src="https://static.igem.org/mediawiki/2014/6/61/Melbourne_Department_of_biochemistry.png" width="252" height="185" alt=""/></p></td><br />
</tr><br />
</table><br />
<br />
<h2>Official suppliers of the iGEM team</h2><br />
<table width="800"><br />
<tr><br />
<td width="365"><table width="800"><br />
<tr><br />
<td width="80"><img src="https://static.igem.org/mediawiki/2014/5/58/Melbourne_Genesearch.png" width="170" height="53" alt=""/></td><br />
<td width="78"><img src="https://static.igem.org/mediawiki/2014/d/df/Melbourne_GenScript.png" width="183" height="56" alt=""/></td><br />
<td width="109"><img src="https://static.igem.org/mediawiki/2014/7/71/Melbourne_New_England_bio_labs.png" width="171" height="67" alt=""/></td><br />
<td width="365"><img src="https://static.igem.org/mediawiki/2014/a/af/Melbourne_Mettler_Toledo.png" width="188" height="111" alt=""/></td><br />
<td width="365"><img src="https://static.igem.org/mediawiki/2014/9/9c/Melbounre_IndieMosh.png" width="149" height="40" alt=""/></td><br />
</tr><br />
</table></td><br />
</tr><br />
</table><br />
<br />
</html></div>Slowehttp://2014.igem.org/Team:Melbourne/Public_OutreachTeam:Melbourne/Public Outreach2014-10-18T01:16:35Z<p>Slowe: </p>
<hr />
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<link href='http://fonts.googleapis.com/css?family=Lato:400,900' rel='stylesheet' type='text/css'><br />
<style><br />
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/* ==========================================================================<br />
HTML5 display definitions<br />
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/**<br />
* Correct `block` display not defined in IE 8/9.<br />
*/<br />
<br />
article,<br />
aside,<br />
details,<br />
figcaption,<br />
figure,<br />
footer,<br />
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main,<br />
nav,<br />
section,<br />
summary {<br />
display: block;<br />
}<br />
<br />
/**<br />
* Correct `inline-block` display not defined in IE 8/9.<br />
*/<br />
<br />
audio,<br />
canvas,<br />
video {<br />
display: inline-block;<br />
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/**<br />
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*/<br />
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audio:not([controls]) {<br />
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*/<br />
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[hidden] {<br />
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/* ==========================================================================<br />
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/**<br />
* 1. Set default font family to sans-serif.<br />
* 2. Prevent iOS text size adjust after orientation change, without disabling<br />
* user zoom.<br />
*/<br />
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html {<br />
font-family: sans-serif; /* 1 */<br />
-ms-text-size-adjust: 100%; /* 2 */<br />
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a:active,<br />
a:hover {<br />
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*/<br />
<br />
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font-size: 2em;<br />
margin: 0.67em 0;<br />
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abbr[title] {<br />
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}<br />
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*/<br />
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b,<br />
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*/<br />
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<br />
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* Address differences between Firefox and other browsers.<br />
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hr {<br />
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*/<br />
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mark {<br />
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}<br />
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*/<br />
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code,<br />
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pre,<br />
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*/<br />
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pre {<br />
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}<br />
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*/<br />
<br />
small {<br />
font-size: 80%;<br />
}<br />
<br />
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* Prevent `sub` and `sup` affecting `line-height` in all browsers.<br />
*/<br />
<br />
sub,<br />
sup {<br />
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*/<br />
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overflow: hidden;<br />
}<br />
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*/<br />
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/**<br />
* Define consistent border, margin, and padding.<br />
*/<br />
<br />
fieldset {<br />
border: 1px solid #c0c0c0;<br />
margin: 0 2px;<br />
padding: 0.35em 0.625em 0.75em;<br />
}<br />
<br />
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* 1. Correct `color` not being inherited in IE 8/9.<br />
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*/<br />
<br />
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border: 0; /* 1 */<br />
padding: 0; /* 2 */<br />
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<br />
/**<br />
* 1. Correct font family not being inherited in all browsers.<br />
* 2. Correct font size not being inherited in all browsers.<br />
* 3. Address margins set differently in Firefox 4+, Safari 5, and Chrome.<br />
*/<br />
<br />
button,<br />
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<br />
/**<br />
* Address Firefox 4+ setting `line-height` on `input` using `!important` in<br />
* the UA stylesheet.<br />
*/<br />
<br />
button,<br />
input {<br />
line-height: normal;<br />
}<br />
<br />
/**<br />
* Address inconsistent `text-transform` inheritance for `button` and `select`.<br />
* All other form control elements do not inherit `text-transform` values.<br />
* Correct `button` style inheritance in Chrome, Safari 5+, and IE 8+.<br />
* Correct `select` style inheritance in Firefox 4+ and Opera.<br />
*/<br />
<br />
button,<br />
select {<br />
text-transform: none;<br />
}<br />
<br />
/**<br />
* 1. Avoid the WebKit bug in Android 4.0.* where (2) destroys native `audio`<br />
* and `video` controls.<br />
* 2. Correct inability to style clickable `input` types in iOS.<br />
* 3. Improve usability and consistency of cursor style between image-type<br />
* `input` and others.<br />
*/<br />
<br />
button,<br />
html input[type="button"], /* 1 */<br />
input[type="reset"],<br />
input[type="submit"] {<br />
-webkit-appearance: button; /* 2 */<br />
cursor: pointer; /* 3 */<br />
}<br />
<br />
/**<br />
* Re-set default cursor for disabled elements.<br />
*/<br />
<br />
button[disabled],<br />
html input[disabled] {<br />
cursor: default;<br />
}<br />
<br />
/**<br />
* 1. Address box sizing set to `content-box` in IE 8/9.<br />
* 2. Remove excess padding in IE 8/9.<br />
*/<br />
<br />
input[type="checkbox"],<br />
input[type="radio"] {<br />
box-sizing: border-box; /* 1 */<br />
padding: 0; /* 2 */<br />
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<br />
/**<br />
* 1. Address `appearance` set to `searchfield` in Safari 5 and Chrome.<br />
* 2. Address `box-sizing` set to `border-box` in Safari 5 and Chrome<br />
* (include `-moz` to future-proof).<br />
*/<br />
<br />
input[type="search"] {<br />
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box-sizing: content-box;<br />
}<br />
<br />
/**<br />
* Remove inner padding and search cancel button in Safari 5 and Chrome<br />
* on OS X.<br />
*/<br />
<br />
input[type="search"]::-webkit-search-cancel-button,<br />
input[type="search"]::-webkit-search-decoration {<br />
-webkit-appearance: none;<br />
}<br />
<br />
/**<br />
* Remove inner padding and border in Firefox 4+.<br />
*/<br />
<br />
button::-moz-focus-inner,<br />
input::-moz-focus-inner {<br />
border: 0;<br />
padding: 0;<br />
}<br />
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/**<br />
* 1. Remove default vertical scrollbar in IE 8/9.<br />
* 2. Improve readability and alignment in all browsers.<br />
*/<br />
<br />
textarea {<br />
overflow: auto; /* 1 */<br />
vertical-align: top; /* 2 */<br />
}<br />
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Tables<br />
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* Remove most spacing between table cells.<br />
*/<br />
<br />
table {<br />
border-collapse: collapse;<br />
border-spacing: 0;<br />
}<br />
<br />
html, body {<br />
color: #111;<br />
background-color: transparent;<br />
width: 100%;<br />
height: 100%;<br />
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display:block;<br />
line-height:normal;<br />
<br />
}<br />
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<img src="https://static.igem.org/mediawiki/2014/9/9b/Melbourne_Banner.png" alt="Banner" height="225" width="1000"><br />
<br />
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<a href="https://2014.igem.org/Team:Melbourne"style="color:#000000"><br />
Home</a> </td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Team"style="color:#000000"><br />
Team</a> </td><br />
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<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Project"style="color:#000000"><br />
Project</a> </td><br />
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<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Public_Outreach"style="color:#000000"> <br />
Outreach</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Achievements"style="color:#000000"><br />
Achievements</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Notebook"style="color:#000000"><br />
Notebook</a></td><br />
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<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Protocols"style=" color:#000000"> <br />
Protocols</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Safety"style="color:#000000"><br />
Safety</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Sponsors"style="color:#000000"><br />
Sponsors</a></td><br />
<br />
<td align="center" height="30px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7><br />
<a href="https://2014.igem.org/Team:Melbourne/Attributions"style="color:#000000"><br />
Attributions</a></td><br />
<br />
<br />
<td align="right"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="46px"></a> </td><br />
</tr><br />
</table><br />
<br />
<br />
<h1>Public Outreach</h1><br />
<h2>&lsquo;Synthetic biology for kids and adults and everyone in between&rsquo; </h2><br />
<p>The University of Melbourne took up the challenge of teaching synthetic biology to all ages, embracing a &lsquo;science for everyone&rsquo; approach. In so doing, we engaged in education outreach activities with kindergarten students from three childcare centres as well as both university and high school students from across Australia.</p><br />
<p>&nbsp; </p><br />
<h3>For kindergarten students </h3><br />
<table width="100%"><br />
<tr><br />
<td width="195" ><img src="https://static.igem.org/mediawiki/2014/4/4b/Melbourne_Adventure_of_E_coli.jpg" width="176" height="264" alt="Adventure of E coli"/></td><br />
<td width="594"><p>We decided to write the first in a series of children&rsquo;s books for pre-schoolers aged 4-6 themed in the spirit of iGEM entitled &lsquo;The Adventures of E. coli&rsquo; for a number of reasons:</p><br />
<ul><br />
<li> Children aged 4-6 are in a key learning stage, during which their interest in science can significantly influence their long-term science education outcomes (Bowman, Donovan, &amp; Burns, 2001, pp. 8-9). </li><br />
<li>There are lots of resources about science for older kids but not so many for kids at such a young age. </li><br />
<li>Picture books provide a springboard for further classroom activities </li><br />
</ul><br />
<p>With the assistance of IndieMosh (Publishers link is here: <A href="http://www.indiemosh.com.au/author-42-iGEM-Melbourne-childrens-biology-science" rel="nofollow">http://www.indiemosh.com.au/author-42-iGEM-Melbourne-childrens-biology-science</A>) – a self-publishing facilitator – we managed to get our book in print and on Amazon, please see <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/</A></p><br />
<p>After we successfully published our book, it was time to take it to the audience. We approached childcare centres across Melbourne and negotiated for our book to be read to pre-schoolers and for our team members to come along and teach the kids about science.</p></td><br />
</tr><br />
</table><p>The book reading was well received and was followed by playing some science-related games, and having a chat about science in groups. Please check out the video we made about the day: </p><br />
<br />
<iframe width="560" height="315" src="//www.youtube.com/embed/RNV_lPKcrSc" frameborder="0" allowfullscreen></iframe><br />
<br />
<p>We believe that through this book, young children can learn about basic concepts and the language of biology, forming a scientific foundation to be built on in future years. Moreover, it is our hope that this book will inspire children to continue to learn about science and to appreciate all the wonders that it holds. </p><br />
<p>The Kindle book is available from here: <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon-ebook/dp/B00OJ691DI/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon-ebook/dp/B00OJ691DI/</A></p><br />
<p>Paperback available on Amazon here: <A href="http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/" rel="nofollow">http://www.amazon.com/Adventures-E-Coli-Bianna-Makogon/dp/1925219208/</A></p><br />
<p>Fixed layout epub (for iPad and Android tablets, phones etc) available on Smashwords here: <A href="http://www.smashwords.com/books/view/484305" rel="nofollow">http://www.smashwords.com/books/view/484305</A></p><br />
<h3>&nbsp;</h3><br />
<h3>For high school students </h3><br />
<p>In an effort to broaden the accessibility of the iGEM competition to those who appeared to be currently underrepresented in the competition (e.g. disadvantaged students, females and Australian high school students), we approached a private high school in Victoria with the following aim: </p><br />
<p>To find a well-resourced, private high school to partner with an iGEM team (e.g. UoM) to field a team consisting of half private high school students/half underrepresented students – who would have their flights to Boston funded by fundraising done primarily by the private high school. </p><br />
<p>After extensive email &amp; phone correspondence spanning several months, the school decided it was not currently in the position to go ahead with the iGEM team proposal due to upcoming infrastructure developments, though would be interested in fielding a team after these are completed (scheduled for 2016). Nonetheless, the school did encourage us that other APS schools would likely be interested in hearing about iGEM and our team concept. </p><br />
<p>Whilst we didn&rsquo;t achieve our ultimate goal, this activity did raise awareness of the iGEM competition with all the key science staff members at the school which requested not to be named on this wiki (for further details please contact our team leader Sean). </p><br />
<p>Finally, we would encourage all Australian teams to work on the following goals: </p><br />
<ul><br />
<li>improve iGEM's accessibility to those of disadvantaged backgrounds </li><br />
<li>increase female representation in science &amp; iGEM </li><br />
<li>increase the participation of Australian/international high school students in the iGEM competition. </li><br />
</ul><br />
<p><BR><br />
In addition to our negotiations with the private high school, we also aimed at widening the participation of high school students in the iGEM competition by collaborating with the University of Sydney on The Strange Nature Writing competition. </p><br />
<p>We spruiked the competition not only on our project flyers which described the iGEM competition, our project and the Strange Nature Writing Competition, but also through our development of a promotional video designed to be shown in high school assemblies as a way of promoting both iGEM and the Strange Nature Writing competition. Please see the video below: </p><br />
<br />
<iframe width="560" height="315" src="//www.youtube.com/embed/ZuSoO3HVMjo" frameborder="0" allowfullscreen></iframe><br />
<br />
<p>Furthermore, we wrote and published articles on Comet.is which is aimed at high school students, university students and recent university graduates, please see 'For university students' for a list of the articles we wrote. </p><br />
<h3>&nbsp;</h3><br />
<h3>For university students </h3><br />
<p>Our main form of outreach toward university students was in writing and publishing articles on a burgeoning and government sponsored website Comet.is which is aimed at high school students, university students and recent graduates. Our articles covered a range of topics from our iGEM experience to considerations of ethical issues of synthetic biology and the potential of synthetic biology. </p><br />
<br />
<td width="195" ><img src="https://static.igem.org/mediawiki/2014/d/d5/Comet.jpg" width="375" height="400" alt="Adventure of E coli"/></td><br />
<br />
</p> Here are some excerpts: </p><br />
<br />
</p> "All this leads me to refer to synthetic biology as a ménage à trois of a discipline, utilizing engineering, biology and computer science to produce a fantastically creative activity." </p><br />
<br />
</p> "Given assistance in the form of grants or human resources, entrepreneurial forums along with student competitions like InnovationACT and iGEM have the chance to inspire entrepreneurialism in students. It is in these opportunities for students to go on entrepreneurial adventures, replete with late nights, red bulls and a shot at winning a grand prize – that the next generation of entrepreneurs is born." </p><br />
<br />
</p> "A prime example of this is in the International Genetically Engineered Machine (iGEM) competition, which I am a team member of for the University of Melbourne this year. Our team stretches across many disciplines; from telecommunications and business to chemical engineering and science communication, even information technology - we are practically the UN of fields and backgrounds." </p><br />
<br />
</p> "Participating in IGEM can definitely be categorized as having an experience and not just joining a team. The competition brings together all the components of completing a project for undergraduate students to be part of, a little like a trial run of a Masters or Doctorate course." </p><br />
<br />
<p>Here are the links to our articles: </p><br />
<p><A href="https://comet.is/article/2960" rel="nofollow">https://comet.is/article/2960</A> <A href="https://comet.is/article/3499/" rel="nofollow">https://comet.is/article/3499/</A> <A href="https://comet.is/article/3381/" rel="nofollow">https://comet.is/article/3381/</A> <A href="https://comet.is/article/3561/" rel="nofollow">https://comet.is/article/3561/</A> <A href="https://comet.is/article/3562/" rel="nofollow">https://comet.is/article/3562/</A> <A href="https://comet.is/article/3694/" rel="nofollow">https://comet.is/article/3694/</A> <A href="http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/" rel="nofollow">http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/</A> <A href="http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/" rel="nofollow">http://blogs.unimelb.edu.au/…/igem-a-small-word-for-a-life…/</A></p><br />
<p>Furthermore, as part of our outreach directed at university students, we combined our recruitment campaign for an IT whiz to help with our wiki, with a presentation on what iGEM was all about. We lecture bashed over several weeks, presenting over 11 times. </p><br />
<br />
<h3><BR><br />
</h3><br />
<h3>For general public </h3><br />
<p>After promoting the iGEM competition to the Victorian State Chair of National Science Week (NSW) - Nick Besley, our team decided to field a stall in &lsquo;Market of the Mind&rsquo; – a NSW event aimed at the general public located in a high foot traffic area alongside the Yarra River in Southbank across the river from Flinders St Station. Luckily, our stall was located next to the bar, which meant there was a lot of foot traffic directed to our happy team members wearing our iGEM team t-shirts. Using a true/false quiz, armed with Ferrero Rochers as rewards for getting all the answers correct, we had over 30 quiz contestants engage with us on the first night and 35+ contestants on the second day. Please see the accompanying video made by the City of Melbourne, which shows our stall/team members from 0:45 <br />
<br />
<p><iframe width="560" height="315" src="//www.youtube.com/embed/ahFI3aFBdNc" frameborder="0" allowfullscreen></iframe><p><br />
<br />
<p><BR><br />
<A href="http://www.scienceweek.net.au/market-of-the-mind/" rel="nofollow">http://www.scienceweek.net.au/market-of-the-mind/</A> <A href="http://re-science.org.au/science-event/market-mind-2649" rel="nofollow">http://re-science.org.au/science-event/market-mind-2649</A></p></html></div>Slowehttp://2014.igem.org/Team:Melbourne/NotebookTeam:Melbourne/Notebook2014-10-18T01:12:39Z<p>Slowe: </p>
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<br />
<h1 >Notebook</h1><br />
<h2>Semester 1</h2><br />
<p><h3>Preliminary work – Lab setup, training, etc.</h3><br />
<p><br><br />
<em>In Semester 1, we began the process of recruiting the team and setting up the lab. Because our lab was new, we needed to order all supplies, secure scientific sponsorships, and establish protocols. The founding team was also able to do some exploratory lab work before the changeover to the full team later in the semester.</em><br><br />
<em>Lab members were recruited from the wider University of Melbourne student community through an advertising campaign, followed by a selection process. The team underwent training during the Easter break and the following weeks.</em><br />
</p><br />
</p><br />
<p>&nbsp; </p><br />
<h3>Week beginning 6 January</h3><br />
<p>Grew stock of competent DH5α E. coli. Cells stored at -80°C.</p><br />
<p>&nbsp;</p><br />
<h3>Week beginning 13 January</h3><br />
<p>Note that the 2013 iGEM Founding Team originally came up with the concept for a star peptide similar to the USP peptide reported on the Project page. This peptide was meant to be unstructured. It contained an RGD motif to support potential binding of the peptide to integrin (hence why it was termed the RGD peptide). Unlike the peptides reported on the project page, the RGD contained a TEV protease recognition sequence. This would have allowed it to be co-expressed with the TEV protease, and therefore cleaved to form a star peptide inside the cell. <br />
</p><br />
<p>The RGD peptide sequence was: MRV LLF LLL SLF MLP AFS RGD RGD GGG AKQ RGG C GGR QKA GGG RGD RGD EQLYFQG RGD RGD GGG AKQ RGG C GGR QKA GGG RGD RGD HHHHHH<br />
<br />
</p><br />
<p>The founding team ordered this peptide's gene to be synthesised and most of the early work in the lab was focused on using it to practice lab techniques. At this stage, we were waiting for the gene to arrive.</p><br />
<h3>Week beginning 20 January</h3><br />
<p><strong> (20-01-14)</strong><br><br />
RGD construct DNA arrived.<br><br />
Completed transformation of DH5α competent cells with heat shock and LB (see Protocols).<br><br />
OD of competent cell growth:<br />
<p><img src="https://static.igem.org/mediawiki/2014/4/4b/Lab_book_picture_001.png" alt="Lab book picture 1"> </p><br />
Digestion was performed using EcoRI (see Protocols).<br><br />
Amount of plasmid was measured.<br><br />
RGD001 – 37.1 ug/uL<br><br />
RGD002 – 51.2 ug/uL<br><br />
RGD003 – 34.5 uG/uL<br><br />
Gel was run. No DNA was visible but the ladder was fine.<br><br />
<strong>(24-01-14)</strong></p><br />
<ul><br />
<li>Redid miniprep. Quantified RGD plasmid again.</li><br />
</ul><br />
<p>RGD001 – 37.1 ug/uL<br><br />
RGD002 – 51.2 ug/uL<br><br />
RGD003 – 34.5 uG/uL</p><br />
<ul><br />
<li>Performed double digest of RGD plasmid with EcoRI and PstI (see Protocols).</li><br />
<li>Performed gel electrophoresis of digested plasmid and undigested plasmid. Plasmids were present at approx 3.5 kb. RGD001 seemed to be the better digested plasmid and best to use for transformation.</li><br />
</ul><br />
<p>&nbsp;</p><br />
<h3>Week beginning 27 January</h3><br />
<ul><br />
<li>Transformed BL21 with RGD. Placed in incubator overnight at 37°C.</li><br />
<li>Ran third gel of transformed DH5α (with RGD).</li><br />
<li>Performed double digests before running another gel to check success.</li><br />
</ul><br />
<p>&nbsp;</p><br />
<h3>Week beginning 3 February</h3><br />
<p>Performed protein induction on cells containing RGD plasmid.<br><br />
NOTE: </p><br />
<ul><br />
<li>Grew 800 mL culture and measured OD (absorbance at 600nm):</li><br />
</ul><br />
<p>0 hrs – 0.184<br><br />
1 hr – 0.322<br><br />
2 hrs – 1.265<br><br />
2.5 hrs – 1.471</p><br />
<ul><br />
<li>Despite overshooting OD target, went ahead with IPTG induction anyway.</li><br />
</ul><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 10 February</h3><br />
<p><strong>Project design/planning</strong><br><br />
Did some project planning based on meeting with advisor Neil O&rsquo;Brien-Simpson:<br><br />
<u>Selenocysteine</u><br><br />
First thought when he saw the project was the liability of the disulfide bond (i.e. tends to fall apart under producing environments).<br><br />
He suggested trying selenocysteine, because selenocysteine bonds are stronger and the amino acids should be sitting around in E. coli ready to use.<br><br />
<u>Comments on AMP</u><br><br />
Suggested a good idea to stick with the Wiradharma, 2012 structure rather than alter too much so as to make comparisons easier.<br><br />
We briefly discussed the mechanism of action of AMPs. As we know, there are several models, including the barrel stave, toroidal poor, and carpet models. In addition, peptide aggregation at the surface of the bacterial cell can lead to membrane thinning, which facilitates pore formation.<br><br />
Neil wasn&rsquo;t sure how the star polymers would work in particular. They may work using the above mechanisms… The disulfide bond may also be broken as the stars enter the cell cytoplasm.<br><br />
<u>Related work</u><br><br />
A researcher by the name of Tam has developed a Multiple Antigenic Peptide (MAP) similar to ours. This peptide includes branched lysine residues. However, the problem with this molecule is that it is very challenging to synthetically produce.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 17 February</h3><br />
<ul><br />
<li>Began new protein expression method (see Protocols). </li><br />
<li>Pelleted cells (see Protocols).</li><br />
</ul><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 24 February</h3><br />
<ul><br />
<li>Transformed DH5α Kan.R resistance (see Protocols). Placed 4 plates on incubator overnight.</li><br />
<li>Transferred cells to 4°C. Colonies grew, suggesting the transformation was successful.</li><br />
</ul><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 3 March</h3><br />
<p><strong>(04-03-14)</strong></p><br />
<ul><br />
<li>To check success of transformation, we ran an SDS-PAGE gel at 150V, 400mA, 45 mins with positive controls and RGD protein (see Protocols):</li><br />
</ul><br />
<p> <img src="https://static.igem.org/mediawiki/2014/f/fa/Lab_book_picture_002.png" alt="Lab book picture 2"></p><br />
<ul><br />
<li>Transformed DH5α colonies with Magainin I.</li><br />
<li>DH5α (MagI) transformed cells were miniprepped (see Protocols). Some of cell pellet was loaded into SDS-PAGE gel with two positive controls for His-tags. Results appeared successful so we continued onto Ni-NTA column purification.</li><br />
<li>Unfortunately, Ni-NTA column purification failed.</li><br />
</ul><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 10 March</h3><br />
<p>Redid process from last week to try to diagnose what went wrong.<br><br />
Double digestion of DH5α (Magainin I) colony miniprep products with EcoRI and PstII.<br><br />
Made and ran DNA gel with double digest at 120V, 100mA, 35 minutes then 135V, 100mA, 15 minutes then 120V 3 minutes.<br><br />
<br />
<p><img src="https://static.igem.org/mediawiki/2014/4/45/Lab_book_picture_003.png" alt="Lab book picture 3"></p><br />
Results were visible for lanes 1 – 4, but digests were not visible.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 17 March</h3><br />
<p><strong><em>(Recruitment of team members)</em></strong></p><br />
<h3>Week beginning 24 March</h3><br />
<p><strong><em>(Recruitment of team members)</em></strong></p><br />
<h3>Week beginning 31 March</h3><br />
<p><strong><em>(Recruitment of team members)</em></strong></p><br />
<h3>Week beginning 7 April</h3><br />
<p><strong><em>(Recruitment of team members)</em></strong></p><br />
<h3>Week beginning 14 April</h3><br />
<p><strong><em>(Recruitment of team members)</em></strong></p><br />
<h3>Week beginning 21 April</h3><br />
<p><strong><em>(Lab introductory training)</em></strong></p><br />
<h3>Week beginning 28 April</h3><br />
<p><strong><em>(Lab setup work)</em></strong></p><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 5 May</h3><br />
<p>Despite negative results on gel of double digest (thought to be due to faulty gel electrophoresis process), we went ahead with protein expression.<br><br />
We also transformed BL21 with pMK-RGD vector (see Protocols) and ran a protein gel with pGEX, pET, pMK RGD and pMK Mag 1 (samples taken from the training week during the break).<br><br />
<br><br />
Conducted miniprep of DH5α pMK-RGD culture and ran DNA gel on the RGD plasmid extracted. </p><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 12 May</h3><br />
<p>Started protein expression on transformed BL21 pMK-RGD.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 19 May</h3><br />
<p>Completed protein expression, but cultures failed to grow.<br><br />
Ran PCR for pMK RGD (reaction volume = 50 uL) with the following new recipe:</p><br />
<ul><br />
<li>10X Thermopod Reaction Buffer – 5 uL</li><br />
<li>10 mM dNTP mixture – 1 uL</li><br />
<li>10 uM forward primer – 1 uL</li><br />
<li>10 uM reverse primer – 1 uL</li><br />
<li>Template DNA – 1 uL</li><br />
<li>Taq DNA polymerase – 0.5 uL</li><br />
<li>Nuclease-free h3O – 40.5 uL</li><br />
</ul><br />
<p>Conditions for the PCR cycle:</p><br />
<ul><br />
<li>95°C – 2 minutes</li><br />
<li>30 cycles:</li><br />
<ul><br />
<li>95°C – 20 seconds</li><br />
<li>60°C – 20 seconds</li><br />
<li>72°C – 20 seconds</li><br />
</ul><br />
<li>72°C – 5 minutes</li><br />
<li>4°C – infinity (hold) <strong></strong></li><br />
</ul><br />
<p>Performed protein induction on PMK-MAG1 .</p><br />
<p>Created a glycerol stock of BL21-RGD and BL21-MAG1 and stored it at -86°C.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 26 May</h3><br />
<p><strong><em>(Exam period – no lab preparation work)</em></strong></p><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 2 June</h3><br />
<p><strong><em>(Exam period – no lab preparation work)</em></strong></p><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 9 June</h3><br />
<p><strong><em>(Exam period – no lab preparation work)</em></strong></p><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 16 June</h3><br />
<p><strong><em>(Exam period – no lab preparation work)</em></strong></p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
<h2>Semester 2 </h2><br />
<p><em>Main project work begins.</em><br><br />
<em>With the lab set up, the team fully formed, and preliminary work completed, we began work on the project. Our focus throughout Semester 2 was on the design of the BioBricks, and ensuring that each of our proteins could be properly expressed in transformed bacteria after cloning and subcloning.</em></p><br />
<h3>&nbsp;</h3><br />
<h3>Week 1 – 23 June</h3><br />
<p><strong>(24-06-14)</strong></p><br />
<ul><br />
<li>Plated BL21(DE3) + Magainin 1 glycerol stock on one plate using &lsquo;spread&rsquo; technique at 12:45pm, placed in 37 degree incubator at 3pm (should be taken out at 11am next day [+20 hours]).</li><br />
</ul><br />
<p>However, the plates had way too much growth, almost resembling a lawn, and couldn&rsquo;t be used due to a need for &lsquo;isolated&rsquo; colonies. After checking lab-book again, realised the glycerol stock was made using culture from already plated colonies and instead of just transformed cells. <br><br />
New glycerol stock was used to plate again, this time using the &lsquo;streak&rsquo; technique.</p><br />
<ul><br />
<li>SDS-PAGE gel with MagI pellet was run. 2 samples were prepared using approximately 100μL of SDS-PAGE Sample Buffer and a small amount of pellet. The samples were prepared using Ash&rsquo;s method of quickly boiling the samples (placing tubes in heat block set at 100(99.9) degrees) to lyse the cells. Unfortunately, the samples thickened and SDS-PAGE could not be run. This may be a result of specific faults in the buffer recipe, so possibly use a RIPA buffer next time.</li><br />
</ul><br />
<h3>&nbsp;</h3><br />
<h3>Week 2 – 30 June</h3><br />
<p><strong>Project design/planning</strong><br><br />
The team agreed to have a multi-track research program. What we are essentially doing at the moment is trying to attack this problem of synthesizing disulfide bonded structures from multiple fronts.<br><br />
Tobias suggested a new concept for ligating peptides, carbohydrates, lipids and other biomolecules to the star peptide structure through native chemical ligation.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week 3– 7 July</h3><br />
<p><strong>Project design/planning</strong><br><br />
Further work on the idea of native chemical ligation (NCL).<br><br />
Summary of progress on shuttling the MAG 1 insert with the periplasmic export tag to the standard iGEM plasmid – </p><br />
<ul><br />
<li>Ran PCR on pMK-RGD using primers to make RGD compatible with pGEX vector</li><br />
<li>Ran PCR product on gel</li><br />
<li>Cut out PCR product from gel and use gel purification kit</li><br />
<li>Ran restriction digestion on PCR insert product of 3a using BamHI and EcoRI</li><br />
<li>Ran PCR purification using Thermo kit on product of 4a</li><br />
<li>Transformed pGEX vector donated by a neighboring lab into DH5a, plate and incubated overnight</li><br />
<li>Miniprepped plasmid</li><br />
<li>Ran restriction digestion on plasmid product of 2b using BamHI and EcoRI</li><br />
</ul><br />
<p>Summary of previous progress on inserting RGD coding region into pGEX vector, and expression in BL21(DE3) cells – </p><br />
<ul><br />
<li>Ran PCR on pMK-RGD using primers to make RGD compatible with pGEX vector</li><br />
<li>Ran PCR product on gel</li><br />
<li>Cut out PCR product from gel and use gel purification kit</li><br />
<li>Ran restriction digestion on PCR insert product of 3a using BamHI and EcoRI</li><br />
<li>Ran PCR purification using Thermo kit on product of 4a</li><br />
<li>Transformed pGEX vector donated by a neighboring lab into DH5a, plate and incubated overnight</li><br />
<li>Miniprepped plasmid</li><br />
<li>Ran restriction digestion on plasmid product of 2b using BamHI and EcoRI</li><br />
</ul><br />
<p>Decided to divide work into streams as previously planned – indicated in lab book as:</p><br />
<ul><br />
<li>pSB1C3-MAG1 subcloning</li><br />
<li>pGEX-RGD cloning</li><br />
<li>Cytoplasmic Star Magainin 1 cloning</li><br />
</ul><br />
<h3>&nbsp;</h3><br />
<h3>Week 4 – 14 July </h3><br />
<p><strong>pSB1C3-MAG1 subcloning – </strong></p><br />
<ul><br />
<li>Ran PCR on pMK-MAG1 using primers to remove TorTSS and add sticky ends for pSB (BBa_K525998). When run on a gel, the PCR product was a very clear band, indicating success.</li><br />
<li>Ran restriction digestion on pMK-MAG1 PCR insert product using SpeI and PstI. </li><br />
<li>Ran PCR on MAG1 using primers to remove TorTSS and add sticky ends for pSB (BBa_K525998) for a second time.</li><br />
</ul><br />
<p><strong>Cytoplasmic Star Magainin 1 cloning –</strong> Plated DH5a cells.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week 5 – 21 July</h3><br />
<p><strong>pSB1C3-MAG1 subcloning – </strong><br><br />
We transformed DH5a with the pSB1C3 (RFP) plasmid provided by iGEM. We then miniprepped the cells. However, the nanodrop suggested that the concentration of the cells was extremely low. Gayle then ligated pSB1C3 with a MAG 1 insert. Because the concentrations were too low to see on a DNA gel, she re-transformed the plasmid to DH5a. The idea is that we should now be able to re-miniprep this DH5a and reclaim the ligated plasmid.<br><br />
<strong>Cytoplasmic Star Magainin 1 cloning – </strong><br><br />
Did restriction digestion using PstI and SpeI on miniprepped T7 promoter containing pSB1C3. Ran the product on a gel, but it came up blank. We had a thorough discussion with Ash and David about why this could be. Ash suggested trying digesting with one enzyme at a time, having several lanes, each with different enzyme combinations. <br><br />
A Google search suggested that there could be a problem with the miniprep procedure. Specifically, there could be some enzymes being left over from the miniprep which, when the samples were heated during the restriction digestion, ate up the DNA.<br><br />
Therefore, Stan designed an experiment to test this. We had 5 conditions: </p><br />
<ul><br />
<li>the PSB1C3 plasmid directly after the miniprep, with no incubation or restriction digestion; </li><br />
<li>the plasmid heated to 37° with restriction enzymes, </li><br />
<li>the plasmid heated to 37° WITHOUT restriction enzymes, </li><br />
<li>a PCR insert meant to go into the plasmid heated to 37° with restriction enzymes (the same restriction enzymes which would allow us to like it that insert into the pSB1C3 plasmid),</li><br />
<li>the PCR insert heated to 37° without restriction enzymes.</li><br />
</ul><br />
<p>So, we're testing the effect of heat and the effect of the restriction enzymes.<br><br />
In the resulting gel, every lane was visible, except for the plasmid which had been double digested. This suggests that there was not anything wrong with the restriction enzymes per se, neither with the buffer nor the heating process. It argues against the hypothesis that there was some DNA degrading enzyme present after the miniprep could have been activated by heat.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week 6 – 28 July</h3><br />
<p><strong>Cytoplasmic Star Magainin 1 cloning – </strong><br><br />
Thawed out the plasmid samples in sample buffer from yesterday and loaded them onto a 16 well agarose gel. Every lane was visible except for the Axygen miniprepped plasmid treated with SpeI. However, the Axygen miniprepped plasmid treated with a double digestion of SpeI and PstI DID show upon the gel. After speaking with David, he suspects that we perhaps neglected to put DNA into that well. <br><br />
Note that these results are in direct contrast to the gel described earlier, where we had an identical plasmid (pSB1C3 containing the P7 promoter), picked from the same plate, using the same miniprep kit, same restriction enzymes (PstI and SpeI), for the same incubation period (2.5-3 hrs). That is, we did exactly the same thing, and this time, it showed up on the gel, whereas before it disappeared.<br><br />
Given that the double digest with SpeI and PstI and EcoR1 and PstI worked, we went ahead and began culturing in 10 mL colonies from the same pSB1C3 plate used in the experiment above. We made 6, 10 mL cultures. To provide a backup, we also picked three colonies which had previously been plated out by Lumi containing GFP, RFP, or YFP, creating 3, 10 mL cultures which may also be miniprepped.<br><br />
Further progress:</p><br />
<ul><br />
<li>Miniprepped 10 mL cultures of pSB containing the p7 promoter. Ran various restriction digests on the miniprep product in preparation for ligation of pSB with our MAG1 PCR insert.<strong></strong></li><br />
<li>Ran the restriction digests from yesterday. Results are very mysterious. Thus, re-ran restriction digests. Results favourably suggest one of the minipreps was successful.<strong></strong></li><br />
<li>Gel extracted pSB1C3 digested with both SpeI and PstI.<strong></strong></li><br />
</ul><br />
<h3>&nbsp;</h3><br />
<h3>Week 7– 4 August</h3><br />
<p><strong>pSB1C3-MAG1 subcloning – </strong>Transformation of pSB1C3 to DH5α (see Protocols).<br><br />
<strong>pGEX-RGD cloning – </strong>Transformation of pGEX to DH5α (see Protocols).<strong> </strong> <br><br />
<strong>Cytoplasmic Star Magainin 1 cloning – </strong> <br><br />
We were originally planning to do the ligation reaction with the SpeI and PstI restriction enzymes AND the PCR insert. However, during that restriction digest of the PCR insert, DNA loading dye was erroneously added. We attempted to gel purify the PCR insert to remove the loading dye. However, for some reason the &quot;PCR insert&quot; ran at a very high molecular weight (approximately 2 kDa). Moreover, there is a strange double band.<br><br />
Because the PCR insert appears to be screwed up, we will have to start from the PCR step again. Planning on ordering new primers which will allow the Magainin 1 insert to be biobrick compatible.<strong> </strong></p><br />
<h3>&nbsp;</h3><br />
<h3>Week 8 – 11 August</h3><br />
<p><strong>pSB1C3-MAG1 subcloning –</strong><strong> </strong></p><br />
<ul><br />
<li>PLASMID: </li><br />
<ul><br />
<li>Stanislav performed a double restriction digest on the linearized plasmid backbone pSB1C3. </li><br />
<li>Performed a miniprep on the 10 mL cultures. Confirmed the success of the miniprep/presence of the plasmid using a DNA gel.</li><br />
</ul><br />
<li>INSERT: </li><br />
<ul><br />
<li>Performed a restriction digestion on previous pMK-MAG1 plasmid stock in order to liberate the MAG 1 insert in preparation for ligation with pSB1C3 and ran a gel on the products. In this gel, a distinct band was observed corresponding to the approximately 2 kDa vector (pMK). However, the 6 kDa insert was not visible. </li><br />
<li>In order to get more of the insert, we propagated more pMK-MAG1 plasmid. </li><br />
</ul><br />
</ul><br />
<p><strong>pGEX-RGD cloning – </strong></p><br />
<ul><br />
<li>Transformed DH5a competent cells with pGEX-6P3 and performed a miniprep to increase the amount of plasmid stock available for ligation. A DNA gel performed on the products suggested the miniprep was successful. </li><br />
<li>A gel performed on PCR products of the RGD insert amplified with restrictions sites added for subcloning into pGEX gave odd results. </li><br />
</ul><br />
<p>Both the PCR products created yesterday and the products done over the summer break showed over 10 bands in each lane. This suggests that there was nonspecific binding, leading to a variety of different molecular weight products. Spoke to advisor Ash who prescribed a new PCR temperature/timing cycle to minimise this problem.</p><br />
<p>Ran the PCR reaction (using Ash&rsquo;s suggested solution) product on a gel. Increasing the annealing temperature in the PCR cycle did decrease the presence of multiple (~10) bands. However, it also led to a general reduction in the intensity of the PCR product's signal on the gel. It's not clear that the changes to the PCR protocol actually reduced nonspecific binding so much as just decreased the reaction yield.</p><br />
<p>As suggested by our supervisor, Cheng, we reran the PCR reaction. This time, in order to eliminate the possibility of nonspecific binding, we cut out the RGD insert from the PMK vector using restriction digestion, gel extract the insert, and then used this insert as the PCR template.</p><br />
<p><strong>Cytoplasmic Star Magainin 1 cloning – </strong><br><br />
We had previously ordered the primers which would both remove the periplasmic export tag and allow for ligation into Biobrick BBa_K525998 of our Magainin 1 Star Peptide insert. The problem with these primers is, however, that after the ligation, there would be the Spe1 restriction site at the 5' end of the protein coding region. Therefore, new primers were designed which would eliminate this problem. The protein coding region will be inserted after the T7 promoter in BBa_K525998. At the 5' end of the insert, XbaI will be used to join the insert to the plasmid's SpeI site. At the 3' end of the insert, PstI will be used to join the insert to the plasmids PstI site. </p><br />
<h3>&nbsp;</h3><br />
<h3>Week 9 – 18 August</h3><br />
<p><strong>pSB1C3-MAG1 subcloning –</strong></p><br />
<ul><br />
<li>INSERT:</li><br />
<ul><br />
<li>Did a double digestion on the pMK-MAG1 plasmid.</li><br />
<li>Ran the double digestion product on a DNA gel and gel-extracted.</li><br />
<li>Ran an analytical amount of the digested linearized PSB1C3 backbone on a DNA gel. The plasmid was clearly visible on the gel (Gel C35).</li><br />
</ul><br />
<li>PLASMID: Did a double digestion on a significant amount of the linearized plasmid backbone and ran the double digestion product of pMK-MAG1 on a DNA gel. However, the results on the DNA gel (Gel C35) were inexplicable. We observed a smear in the lanes containing pMK-MAG1, not the clear bands we expected. Moreover, the insert was not visible at all in the lower regions of the gel.</li><br />
</ul><br />
<p>Due to the anomalous results, we designed an experiment performing a series of restriction digestions on the pMK plasmid containing the Magainin 1 insert. Several controls, including single digestions and digestions using newer tubes of restriction enzymes EcoRI and PstI were performed.<br><br />
<strong>pGEX-RGD cloning –</strong><br><br />
It was discovered that there was a mistake in the PCR primers we have been using up to this date. Specifically, the PCR primers amplified a segment of the DNA including the periplasmic export tag preceding the RGD protein coding region. Therefore, new primers had to be designed for PCR on the RGD insert.<br><br />
Nonetheless, we did a double digestion on the pMK-RGD plasmid, making sure that the volume of the reaction is quite large so that the insert band was be clearly visible on the DNA gel. This measure is a backup in the event that the PCR primers are not specific in their binding to the template. If the primers are non-specific, then we will use the isolated insert as the template.</p><br />
<ul><br />
<li>PLASMID: Ran an analytical amount of the gel-extracted digested pGEX vector on a gel (Gel C35) and confirmed successfμL gel-extraction.</li><br />
<li>INSERT: Ran the restriction digestion product (PMK-RGD) on a DNA gel. However, strangely we did not get the results we expected. Instead of seeing two bands, one corresponding to the vector in one to the insert, we saw approximately 5 bands. This suggests that the restriction digestion failed, and we might be seeing a multiple bands associated with the undigested plasmid.</li><br />
</ul><br />
<p><strong>Cytoplasmic Star Magainin 1 cloning – </strong><br><br />
Completed the PCR reaction to amplify the plasmid in preparation for ligation and ran on a gel. We have previously digested the destination plasmid using the relevant restriction enzymes. We did one final check to ensure that the gel extraction worked by running a sample of the product on another gel.<br><br />
A double digestion was completed on a portion of the gel extracted PCR insert using XbaI and PstI, two restrictions sites which will allow us to ligate this insert with the pSB1C3 plasmid (containing the T7 promoter) at the biobrick suffix. The PCR insert was then PCR purified.<strong></strong><br><br />
A ligation procedure was performed between pSB1C3 in the PCR insert. The ligation product was transformed to DH5a.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week 10 – 25 August</h3><br />
<p><strong>pSB1C3-MAG1 subcloning –</strong><br><br />
A gel run on the restriction digestion experiment last week revealed that there was something wrong with our old EcoRI/PstI when used in combination. <br><br />
We designed an experiment using gel electrophoresis of a combination of different enzymes, used on the same plasmid, repeated with our old and new enzymes. We found that with the NEW enzymes, the double digestion works, and with the old enzymes it doesn't work.<br><br />
Strangely enough, it looks like the single digests with the old enzymes still work. It's as if there's some interaction effect when the old Eco and Post are used together where things go bonkers.<br><br />
This experiment has also revealed a limitation of our cloning strategy. It turns out that there is actually a PstI cutting site in pMK. Hence, you will see that there actually more than two bands in the double digestions (and if you look closely, two bands in the single digestions with PstI). All is not lost, however, as we can still double digest pMK-MAG1 using different restriction enzymes, bypassing PstI. We can use EcoRI and SpeI, and this should work (there are no EcoRI or SpeI sites in pMK).<br><br />
This sub cloning experiment was started from the restriction digestion point again. This time, however, we digested the destination vector (pSB1C3) and insert (contained in pMK-MAG1) with the NEW tubes of EcoRI and PstI. A gel was run on these products. Both the destination vector, pSB1C3, and the gel isolated insert were clearly visible on the gel.<br><br />
A ligation reaction between the Mag1 insert and the pSB1C3 linearized vector was carried out. The ligation product was transformed to DH5α and plated to chloramphenicol-containing plates.<br><br />
<strong>pGEX-RGD cloning – </strong>A gel was run on the PCR product (previously carried out). However, it appeared that the PCR reaction was inefficient; the product had a very feint band on the gel. After gel extraction of the product, nanodrop measurements suggested that the concentration was in the range 5-10 ng/uL. Because the ultimate goal of this PCR reaction was to create a lot of insert for a ligation later on, small amounts of insert DNA were not acceptable. <br><br />
A second PCR reaction was run in order to hopefully increase the yield of the insert. The number of cycles was increased to 35 from 30. In a second condition, the gel extracted PCR product from the last reaction was used as the template.<br><br />
The products of the PCR reaction of the RGD insert were run on a gel and were clearly visible at the correct molecular weight.<br><br />
A restriction digestion of both pGEX-6P-3 and the PCR-amplified RGD insert was carried out. EcoRI and BamHI were used on both vector and insert to create complementary sticky ends. The reaction was completed and the products placed in the -20 freezer.<br><br />
<strong>Cytoplasmic Star Magainin 1 cloning – </strong>Minipreps were performed on colonies containing the ligation and the products were run on a DNA gel (Gel C43). However, no DNA was visible on the gel, suggesting that the minipreps had failed. This is likely because the culture conditions of the miniprep were suboptimal. However, another gel showed that the PCR purified insert ran clearly on the gel. Thus, we can combine that insert with the vector and likely produce a clone again.<strong></strong><br><br />
The ligation of the destination vector in the PCR amplified insert was repeated today. The ligation product was then transformed to DH5α. The ligation reaction to get the Magainin 1 insert into pSB1C3 was found to be successful. The MAG 1 insert was mixed with the destination vector pSB1C3 in a ligation reaction. The ligated product was transformed to DH5α and plated overnight. Colonies were picked from agar plate and then placed in a PCR reaction (PCR-based colony screening). The primers used in the PCR were specific to the Magainin 1 insert. <br><br />
Performing a PCR on the colonies led to a propagation of the insert. This suggests that the colonies contained the Magainin 1 insert. And, because the colonies were chloramphenicol resistant, they should also contain the pSB1C3 vector. Thus, it appears that we have the vector with the insert! </p><br />
<h3>&nbsp;</h3><br />
<h3>Week 11 – 1 September</h3><br />
<p><strong>pSB1C3-MAG1 subcloning – </strong>From the plate of DH5α transformed with the ligation product, PCR colony screening was performed. Only one colony showed successful transformation on a DNA gel. This culture later showed no growth, and transformation was suspected to have failed. Because of this, a second transformation was carried out using the ligation product used for the previous transformation/plating, and appeared to be successful. Glycerol stocks were made of the cultures from the second transformation.<br><br />
Later, more colonies were miniprepped, double digested, and ran on a gel. Strangely, as in previous gels, the only thing visible on the gel was a very faint band with molecular weight greater than 3000. It is unclear what that band corresponds to. Because the colony screening failed in this manner across many colonies and two different transformations, it suggests that there something went wrong during the ligation reaction or earlier.<br><br />
<strong>pGEX-RGD cloning –</strong><br><br />
The insert for the pGEX RGD stream was PCR purified and then run on a gel. For unknown reasons, the PCR purification product was not visible on the gel. The nanodrop suggested that the concentration was 15 ng/µL. 10 µL of the product were run on the gel. It therefore should have been visible.<br><br />
Note, however, that we observed a strange phenomenon where the DNA/loading dye was floating out of the well once loaded. After trying to load two wells, we eventually increased the loading dye amount to 5 µL. It didn't seem as if the DNA was floating out of the well this time, but it may still have happened.<br><br />
The final, restriction digested, PCR purified insert was again run on a gel. This time, the gel was run with 25 µL of the insert. Inexplicably, on the gel we observed a very high molecular weight band (greater than 3000 kDa). This could possibly be contamination from the pGEX vector. In any case, the insert is not pure or visible on the gel, which suggests that we need to go back several steps and regenerate it.<br><br />
<strong>Cytoplasmic Star Magainin 1 cloning – </strong>Made glycerol stocks of several cultures containing colonies which contain the correct clone (as verified by the colony screening). Miniprepped the cultures, double digested the resulting plasmid with EcoRI and PstI, and ran the product on a gel (A90). Fortunately, most of our plasmids were shown to have been successfully digested such that the vector and the insert were both visible as two distinct bands at the correct molecular weight on the gel. The plasmids were transformed to BL21(DE3) cells and SHuffle T7 cells.<br><br />
Bacterial colonies were visible on chloramphenicol containing agar plates with the transformation product. Thus, it appears that this plasmid is now ready for protein expression.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week 12 – 8 September</h3><br />
<p>This week, agar swabs of bacteria containing three versions of the SUMO protease arrived from TU Delft. These bacteria were immediately plated out onto agar plates with no antibiotics. They were also plated onto plates with the antibiotics corresponding to the resistance of each plasmid.<br><br />
Work on Delft&rsquo;s SUMO protease is indicated by the stream label &lsquo;Delft&rsquo;.<br><br />
<strong>pGEX-RGD cloning – </strong>Performed a double digest on the remaining sample of the PCR purified PCR product. However, when the entirety of this restriction digest was run on a gel, it mysteriously disappeared. This may be a problem with our apparatus, which has been acting up lately.<br><br />
The PCR reaction to amplify the insert was carried out. Because we've experienced very low yields from previous PCR reactions (of the scale of 40 ng/uL), this time we increased the cycles from 35 to 45. Also, we used the PCR product from last time as the template. The reaction appeared to complete successfully and we ran the products on a DNA gel, followed by a double digest with EcoRI and PstI. However, the gel was anomalous. Although the raw PCR product and the first PCR purification product were visible, the product of the restriction digest disappeared. This could possibly be due to Star activity, and appropriate follow-up experiments need to be run.<br><br />
<strong>Cytoplasmic Star Magainin 1 cloning – </strong><br><br />
Protein expression turned out to be complicated when we discovered that a chloramphenicol-containing petri dish with a sham transformation showed colony growth. These colonies should not have appeared on the plate because the plate had this antibiotic resistance and BL21(DE3) does not inherently have resistance to chloramphenicol.<br><br />
In order to troubleshoot, an experiment was set up with several controls, including conditions where a new chloramphenicol stock solution was made up and used, as well as conditions where different cell lines, separate from BL21(DE3), were plated out.<strong></strong><br><br />
We discovered that the particular substrain of BL21(DE3) that we're using, BL21(DE3)_pLysS, is chloramphenicol resistant. The resistance comes from the pLysS plasmid. The pLysS is supposed to inhibit constitutive expression of the T7 polymerase. This is better for expression of toxic proteins, because it stops leaky expression of the toxic gene (which is why we chose this strain).<br><br />
We confirmed this with a massive experiment with 12 controls. DH5a cells did not grow on the chloramphenicol plates, but did grow on agar plates with no antibiotic<br><br />
Our BL21(DE3) cells grew happily on the chloramphenicol plates, even though there was no plasmid in them. The cells did not grow on ampicillin plates.<br><br />
Fortunately, our competent SHuffle T7 cells are not resistant to chloramphenicol. Therefore, we proceeded with protein expression (see Protocols).<br><br />
We then discovered there was an error in the transformation, in that an incorrect plasmid had been transformed. <br><br />
Also, sequencing data for two of the colonies came back. Unfortunately, the sequencing reaction failed to produce a sufficiently long sequence. The primer/plasmid concentrations will be optimised for the next run.<br><br />
<strong>TU Delft – </strong><br><br />
Today, all of the plates without antibiotics showed proliferative growth. Only one out of three plates with antibiotics showed growth. This was the gene for pBAD-UlpI-HIS6-TT. Several colonies from this plate were picked and cultured in 6 mL of LB overnight in preparation for plasmid miniprep.<br><br />
For the remaining plates, colonies were picked and streaked onto antibiotic-containing plates to hopefully get some growth. Colonies which were plated to antibiotic-containing plates showed growth on these new plates.<br><br />
Last weekend, the FG gene/MAP peptide gene had been transformed to SHuffle cells and plated onto chloramphenicol-containing plates. This week, protein expression was carried out. </p><br />
<h3>&nbsp;</h3><br />
<h3>Week 13 – 15 September</h3><br />
<p>Continuing to attempt to subclone the RGD gene to both pGEX-6P3 and pSB1C3. Discovered that the reason our small ~300 bp PCR fragments were not visible was because we were using a 1% gel, 75 V run. We found that we should have been using a 2% gel at 150 V.<br><br />
We optimized our PCR reaction for the RGDàpGEX cloning task. We used 45 cycles and the product of the previous reaction as the template.<br><br />
Over the weekend, we expressed all our BioBricks. The FG repeats gene/MAP protein and the Star Magainin 1 were expressed in BL21(DE3) and Shuffle cells. We had difficulty running a western blot on the crude whole cell lysate from the weekend, with nothing showing up during the ECL image development.<br><br />
We will continue to troubleshoot this issue.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week 14– 22 September</h3><br />
<p>The Western Blot issue was somewhat resolved by using all of our parent lab&rsquo;s reagents. It appears that our technique is correct, and there must have been an issue with our reagents or buffers. We suspect our PVDF membrane either has expired or is otherwise off. However, we will run duplicates of each gel from now on in order to determine the cause of our previous issues.<br><br />
We continued the RGD cloning. We were having technical difficulties with the RGD to pSB1C3 operation. While the PCR product is visible on a gel, the gel extracted product is not. It is likely because the yield of the PCR is too low, which means that because of product loss during gel extraction, we don&rsquo;t have enough DNA left to see on the subseqeuent gel.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week 15 – 29 September</h3><br />
<p>Successfully optimized the RGD to pSB1C3 PCR reaction by stepping through different annealing temperatures. By lowering the annealing temperature by 4 C, we were able to improve yield of the reaction. <br><br />
We thought we might be able to perform the ligation, but when double digesting the linear magainin 1 gene in pSB1C3 with AgeI and PstI, three bands showed up on the blot. There should only be two. It is not clear why this is, and it may be due to contamination of the linear mag 1 plasmid. Although we could put more resources into this, we decided given the limited time, we would focus our efforts on other tasks, including protein expression and characterization and the remaining cloning task.<br><br />
Having prepared all the vectors and inserts, we performed a ligation between RGD and pGEX-6P3 and RGD and pSB1C3.<br><br />
The Western Blots from last week were inconclusive. There appeared to be his-tagged proteins in some of the cultures, but the staining was splotchy, not a clear band. Also, it was not clear which lanes had the staining.<br><br />
On our advisors&rsquo; advice (H.C. Cheng), we performed a mini-scale his-tag purification on all cultures using Ni-NTA beads in Eppendorf tubes. We will run western blots on these purified samples next week.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week 16 – 6 October</h3><br />
<p>The results of the mini-purification were positive, showing his-tag staining in a western blot in the cultures. We decided to culture more bacteria as the mini purification did not give us enough sample last time for downstream analysis, as we had run out of cell pellet.<br><br />
After the re-expression, we did a second mini-purification, this time using more (300 uL) of Ni-NTA beads.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week 17 – 13 October</h3><br />
<p>The second mini-purification purification appeared to work well on a his-tag Western Blot. We therefore set out to characterise our protein with mass spec, yielding the in-gel digestion results on the results page.</p><br />
</html></div>Slowehttp://2014.igem.org/Team:Melbourne/NotebookTeam:Melbourne/Notebook2014-10-18T01:11:58Z<p>Slowe: </p>
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<br />
<h1 >Notebook</h1><br />
<h2>Semester 1</h2><br />
<p><h3>Preliminary work – Lab setup, training, etc.</h3><br />
<p><br><br />
<em>In Semester 1, we began the process of recruiting the team and setting up the lab. Because our lab was new, we needed to order all supplies, secure scientific sponsorships, and establish protocols. The founding team was also able to do some exploratory lab work before the changeover to the full team later in the semester.</em><br><br />
<em>Lab members were recruited from the wider University of Melbourne student community through an advertising campaign, followed by a selection process. The team underwent training during the Easter break and the following weeks.</em><br />
</p><br />
</p><br />
<p>&nbsp; </p><br />
<h3>Week beginning 6 January</h3><br />
<p>Grew stock of competent DH5α E. coli. Cells stored at -80°C.</p><br />
<p>&nbsp;</p><br />
<h3>Week beginning 13 January</h3><br />
<p>Note that the 2014 founding team originally came up with the concept for a star peptide similar to the USP peptide reported on the Project page. This peptide was meant to be unstructured. It contained an RGD motif to support potential binding of the peptide to integrin (hence why it was termed the RGD peptide). Unlike the peptides reported on the project page, the RGD contained a TEV protease recognition sequence. This would have allowed it to be co-expressed with the TEV protease, and therefore cleaved to form a star peptide inside the cell. <br />
</p><br />
<p>The RGD peptide sequence was: MRV LLF LLL SLF MLP AFS RGD RGD GGG AKQ RGG C GGR QKA GGG RGD RGD EQLYFQG RGD RGD GGG AKQ RGG C GGR QKA GGG RGD RGD HHHHHH<br />
<br />
</p><br />
<p>The founding team ordered this peptide's gene to be synthesised and most of the early work in the lab was focused on using it to practice lab techniques.</p><br />
<h3>Week beginning 20 January</h3><br />
<p><strong> (20-01-14)</strong><br><br />
RGD construct DNA arrived.<br><br />
Completed transformation of DH5α competent cells with heat shock and LB (see Protocols).<br><br />
OD of competent cell growth:<br />
<p><img src="https://static.igem.org/mediawiki/2014/4/4b/Lab_book_picture_001.png" alt="Lab book picture 1"> </p><br />
Digestion was performed using EcoRI (see Protocols).<br><br />
Amount of plasmid was measured.<br><br />
RGD001 – 37.1 ug/uL<br><br />
RGD002 – 51.2 ug/uL<br><br />
RGD003 – 34.5 uG/uL<br><br />
Gel was run. No DNA was visible but the ladder was fine.<br><br />
<strong>(24-01-14)</strong></p><br />
<ul><br />
<li>Redid miniprep. Quantified RGD plasmid again.</li><br />
</ul><br />
<p>RGD001 – 37.1 ug/uL<br><br />
RGD002 – 51.2 ug/uL<br><br />
RGD003 – 34.5 uG/uL</p><br />
<ul><br />
<li>Performed double digest of RGD plasmid with EcoRI and PstI (see Protocols).</li><br />
<li>Performed gel electrophoresis of digested plasmid and undigested plasmid. Plasmids were present at approx 3.5 kb. RGD001 seemed to be the better digested plasmid and best to use for transformation.</li><br />
</ul><br />
<p>&nbsp;</p><br />
<h3>Week beginning 27 January</h3><br />
<ul><br />
<li>Transformed BL21 with RGD. Placed in incubator overnight at 37°C.</li><br />
<li>Ran third gel of transformed DH5α (with RGD).</li><br />
<li>Performed double digests before running another gel to check success.</li><br />
</ul><br />
<p>&nbsp;</p><br />
<h3>Week beginning 3 February</h3><br />
<p>Performed protein induction on cells containing RGD plasmid.<br><br />
NOTE: </p><br />
<ul><br />
<li>Grew 800 mL culture and measured OD (absorbance at 600nm):</li><br />
</ul><br />
<p>0 hrs – 0.184<br><br />
1 hr – 0.322<br><br />
2 hrs – 1.265<br><br />
2.5 hrs – 1.471</p><br />
<ul><br />
<li>Despite overshooting OD target, went ahead with IPTG induction anyway.</li><br />
</ul><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 10 February</h3><br />
<p><strong>Project design/planning</strong><br><br />
Did some project planning based on meeting with advisor Neil O&rsquo;Brien-Simpson:<br><br />
<u>Selenocysteine</u><br><br />
First thought when he saw the project was the liability of the disulfide bond (i.e. tends to fall apart under producing environments).<br><br />
He suggested trying selenocysteine, because selenocysteine bonds are stronger and the amino acids should be sitting around in E. coli ready to use.<br><br />
<u>Comments on AMP</u><br><br />
Suggested a good idea to stick with the Wiradharma, 2012 structure rather than alter too much so as to make comparisons easier.<br><br />
We briefly discussed the mechanism of action of AMPs. As we know, there are several models, including the barrel stave, toroidal poor, and carpet models. In addition, peptide aggregation at the surface of the bacterial cell can lead to membrane thinning, which facilitates pore formation.<br><br />
Neil wasn&rsquo;t sure how the star polymers would work in particular. They may work using the above mechanisms… The disulfide bond may also be broken as the stars enter the cell cytoplasm.<br><br />
<u>Related work</u><br><br />
A researcher by the name of Tam has developed a Multiple Antigenic Peptide (MAP) similar to ours. This peptide includes branched lysine residues. However, the problem with this molecule is that it is very challenging to synthetically produce.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 17 February</h3><br />
<ul><br />
<li>Began new protein expression method (see Protocols). </li><br />
<li>Pelleted cells (see Protocols).</li><br />
</ul><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 24 February</h3><br />
<ul><br />
<li>Transformed DH5α Kan.R resistance (see Protocols). Placed 4 plates on incubator overnight.</li><br />
<li>Transferred cells to 4°C. Colonies grew, suggesting the transformation was successful.</li><br />
</ul><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 3 March</h3><br />
<p><strong>(04-03-14)</strong></p><br />
<ul><br />
<li>To check success of transformation, we ran an SDS-PAGE gel at 150V, 400mA, 45 mins with positive controls and RGD protein (see Protocols):</li><br />
</ul><br />
<p> <img src="https://static.igem.org/mediawiki/2014/f/fa/Lab_book_picture_002.png" alt="Lab book picture 2"></p><br />
<ul><br />
<li>Transformed DH5α colonies with Magainin I.</li><br />
<li>DH5α (MagI) transformed cells were miniprepped (see Protocols). Some of cell pellet was loaded into SDS-PAGE gel with two positive controls for His-tags. Results appeared successful so we continued onto Ni-NTA column purification.</li><br />
<li>Unfortunately, Ni-NTA column purification failed.</li><br />
</ul><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 10 March</h3><br />
<p>Redid process from last week to try to diagnose what went wrong.<br><br />
Double digestion of DH5α (Magainin I) colony miniprep products with EcoRI and PstII.<br><br />
Made and ran DNA gel with double digest at 120V, 100mA, 35 minutes then 135V, 100mA, 15 minutes then 120V 3 minutes.<br><br />
<br />
<p><img src="https://static.igem.org/mediawiki/2014/4/45/Lab_book_picture_003.png" alt="Lab book picture 3"></p><br />
Results were visible for lanes 1 – 4, but digests were not visible.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 17 March</h3><br />
<p><strong><em>(Recruitment of team members)</em></strong></p><br />
<h3>Week beginning 24 March</h3><br />
<p><strong><em>(Recruitment of team members)</em></strong></p><br />
<h3>Week beginning 31 March</h3><br />
<p><strong><em>(Recruitment of team members)</em></strong></p><br />
<h3>Week beginning 7 April</h3><br />
<p><strong><em>(Recruitment of team members)</em></strong></p><br />
<h3>Week beginning 14 April</h3><br />
<p><strong><em>(Recruitment of team members)</em></strong></p><br />
<h3>Week beginning 21 April</h3><br />
<p><strong><em>(Lab introductory training)</em></strong></p><br />
<h3>Week beginning 28 April</h3><br />
<p><strong><em>(Lab setup work)</em></strong></p><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 5 May</h3><br />
<p>Despite negative results on gel of double digest (thought to be due to faulty gel electrophoresis process), we went ahead with protein expression.<br><br />
We also transformed BL21 with pMK-RGD vector (see Protocols) and ran a protein gel with pGEX, pET, pMK RGD and pMK Mag 1 (samples taken from the training week during the break).<br><br />
<br><br />
Conducted miniprep of DH5α pMK-RGD culture and ran DNA gel on the RGD plasmid extracted. </p><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 12 May</h3><br />
<p>Started protein expression on transformed BL21 pMK-RGD.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 19 May</h3><br />
<p>Completed protein expression, but cultures failed to grow.<br><br />
Ran PCR for pMK RGD (reaction volume = 50 uL) with the following new recipe:</p><br />
<ul><br />
<li>10X Thermopod Reaction Buffer – 5 uL</li><br />
<li>10 mM dNTP mixture – 1 uL</li><br />
<li>10 uM forward primer – 1 uL</li><br />
<li>10 uM reverse primer – 1 uL</li><br />
<li>Template DNA – 1 uL</li><br />
<li>Taq DNA polymerase – 0.5 uL</li><br />
<li>Nuclease-free h3O – 40.5 uL</li><br />
</ul><br />
<p>Conditions for the PCR cycle:</p><br />
<ul><br />
<li>95°C – 2 minutes</li><br />
<li>30 cycles:</li><br />
<ul><br />
<li>95°C – 20 seconds</li><br />
<li>60°C – 20 seconds</li><br />
<li>72°C – 20 seconds</li><br />
</ul><br />
<li>72°C – 5 minutes</li><br />
<li>4°C – infinity (hold) <strong></strong></li><br />
</ul><br />
<p>Performed protein induction on PMK-MAG1 .</p><br />
<p>Created a glycerol stock of BL21-RGD and BL21-MAG1 and stored it at -86°C.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 26 May</h3><br />
<p><strong><em>(Exam period – no lab preparation work)</em></strong></p><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 2 June</h3><br />
<p><strong><em>(Exam period – no lab preparation work)</em></strong></p><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 9 June</h3><br />
<p><strong><em>(Exam period – no lab preparation work)</em></strong></p><br />
<h3>&nbsp;</h3><br />
<h3>Week beginning 16 June</h3><br />
<p><strong><em>(Exam period – no lab preparation work)</em></strong></p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
<h2>Semester 2 </h2><br />
<p><em>Main project work begins.</em><br><br />
<em>With the lab set up, the team fully formed, and preliminary work completed, we began work on the project. Our focus throughout Semester 2 was on the design of the BioBricks, and ensuring that each of our proteins could be properly expressed in transformed bacteria after cloning and subcloning.</em></p><br />
<h3>&nbsp;</h3><br />
<h3>Week 1 – 23 June</h3><br />
<p><strong>(24-06-14)</strong></p><br />
<ul><br />
<li>Plated BL21(DE3) + Magainin 1 glycerol stock on one plate using &lsquo;spread&rsquo; technique at 12:45pm, placed in 37 degree incubator at 3pm (should be taken out at 11am next day [+20 hours]).</li><br />
</ul><br />
<p>However, the plates had way too much growth, almost resembling a lawn, and couldn&rsquo;t be used due to a need for &lsquo;isolated&rsquo; colonies. After checking lab-book again, realised the glycerol stock was made using culture from already plated colonies and instead of just transformed cells. <br><br />
New glycerol stock was used to plate again, this time using the &lsquo;streak&rsquo; technique.</p><br />
<ul><br />
<li>SDS-PAGE gel with MagI pellet was run. 2 samples were prepared using approximately 100μL of SDS-PAGE Sample Buffer and a small amount of pellet. The samples were prepared using Ash&rsquo;s method of quickly boiling the samples (placing tubes in heat block set at 100(99.9) degrees) to lyse the cells. Unfortunately, the samples thickened and SDS-PAGE could not be run. This may be a result of specific faults in the buffer recipe, so possibly use a RIPA buffer next time.</li><br />
</ul><br />
<h3>&nbsp;</h3><br />
<h3>Week 2 – 30 June</h3><br />
<p><strong>Project design/planning</strong><br><br />
The team agreed to have a multi-track research program. What we are essentially doing at the moment is trying to attack this problem of synthesizing disulfide bonded structures from multiple fronts.<br><br />
Tobias suggested a new concept for ligating peptides, carbohydrates, lipids and other biomolecules to the star peptide structure through native chemical ligation.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week 3– 7 July</h3><br />
<p><strong>Project design/planning</strong><br><br />
Further work on the idea of native chemical ligation (NCL).<br><br />
Summary of progress on shuttling the MAG 1 insert with the periplasmic export tag to the standard iGEM plasmid – </p><br />
<ul><br />
<li>Ran PCR on pMK-RGD using primers to make RGD compatible with pGEX vector</li><br />
<li>Ran PCR product on gel</li><br />
<li>Cut out PCR product from gel and use gel purification kit</li><br />
<li>Ran restriction digestion on PCR insert product of 3a using BamHI and EcoRI</li><br />
<li>Ran PCR purification using Thermo kit on product of 4a</li><br />
<li>Transformed pGEX vector donated by a neighboring lab into DH5a, plate and incubated overnight</li><br />
<li>Miniprepped plasmid</li><br />
<li>Ran restriction digestion on plasmid product of 2b using BamHI and EcoRI</li><br />
</ul><br />
<p>Summary of previous progress on inserting RGD coding region into pGEX vector, and expression in BL21(DE3) cells – </p><br />
<ul><br />
<li>Ran PCR on pMK-RGD using primers to make RGD compatible with pGEX vector</li><br />
<li>Ran PCR product on gel</li><br />
<li>Cut out PCR product from gel and use gel purification kit</li><br />
<li>Ran restriction digestion on PCR insert product of 3a using BamHI and EcoRI</li><br />
<li>Ran PCR purification using Thermo kit on product of 4a</li><br />
<li>Transformed pGEX vector donated by a neighboring lab into DH5a, plate and incubated overnight</li><br />
<li>Miniprepped plasmid</li><br />
<li>Ran restriction digestion on plasmid product of 2b using BamHI and EcoRI</li><br />
</ul><br />
<p>Decided to divide work into streams as previously planned – indicated in lab book as:</p><br />
<ul><br />
<li>pSB1C3-MAG1 subcloning</li><br />
<li>pGEX-RGD cloning</li><br />
<li>Cytoplasmic Star Magainin 1 cloning</li><br />
</ul><br />
<h3>&nbsp;</h3><br />
<h3>Week 4 – 14 July </h3><br />
<p><strong>pSB1C3-MAG1 subcloning – </strong></p><br />
<ul><br />
<li>Ran PCR on pMK-MAG1 using primers to remove TorTSS and add sticky ends for pSB (BBa_K525998). When run on a gel, the PCR product was a very clear band, indicating success.</li><br />
<li>Ran restriction digestion on pMK-MAG1 PCR insert product using SpeI and PstI. </li><br />
<li>Ran PCR on MAG1 using primers to remove TorTSS and add sticky ends for pSB (BBa_K525998) for a second time.</li><br />
</ul><br />
<p><strong>Cytoplasmic Star Magainin 1 cloning –</strong> Plated DH5a cells.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week 5 – 21 July</h3><br />
<p><strong>pSB1C3-MAG1 subcloning – </strong><br><br />
We transformed DH5a with the pSB1C3 (RFP) plasmid provided by iGEM. We then miniprepped the cells. However, the nanodrop suggested that the concentration of the cells was extremely low. Gayle then ligated pSB1C3 with a MAG 1 insert. Because the concentrations were too low to see on a DNA gel, she re-transformed the plasmid to DH5a. The idea is that we should now be able to re-miniprep this DH5a and reclaim the ligated plasmid.<br><br />
<strong>Cytoplasmic Star Magainin 1 cloning – </strong><br><br />
Did restriction digestion using PstI and SpeI on miniprepped T7 promoter containing pSB1C3. Ran the product on a gel, but it came up blank. We had a thorough discussion with Ash and David about why this could be. Ash suggested trying digesting with one enzyme at a time, having several lanes, each with different enzyme combinations. <br><br />
A Google search suggested that there could be a problem with the miniprep procedure. Specifically, there could be some enzymes being left over from the miniprep which, when the samples were heated during the restriction digestion, ate up the DNA.<br><br />
Therefore, Stan designed an experiment to test this. We had 5 conditions: </p><br />
<ul><br />
<li>the PSB1C3 plasmid directly after the miniprep, with no incubation or restriction digestion; </li><br />
<li>the plasmid heated to 37° with restriction enzymes, </li><br />
<li>the plasmid heated to 37° WITHOUT restriction enzymes, </li><br />
<li>a PCR insert meant to go into the plasmid heated to 37° with restriction enzymes (the same restriction enzymes which would allow us to like it that insert into the pSB1C3 plasmid),</li><br />
<li>the PCR insert heated to 37° without restriction enzymes.</li><br />
</ul><br />
<p>So, we're testing the effect of heat and the effect of the restriction enzymes.<br><br />
In the resulting gel, every lane was visible, except for the plasmid which had been double digested. This suggests that there was not anything wrong with the restriction enzymes per se, neither with the buffer nor the heating process. It argues against the hypothesis that there was some DNA degrading enzyme present after the miniprep could have been activated by heat.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week 6 – 28 July</h3><br />
<p><strong>Cytoplasmic Star Magainin 1 cloning – </strong><br><br />
Thawed out the plasmid samples in sample buffer from yesterday and loaded them onto a 16 well agarose gel. Every lane was visible except for the Axygen miniprepped plasmid treated with SpeI. However, the Axygen miniprepped plasmid treated with a double digestion of SpeI and PstI DID show upon the gel. After speaking with David, he suspects that we perhaps neglected to put DNA into that well. <br><br />
Note that these results are in direct contrast to the gel described earlier, where we had an identical plasmid (pSB1C3 containing the P7 promoter), picked from the same plate, using the same miniprep kit, same restriction enzymes (PstI and SpeI), for the same incubation period (2.5-3 hrs). That is, we did exactly the same thing, and this time, it showed up on the gel, whereas before it disappeared.<br><br />
Given that the double digest with SpeI and PstI and EcoR1 and PstI worked, we went ahead and began culturing in 10 mL colonies from the same pSB1C3 plate used in the experiment above. We made 6, 10 mL cultures. To provide a backup, we also picked three colonies which had previously been plated out by Lumi containing GFP, RFP, or YFP, creating 3, 10 mL cultures which may also be miniprepped.<br><br />
Further progress:</p><br />
<ul><br />
<li>Miniprepped 10 mL cultures of pSB containing the p7 promoter. Ran various restriction digests on the miniprep product in preparation for ligation of pSB with our MAG1 PCR insert.<strong></strong></li><br />
<li>Ran the restriction digests from yesterday. Results are very mysterious. Thus, re-ran restriction digests. Results favourably suggest one of the minipreps was successful.<strong></strong></li><br />
<li>Gel extracted pSB1C3 digested with both SpeI and PstI.<strong></strong></li><br />
</ul><br />
<h3>&nbsp;</h3><br />
<h3>Week 7– 4 August</h3><br />
<p><strong>pSB1C3-MAG1 subcloning – </strong>Transformation of pSB1C3 to DH5α (see Protocols).<br><br />
<strong>pGEX-RGD cloning – </strong>Transformation of pGEX to DH5α (see Protocols).<strong> </strong> <br><br />
<strong>Cytoplasmic Star Magainin 1 cloning – </strong> <br><br />
We were originally planning to do the ligation reaction with the SpeI and PstI restriction enzymes AND the PCR insert. However, during that restriction digest of the PCR insert, DNA loading dye was erroneously added. We attempted to gel purify the PCR insert to remove the loading dye. However, for some reason the &quot;PCR insert&quot; ran at a very high molecular weight (approximately 2 kDa). Moreover, there is a strange double band.<br><br />
Because the PCR insert appears to be screwed up, we will have to start from the PCR step again. Planning on ordering new primers which will allow the Magainin 1 insert to be biobrick compatible.<strong> </strong></p><br />
<h3>&nbsp;</h3><br />
<h3>Week 8 – 11 August</h3><br />
<p><strong>pSB1C3-MAG1 subcloning –</strong><strong> </strong></p><br />
<ul><br />
<li>PLASMID: </li><br />
<ul><br />
<li>Stanislav performed a double restriction digest on the linearized plasmid backbone pSB1C3. </li><br />
<li>Performed a miniprep on the 10 mL cultures. Confirmed the success of the miniprep/presence of the plasmid using a DNA gel.</li><br />
</ul><br />
<li>INSERT: </li><br />
<ul><br />
<li>Performed a restriction digestion on previous pMK-MAG1 plasmid stock in order to liberate the MAG 1 insert in preparation for ligation with pSB1C3 and ran a gel on the products. In this gel, a distinct band was observed corresponding to the approximately 2 kDa vector (pMK). However, the 6 kDa insert was not visible. </li><br />
<li>In order to get more of the insert, we propagated more pMK-MAG1 plasmid. </li><br />
</ul><br />
</ul><br />
<p><strong>pGEX-RGD cloning – </strong></p><br />
<ul><br />
<li>Transformed DH5a competent cells with pGEX-6P3 and performed a miniprep to increase the amount of plasmid stock available for ligation. A DNA gel performed on the products suggested the miniprep was successful. </li><br />
<li>A gel performed on PCR products of the RGD insert amplified with restrictions sites added for subcloning into pGEX gave odd results. </li><br />
</ul><br />
<p>Both the PCR products created yesterday and the products done over the summer break showed over 10 bands in each lane. This suggests that there was nonspecific binding, leading to a variety of different molecular weight products. Spoke to advisor Ash who prescribed a new PCR temperature/timing cycle to minimise this problem.</p><br />
<p>Ran the PCR reaction (using Ash&rsquo;s suggested solution) product on a gel. Increasing the annealing temperature in the PCR cycle did decrease the presence of multiple (~10) bands. However, it also led to a general reduction in the intensity of the PCR product's signal on the gel. It's not clear that the changes to the PCR protocol actually reduced nonspecific binding so much as just decreased the reaction yield.</p><br />
<p>As suggested by our supervisor, Cheng, we reran the PCR reaction. This time, in order to eliminate the possibility of nonspecific binding, we cut out the RGD insert from the PMK vector using restriction digestion, gel extract the insert, and then used this insert as the PCR template.</p><br />
<p><strong>Cytoplasmic Star Magainin 1 cloning – </strong><br><br />
We had previously ordered the primers which would both remove the periplasmic export tag and allow for ligation into Biobrick BBa_K525998 of our Magainin 1 Star Peptide insert. The problem with these primers is, however, that after the ligation, there would be the Spe1 restriction site at the 5' end of the protein coding region. Therefore, new primers were designed which would eliminate this problem. The protein coding region will be inserted after the T7 promoter in BBa_K525998. At the 5' end of the insert, XbaI will be used to join the insert to the plasmid's SpeI site. At the 3' end of the insert, PstI will be used to join the insert to the plasmids PstI site. </p><br />
<h3>&nbsp;</h3><br />
<h3>Week 9 – 18 August</h3><br />
<p><strong>pSB1C3-MAG1 subcloning –</strong></p><br />
<ul><br />
<li>INSERT:</li><br />
<ul><br />
<li>Did a double digestion on the pMK-MAG1 plasmid.</li><br />
<li>Ran the double digestion product on a DNA gel and gel-extracted.</li><br />
<li>Ran an analytical amount of the digested linearized PSB1C3 backbone on a DNA gel. The plasmid was clearly visible on the gel (Gel C35).</li><br />
</ul><br />
<li>PLASMID: Did a double digestion on a significant amount of the linearized plasmid backbone and ran the double digestion product of pMK-MAG1 on a DNA gel. However, the results on the DNA gel (Gel C35) were inexplicable. We observed a smear in the lanes containing pMK-MAG1, not the clear bands we expected. Moreover, the insert was not visible at all in the lower regions of the gel.</li><br />
</ul><br />
<p>Due to the anomalous results, we designed an experiment performing a series of restriction digestions on the pMK plasmid containing the Magainin 1 insert. Several controls, including single digestions and digestions using newer tubes of restriction enzymes EcoRI and PstI were performed.<br><br />
<strong>pGEX-RGD cloning –</strong><br><br />
It was discovered that there was a mistake in the PCR primers we have been using up to this date. Specifically, the PCR primers amplified a segment of the DNA including the periplasmic export tag preceding the RGD protein coding region. Therefore, new primers had to be designed for PCR on the RGD insert.<br><br />
Nonetheless, we did a double digestion on the pMK-RGD plasmid, making sure that the volume of the reaction is quite large so that the insert band was be clearly visible on the DNA gel. This measure is a backup in the event that the PCR primers are not specific in their binding to the template. If the primers are non-specific, then we will use the isolated insert as the template.</p><br />
<ul><br />
<li>PLASMID: Ran an analytical amount of the gel-extracted digested pGEX vector on a gel (Gel C35) and confirmed successfμL gel-extraction.</li><br />
<li>INSERT: Ran the restriction digestion product (PMK-RGD) on a DNA gel. However, strangely we did not get the results we expected. Instead of seeing two bands, one corresponding to the vector in one to the insert, we saw approximately 5 bands. This suggests that the restriction digestion failed, and we might be seeing a multiple bands associated with the undigested plasmid.</li><br />
</ul><br />
<p><strong>Cytoplasmic Star Magainin 1 cloning – </strong><br><br />
Completed the PCR reaction to amplify the plasmid in preparation for ligation and ran on a gel. We have previously digested the destination plasmid using the relevant restriction enzymes. We did one final check to ensure that the gel extraction worked by running a sample of the product on another gel.<br><br />
A double digestion was completed on a portion of the gel extracted PCR insert using XbaI and PstI, two restrictions sites which will allow us to ligate this insert with the pSB1C3 plasmid (containing the T7 promoter) at the biobrick suffix. The PCR insert was then PCR purified.<strong></strong><br><br />
A ligation procedure was performed between pSB1C3 in the PCR insert. The ligation product was transformed to DH5a.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week 10 – 25 August</h3><br />
<p><strong>pSB1C3-MAG1 subcloning –</strong><br><br />
A gel run on the restriction digestion experiment last week revealed that there was something wrong with our old EcoRI/PstI when used in combination. <br><br />
We designed an experiment using gel electrophoresis of a combination of different enzymes, used on the same plasmid, repeated with our old and new enzymes. We found that with the NEW enzymes, the double digestion works, and with the old enzymes it doesn't work.<br><br />
Strangely enough, it looks like the single digests with the old enzymes still work. It's as if there's some interaction effect when the old Eco and Post are used together where things go bonkers.<br><br />
This experiment has also revealed a limitation of our cloning strategy. It turns out that there is actually a PstI cutting site in pMK. Hence, you will see that there actually more than two bands in the double digestions (and if you look closely, two bands in the single digestions with PstI). All is not lost, however, as we can still double digest pMK-MAG1 using different restriction enzymes, bypassing PstI. We can use EcoRI and SpeI, and this should work (there are no EcoRI or SpeI sites in pMK).<br><br />
This sub cloning experiment was started from the restriction digestion point again. This time, however, we digested the destination vector (pSB1C3) and insert (contained in pMK-MAG1) with the NEW tubes of EcoRI and PstI. A gel was run on these products. Both the destination vector, pSB1C3, and the gel isolated insert were clearly visible on the gel.<br><br />
A ligation reaction between the Mag1 insert and the pSB1C3 linearized vector was carried out. The ligation product was transformed to DH5α and plated to chloramphenicol-containing plates.<br><br />
<strong>pGEX-RGD cloning – </strong>A gel was run on the PCR product (previously carried out). However, it appeared that the PCR reaction was inefficient; the product had a very feint band on the gel. After gel extraction of the product, nanodrop measurements suggested that the concentration was in the range 5-10 ng/uL. Because the ultimate goal of this PCR reaction was to create a lot of insert for a ligation later on, small amounts of insert DNA were not acceptable. <br><br />
A second PCR reaction was run in order to hopefully increase the yield of the insert. The number of cycles was increased to 35 from 30. In a second condition, the gel extracted PCR product from the last reaction was used as the template.<br><br />
The products of the PCR reaction of the RGD insert were run on a gel and were clearly visible at the correct molecular weight.<br><br />
A restriction digestion of both pGEX-6P-3 and the PCR-amplified RGD insert was carried out. EcoRI and BamHI were used on both vector and insert to create complementary sticky ends. The reaction was completed and the products placed in the -20 freezer.<br><br />
<strong>Cytoplasmic Star Magainin 1 cloning – </strong>Minipreps were performed on colonies containing the ligation and the products were run on a DNA gel (Gel C43). However, no DNA was visible on the gel, suggesting that the minipreps had failed. This is likely because the culture conditions of the miniprep were suboptimal. However, another gel showed that the PCR purified insert ran clearly on the gel. Thus, we can combine that insert with the vector and likely produce a clone again.<strong></strong><br><br />
The ligation of the destination vector in the PCR amplified insert was repeated today. The ligation product was then transformed to DH5α. The ligation reaction to get the Magainin 1 insert into pSB1C3 was found to be successful. The MAG 1 insert was mixed with the destination vector pSB1C3 in a ligation reaction. The ligated product was transformed to DH5α and plated overnight. Colonies were picked from agar plate and then placed in a PCR reaction (PCR-based colony screening). The primers used in the PCR were specific to the Magainin 1 insert. <br><br />
Performing a PCR on the colonies led to a propagation of the insert. This suggests that the colonies contained the Magainin 1 insert. And, because the colonies were chloramphenicol resistant, they should also contain the pSB1C3 vector. Thus, it appears that we have the vector with the insert! </p><br />
<h3>&nbsp;</h3><br />
<h3>Week 11 – 1 September</h3><br />
<p><strong>pSB1C3-MAG1 subcloning – </strong>From the plate of DH5α transformed with the ligation product, PCR colony screening was performed. Only one colony showed successful transformation on a DNA gel. This culture later showed no growth, and transformation was suspected to have failed. Because of this, a second transformation was carried out using the ligation product used for the previous transformation/plating, and appeared to be successful. Glycerol stocks were made of the cultures from the second transformation.<br><br />
Later, more colonies were miniprepped, double digested, and ran on a gel. Strangely, as in previous gels, the only thing visible on the gel was a very faint band with molecular weight greater than 3000. It is unclear what that band corresponds to. Because the colony screening failed in this manner across many colonies and two different transformations, it suggests that there something went wrong during the ligation reaction or earlier.<br><br />
<strong>pGEX-RGD cloning –</strong><br><br />
The insert for the pGEX RGD stream was PCR purified and then run on a gel. For unknown reasons, the PCR purification product was not visible on the gel. The nanodrop suggested that the concentration was 15 ng/µL. 10 µL of the product were run on the gel. It therefore should have been visible.<br><br />
Note, however, that we observed a strange phenomenon where the DNA/loading dye was floating out of the well once loaded. After trying to load two wells, we eventually increased the loading dye amount to 5 µL. It didn't seem as if the DNA was floating out of the well this time, but it may still have happened.<br><br />
The final, restriction digested, PCR purified insert was again run on a gel. This time, the gel was run with 25 µL of the insert. Inexplicably, on the gel we observed a very high molecular weight band (greater than 3000 kDa). This could possibly be contamination from the pGEX vector. In any case, the insert is not pure or visible on the gel, which suggests that we need to go back several steps and regenerate it.<br><br />
<strong>Cytoplasmic Star Magainin 1 cloning – </strong>Made glycerol stocks of several cultures containing colonies which contain the correct clone (as verified by the colony screening). Miniprepped the cultures, double digested the resulting plasmid with EcoRI and PstI, and ran the product on a gel (A90). Fortunately, most of our plasmids were shown to have been successfully digested such that the vector and the insert were both visible as two distinct bands at the correct molecular weight on the gel. The plasmids were transformed to BL21(DE3) cells and SHuffle T7 cells.<br><br />
Bacterial colonies were visible on chloramphenicol containing agar plates with the transformation product. Thus, it appears that this plasmid is now ready for protein expression.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week 12 – 8 September</h3><br />
<p>This week, agar swabs of bacteria containing three versions of the SUMO protease arrived from TU Delft. These bacteria were immediately plated out onto agar plates with no antibiotics. They were also plated onto plates with the antibiotics corresponding to the resistance of each plasmid.<br><br />
Work on Delft&rsquo;s SUMO protease is indicated by the stream label &lsquo;Delft&rsquo;.<br><br />
<strong>pGEX-RGD cloning – </strong>Performed a double digest on the remaining sample of the PCR purified PCR product. However, when the entirety of this restriction digest was run on a gel, it mysteriously disappeared. This may be a problem with our apparatus, which has been acting up lately.<br><br />
The PCR reaction to amplify the insert was carried out. Because we've experienced very low yields from previous PCR reactions (of the scale of 40 ng/uL), this time we increased the cycles from 35 to 45. Also, we used the PCR product from last time as the template. The reaction appeared to complete successfully and we ran the products on a DNA gel, followed by a double digest with EcoRI and PstI. However, the gel was anomalous. Although the raw PCR product and the first PCR purification product were visible, the product of the restriction digest disappeared. This could possibly be due to Star activity, and appropriate follow-up experiments need to be run.<br><br />
<strong>Cytoplasmic Star Magainin 1 cloning – </strong><br><br />
Protein expression turned out to be complicated when we discovered that a chloramphenicol-containing petri dish with a sham transformation showed colony growth. These colonies should not have appeared on the plate because the plate had this antibiotic resistance and BL21(DE3) does not inherently have resistance to chloramphenicol.<br><br />
In order to troubleshoot, an experiment was set up with several controls, including conditions where a new chloramphenicol stock solution was made up and used, as well as conditions where different cell lines, separate from BL21(DE3), were plated out.<strong></strong><br><br />
We discovered that the particular substrain of BL21(DE3) that we're using, BL21(DE3)_pLysS, is chloramphenicol resistant. The resistance comes from the pLysS plasmid. The pLysS is supposed to inhibit constitutive expression of the T7 polymerase. This is better for expression of toxic proteins, because it stops leaky expression of the toxic gene (which is why we chose this strain).<br><br />
We confirmed this with a massive experiment with 12 controls. DH5a cells did not grow on the chloramphenicol plates, but did grow on agar plates with no antibiotic<br><br />
Our BL21(DE3) cells grew happily on the chloramphenicol plates, even though there was no plasmid in them. The cells did not grow on ampicillin plates.<br><br />
Fortunately, our competent SHuffle T7 cells are not resistant to chloramphenicol. Therefore, we proceeded with protein expression (see Protocols).<br><br />
We then discovered there was an error in the transformation, in that an incorrect plasmid had been transformed. <br><br />
Also, sequencing data for two of the colonies came back. Unfortunately, the sequencing reaction failed to produce a sufficiently long sequence. The primer/plasmid concentrations will be optimised for the next run.<br><br />
<strong>TU Delft – </strong><br><br />
Today, all of the plates without antibiotics showed proliferative growth. Only one out of three plates with antibiotics showed growth. This was the gene for pBAD-UlpI-HIS6-TT. Several colonies from this plate were picked and cultured in 6 mL of LB overnight in preparation for plasmid miniprep.<br><br />
For the remaining plates, colonies were picked and streaked onto antibiotic-containing plates to hopefully get some growth. Colonies which were plated to antibiotic-containing plates showed growth on these new plates.<br><br />
Last weekend, the FG gene/MAP peptide gene had been transformed to SHuffle cells and plated onto chloramphenicol-containing plates. This week, protein expression was carried out. </p><br />
<h3>&nbsp;</h3><br />
<h3>Week 13 – 15 September</h3><br />
<p>Continuing to attempt to subclone the RGD gene to both pGEX-6P3 and pSB1C3. Discovered that the reason our small ~300 bp PCR fragments were not visible was because we were using a 1% gel, 75 V run. We found that we should have been using a 2% gel at 150 V.<br><br />
We optimized our PCR reaction for the RGDàpGEX cloning task. We used 45 cycles and the product of the previous reaction as the template.<br><br />
Over the weekend, we expressed all our BioBricks. The FG repeats gene/MAP protein and the Star Magainin 1 were expressed in BL21(DE3) and Shuffle cells. We had difficulty running a western blot on the crude whole cell lysate from the weekend, with nothing showing up during the ECL image development.<br><br />
We will continue to troubleshoot this issue.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week 14– 22 September</h3><br />
<p>The Western Blot issue was somewhat resolved by using all of our parent lab&rsquo;s reagents. It appears that our technique is correct, and there must have been an issue with our reagents or buffers. We suspect our PVDF membrane either has expired or is otherwise off. However, we will run duplicates of each gel from now on in order to determine the cause of our previous issues.<br><br />
We continued the RGD cloning. We were having technical difficulties with the RGD to pSB1C3 operation. While the PCR product is visible on a gel, the gel extracted product is not. It is likely because the yield of the PCR is too low, which means that because of product loss during gel extraction, we don&rsquo;t have enough DNA left to see on the subseqeuent gel.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week 15 – 29 September</h3><br />
<p>Successfully optimized the RGD to pSB1C3 PCR reaction by stepping through different annealing temperatures. By lowering the annealing temperature by 4 C, we were able to improve yield of the reaction. <br><br />
We thought we might be able to perform the ligation, but when double digesting the linear magainin 1 gene in pSB1C3 with AgeI and PstI, three bands showed up on the blot. There should only be two. It is not clear why this is, and it may be due to contamination of the linear mag 1 plasmid. Although we could put more resources into this, we decided given the limited time, we would focus our efforts on other tasks, including protein expression and characterization and the remaining cloning task.<br><br />
Having prepared all the vectors and inserts, we performed a ligation between RGD and pGEX-6P3 and RGD and pSB1C3.<br><br />
The Western Blots from last week were inconclusive. There appeared to be his-tagged proteins in some of the cultures, but the staining was splotchy, not a clear band. Also, it was not clear which lanes had the staining.<br><br />
On our advisors&rsquo; advice (H.C. Cheng), we performed a mini-scale his-tag purification on all cultures using Ni-NTA beads in Eppendorf tubes. We will run western blots on these purified samples next week.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week 16 – 6 October</h3><br />
<p>The results of the mini-purification were positive, showing his-tag staining in a western blot in the cultures. We decided to culture more bacteria as the mini purification did not give us enough sample last time for downstream analysis, as we had run out of cell pellet.<br><br />
After the re-expression, we did a second mini-purification, this time using more (300 uL) of Ni-NTA beads.</p><br />
<h3>&nbsp;</h3><br />
<h3>Week 17 – 13 October</h3><br />
<p>The second mini-purification purification appeared to work well on a his-tag Western Blot. We therefore set out to characterise our protein with mass spec, yielding the in-gel digestion results on the results page.</p><br />
</html></div>Slowehttp://2014.igem.org/Team:Melbourne/ProjectTeam:Melbourne/Project2014-10-18T00:53:18Z<p>Slowe: </p>
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<h1>Project and results</h1><br />
<br />
<p>Read about our experimental work below, or jump to our theoretical collaboration with the <A HREF="#Oxford">University of Oxford</A><br />
<br />
<h2>Introduction and theory</h2><br />
<p>Synthetic peptide chemists have long produced peptide-based materials <em>in vitro</em>. Star-shaped peptides are a promising type of biomaterial being explored in the field of nanomedicine (Sulistio et al., 2012). Star peptides can have several biomedical uses, such as acting as drug delivery vehicles (Sulistio et al., 2011) or linkers for other biomacromolecules. Star peptides generally take the form of several linear peptide arms linked together in a central core. One way of linking these linear peptide arms together is to used covalent bonds such as those in disulfides. Typically, disulfide bonds are formed synthetically by taking several linear arms and treating them with an oxidant <em>in vitro</em>. Here, we introduce a new approach to forming star peptides using <em>E. coli</em> and synthetic biology. Thus, we aimed to show how the peptides synthesis and disulfide bond forming machinery of <em>E. coli</em> can be used to form disulfide linked star peptides and key star peptide precursors.</p><br />
<p>&nbsp;</p><br />
<h2>Synthesis approach</h2><br />
<p><em>E. coli</em> naturally possesses the capacity to form disulfide bonds. In native strains, disulfide bonds are naturally formed by an array of enzymes which are part of the Dsb family (e.g. DsbA and DsbC) (Kadokura et al., 2003, Kadokura and Beckwith, 2009). Normally, these enzymes are found in the oxidizing periplasm of the cell. However, several new strains of <em>E. coli</em> have recently been engineered which contain an oxidizing cytoplasm conducive to disulfide bond formation. One example of this is the SHuffle cell line (Lobstein et al., 2012). The cell line contains mutations to key enzymes responsible for the reducing nature of the cytoplasm, namely thioredoxin reductase (<em>trxB</em>) and glutathione reductase (<em>gor</em>). Furthermore, the Shuffle cell line over expresses the disulfide bond isomerase DsbC to the cytoplasm. Together, these mutations allow SHuffle to fold disulfide-bonded proteins in the cytoplasm at a higher success rate compared to non-mutants. We aimed to take advantage of the disulfide bond forming capabilities of this strain of <em>E. coli</em> to synthesize star peptides in cells. As shown in the figure below, the synthesis steps may proceed as follows:</p><br />
<p>&nbsp;</p><br />
<ol><br />
<li> Express a short peptide containing two cysteine residues either to the <em>E. coli</em> periplasm or the cytoplasm of a <em>trxB gor </em>mutant.</li><br />
<li><em>E. coli</em> disulfide bond forming enzymes fold the peptide into a hairpin loop structure.<br />
</li><br />
<li>Cut the loop at a protease recognition site engineered into the peptide. This may be done by extracting the folded peptide from the cell and treating it with the protease in vitro. Alternatively, the protease may be co-expressed in the cell to allow for in vivo cleavage.<br />
</li><br />
</ol><br />
<p>&nbsp;</p><br />
<table align="center"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9d/Project_concept-v2.png" width="480" height="600" alt=""/></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<p>This synthesis approach has several benefits over purely <em>in vitro</em> approaches. Firstly, the exact peptide sequence can be precisely programmed into <em>E. coli</em> using recombinant DNA synthesis. Secondly, by performing the disulfide bond formation in cells and optionally the proteolytic cleavage, several synthesis steps which would need to be performed <em>in vitro</em> are eliminated. From a scale up perspective, this would eliminate entire unit operations which would otherwise be required to produce this product. Given these benefits, in the current study, we aimed to express a star peptide precursor to the cytoplasm of SHuffle cells, which would later be extracted and externally digested with the star-forming protease. In order to achieve this, we first designed several star peptides which might be amenable to this synthetic strategy. These peptides are described below.</p><br />
<p>&nbsp;</p><br />
<h2>Rationally designed peptides</h2><br />
<p>There are two approaches to functionalising star peptides. In the first, the identity of the arms can be chosen to be bioactive peptide molecules. This is the simplest approach to producing a biologically-relevant star. In the second, the arms can be functionalised by ligating molecules to them at any point. We used both of these approaches to design two separate peptides.</p><br />
<h3>Magainin 1 star and linear peptides</h3><br />
<p>Our first strategy was to make a star peptide using antimicrobial peptides(AMPs) as building blocks. AMPs are small, approximately 50 residue peptides secreted by some bacterial and eukaryotic cells which selectively kill microbial cells. It is thought that AMPs work by forming pores in the membrane of prokaryotic cells (Brogden, 2005). AMPs have been recombinantly expressed in a number of organisms, including <em>E. coli</em> (for a review, see Li, 2011) and <em>B. Subtilis</em> (Chen et al., 2009, Yu et al., 2013).</p><br />
<p>Our concept was to design a star peptide with antimicrobial peptide arms. Wiradharma et al. (2012) first showed that placing linear antimicrobial peptides in a star configuration could lead to enhanced antimicrobial activity and decreased hemolytic activity. Although it is unclear why this is the case, it may be due to the ability of neighbouring antimicrobial peptide arms to interact with each other to synergistically rupture the membrane.<br><br />
While Wiradharma used a synthetic peptide sequence, we designed a peptide using the naturally occurring AMP, Magainin 1 (Zasloff, 1987). Magainin 1 peptides will be placed to the ends of each star arm.</p><br />
<br />
<p>Magainin 1 was chosen because the Magainins are one of the major classes of antimicrobial peptides, being well studied and characterised. In addition, we were concerned that tethering the antimicrobial peptide to the star peptide might interfere with its antimicrobial activity. Magainin 1, however, has previously been tethered to surfaces, where it has imparted the surfaces with microbicidal properties (Glinel et al., 2008; Humblot et al., 2009). We surmised that if Magainin 1 maintained its activity while anchored to surfaces, it may also maintain its activity while anchored to a star peptide.</p><br />
<p>The sequence for Magainin 1 is: GIGKFLHSAGKFGKAFVGEIMKS.</p><br />
<p>When attached to a star peptide it will have the following structure:</p><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/MAGAININ_1_peptide.png" width="720" height="300" alt=""/><br />
<p>There are several design elements to note:</p><br />
<ul><br />
<li><br />
The antimicrobial peptide star will be expressed with a SUMO fusion protein. This is because without the fusion, it is likely that the peptide would be toxic to the host cell.</li><br />
<li>The peptide includes a Factor X cutting site between the two cysteines for eventual proteolytic cleavage and formation of the star peptide.</li><br />
</ul><br />
<p>In addition to the star peptide Magainin 1, we synthesised a gene for a linear Magainin 1 peptide as well. This is identical to the construct above, except that there is only one Magainin 1 peptide attached to the SUMO fusion.</p><br />
<br />
<h3>Unstructured Peptide (USP) Construct</h3><br />
<p>In the second approach, we designed a peptide which can be functionalised using chemical approaches. This peptide was designed to have flexible, unstructured arms and was termed the USP construct. Unlike the Magainin 1 star, the arms of this peptide serve not as active peptides themselves, but as inert structural linkers.</p><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/USPI_Peptide.png" width="720" height="216" alt=""/><br />
<p>The arms were designed with the following elements in mind:</p><br />
<ul><br />
<li><br />
<b>Lack of structure.</b> The arms were designed with a bioinspired approach, using the FxFG motif of nucleoporins (where x is a variable amino acid residue). Such segments naturally repeat in nucleoporins and are thought to lead to disorder/lack of stable secondary structure. Nucleoporins are found in mammalian cells, serving as flexible brushes around nuclear pores (Ader et al., 2010).<br />
</li><br />
<li><b>Water-soluble.</b> The arms were designed with several charged amino acids to improve solubility.<br />
</li><br />
<li><b>Designed to form a disulfide bond.</b> Although it is difficult to rationally ensure that the disulfide bond will form between two cysteines in our peptide, we incorporated a beta turn between the two cysteines which may encourage the peptide to fold at the apex of the hairpin loop. This may bring the cysteines into closer proximity, providing more probable bond formation.</li></ul><br />
<br />
<p>The ultimate utility of this peptide lies in its ability to be functionalised with other biomacromolecules. For example, the technique of Native Chemical Ligation(NCL) can be used to join peptides, proteins, and other ligands to the arms (Dawson et al., 1994). The idea of attaching enzymes to the star peptide was explored by the University of Oxford iGEM 2014 team in a collaborative effort between our two teams (See Supplementary Project Work at the end of this page). </p><br />
<br />
<p>&nbsp;</p><br />
<h2>Expression system</h2><br />
<p>In order to successfully express our constructs, we designed our protein expression vectors to include a fusion protein. The fusion protein was necessary for two reasons. First, some of our constructs are very small (e.g. the non-star Magainin 1), and expression levels of very small peptides can be difficult without a fusion partner. Second, two of our constructs code for antimicrobial peptides. Without a fusion partner, it is likely that these genes would be toxic to their hosts upon induction.</p><br />
<p>To find a suitable expression system, we looked towards the Registry of Standard Parts. We used the SUMO protein expression system designed by TU Delft 2014 (for example, see<a target="_blank" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1022101">http://parts.igem.org/wiki/index.php?title=Part:BBa_K1022101</a>). This system essentially consists of a N-terminal HIS-tag followed by the SUMO protein (also known as UlpI).</p><br />
<p><br />
The strategy of expressing toxic AMPs using SUMO has been successfully reported in the literature (Bommarius et al., 2010). We surmise that the SUMO protein could inhibit the antimicrobial activity of single, linear peptides, and that it may also inhibit the activity of our star antimicrobial peptide.</p><br />
<p><br />
SUMO as a fusion protein also has the benefit of leaving no residues at the C-terminal end of the cleavage site. This means that upon cleavage with the SUMO protease, the native protein can be recovered. In our case, this means that one of the arms of the star can be designed without the need to take into account the addition of any amino acid residues left behind by the protease.<br><br />
We used the SUMO peptide sequence reported by TU Delft. However, our construct contained the following unique features:</p><br />
<ol><br />
<li> Standardisation of the biobrick by substituting the T7 promoter and RBS (the origins of which are both not specified in the Delft documentation) with the standard T7 promoter and RBS BioBrick<a target="_blank" href="http://parts.igem.org/Part:BBa_K525998">BBa_K525998</a>. In addition to supporting the principle of standardization, using the well-characterized promoter BioBrick should help assure expression levels.<br><br />
</li><br />
<li>Biobrick BBa_K1022101 lacks a terminator sequence (this was presumably because the part was meant to be integrated into a larger genetic construct with a terminator). A terminator from the registry of standard parts was added (specifically, the wild type terminator from T7 bacteriophage,<a target="_blank" href="http://parts.igem.org/Part:BBa_K731721">BBa_K731721</a>).<br />
</li><br />
<li>The original biobrick BBa_K1022101 codes for three amino acid residues before the HIS-tag (ASM), which appeared to be redundant. Correspondence with the 2013 TU Delft team suggested that these residues were unnecessary and appear to be cleaved within the cell as part of the cells post-translational modifications. However, their presence complicates the addition of additional tags at the N-terminus of the protein (e.g. periplasmic export tags), and therefore were not included.<br><br />
</li><br />
<li>The SUMO sequence was codon optimised for E. coli during synthesis. As the Delft documentation did not specify whether the gene was codon optimal, codon optomisation was undertaken to potentially improve expression levels. The linear Magainin 1 construct, however, was not codon optimised in the SUMO region in order to provide a control condition.</li><br />
</ol><br />
<p>To summarise, the protein expression devices used in our project to the following form:</p><br />
<img src="https://static.igem.org/mediawiki/2014/d/d0/Gene_circuit_export.png" width="720" height="91" alt=""/></td><br />
<p>The protein coding region consisted of a 6x-HIS tag followed by the SUMO protein and the relevant star or linear peptide.</p><br />
<p>&nbsp;</p><br />
<h2>Plasmid preparation: Cloning and acquisition of the genetic constructs</h2><br />
<h3>Magainin 1 Star Peptide</h3><br />
<p>This construct was synthesised in the standard shipping plasmid from Life Technologies- plasmid pMK. We had originally planned to express our protein to the periplasm of E. coli, and therefore had included in the synthesis a periplasmic export tag, the TorTss signal sequence (Steiner et al., 2006).</p><br />
<p><br />
Cytoplasmic and periplasmic expression may both allow for disulfide bond formation in the cell. We decided, however, to focus on cytoplasmic expression. There are distinct advantages to cytoplasmic expression (e.g. the absence of several periplasmic proteases and potentially higher expression levels (Baneyx, 1999)).</p><br />
<p><br />
Another reason the cytoplasm was chosen was to allow us to test the effects of an oxidising versus reducing intracellular environment on disulfide bond formation. We planned to express the construct in both SHuffle T7 cells (oxidising cytoplasm) and BL21(DE3) (reducing cytoplasm) to probe whether there was a difference in disulfide bond formation. Therefore, we needed to remove the periplasmic export tag from the gene.</p><br />
<p><br />
To do this, we use the following cloning strategy (noting that the top row represents the gene initially synthesised in plasmid pMK):</p><br />
<img src="https://static.igem.org/mediawiki/2014/2/2c/Melbourne_cloning_strategy_diagram.jpg" width="900" height="437" alt=""/><br />
<p>The gene was inserted into plasmid pSB1C3 containing the T7 promoter and ribosome binding site, BBa_K525998. It was inserted after the promoter and before the BioBrick suffix. This was accomplished by digesting the destination vector with SpeI and PstI. At the same time, PCR was used to amplify the segment of the gene containing the SUMO fusion and the Magainin 1 Star peptide, adding XbaI and keeping the PstI site existing in the gene.</p><br />
<p><br />
Digestion of the PCR product then allowed for ligation of the insert and destination vector using the PstI sites and XbaI and SpeI (XbaI and SpeI have compatible sticky ends). Note that after the ligation, there will be a scar in the gene where the XbaI and the SpeI sites were ligated.</p><br />
<p><br />
The ligation appeared to be successful. The ligation mixture was transformed to DH5α competent cells and plated onto chloramphenicol-containing agar plates.</p><br />
<p><br />
Plasmid from 5 colonies (C1, C2, C3, C4, and C5) was cultured, extracted, and then digested with EcoRI and PstI.</p><br />
<p><br />
As evident in the figure below, at least 4 of the colonies appeared to contain the insert. The empty, linearised pSB1C3 backbone ran at approximately the correct size (2070 bps), as did the insert (740 bps).</p><br />
<img src="https://static.igem.org/mediawiki/2014/9/95/Magainin_1_subcloning.png" width="353" height="400" alt=""/><br />
<p>N.B. MW marker is the 100 bp Ladder from Axygen. * indicates the colony picked for sequencing and eventual transformation to the expression cell lines.</p><br />
<p>The DNA from Colony C2 was confirmed using Sanger sequencing at the Australian Genome Research Facility. This DNA appears in the registry of standard parts as BioBrick <a target="_blank" href="http://parts.igem.org/Part:BBa_K1394000">BBa_K1394000</a>.</p><br />
<p><br />
For the USP peptide and the linear Magainin 1 peptide, the genes were synthesised by GenScript and delivered in pSB1C3. The expression vectors had identical gene regulatory elements to that used for the Magainin 1 Star peptide. They only differed in the codon optimisation used, and they also lacked the assembly scar described above.</p><br />
<br />
<h2><br />
Protein expression and characterisation<br />
</h2><br />
<p><br />
After acquiring our genes, the project moved to protein expression and purification. We expressed both star peptides (Magainin 1 Star Peptide and the USP peptide) in both the SHuffle T7 and BL21(DE3) cell lines, both sourced from New England Biolabs. As the Linear Magainin 1 peptide does not have any special disulfide bonding<br />
requirements, we expressed it in BL21(DE3).<br />
</p><br />
<p><br />
The plasmids were transformed to the expression cells, and a single colony was cultured and induced overnight at 17 °C. A whole cell sample both before<br />
IPTG induction (-IPTG) and after the induction period (+IPTG) were boiled in SDS-PAGE sample buffer and loaded on a 15% tris-glycine gel. The whole cell<br />
Coomassie stained gel is shown below alongside the NEB P7712S molecular weight marker.<br />
</p><br />
<br><br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/2/24/Whole_cell_CS.png" height="330" width="439"/><br />
</p><br />
<br><br />
<p><br />
From theoretical prediction of the peptide masses, we would expect the following distribution of molecular weights:<br />
</p><br />
<br><br />
<table border="1" cellpadding="0" cellspacing="0" align="center"><br />
<tbody><br />
<tr><br />
<td width="156"><br />
<p align="center"><br />
Magainin 1 Star Peptide (Mag1 Star)<br />
</p><br />
</td><br />
<td width="145"><br />
<p align="center"><br />
USP<br />
</p><br />
</td><br />
<td width="156"><br />
<p align="center"><br />
Linear Magainin 1 (Linear Mag1)<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="156"><br />
<p align="center"><br />
22.15 kDa<br />
</p><br />
</td><br />
<td width="145"><br />
<p align="center"><br />
29.3 kDa<br />
</p><br />
</td><br />
<td width="156"><br />
<p align="center"><br />
14.32 kDa<br />
</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<br><br />
<p><br />
We looked for bands corresponding to overexpression of the proteins. We observed a thick band, post IPTG around 32 kDa in the BL21(DE3) cells carrying the<br />
USP plasmid. However, we saw this same band appearing in the Linear Magainin 1 lane but the Linear Magainin 1 protein should have a much lower molecular<br />
weight than what was observed. Therefore, we did not assume that we had overexpression overexpression of the USP 1 protein.<br />
</p><br />
<p><br />
We also noted bands in all post-IPTG lanes around 17 kDa. This would be roughly consistent with both the Linear Magainin 1 and Magainin 1 Star Peptide,<br />
noting that small proteins may not run at their expected molecular weight. Again, the analysis is complicated by the fact that the band also appears in the<br />
lane corresponding to the larger molecular weight protein, USP I. Given this ambiguity, we decided to purify all the protein in the sample using Ni-NTA<br />
purification.<br />
</p><br />
<h3><br />
Purification and further characterisation<br />
</h3><br />
<p><br />
A small-scale purification was carried out according to our <a href="https://static.igem.org/mediawiki/2014/e/e1/MELBOURNE_D1V1_Column_Purification_of_His-Tagged_Proteins.pdf">protocol</a>. The cells were lysed with iodoacetamide, an alkylating agent, added to the lysis buffer. Iodoacetamide blocks all free cysteines on proteins with a short alkyl group. This was added<br />
because we wanted to determine if a disulphide bond had formed inside the cell. If we did not block free cysteines, then any bond that had formed could be<br />
attributed to spontaneous/air oxidation of disulfides outside the cell.<br />
</p><br />
<p><br />
After purification, we ran a concurrent Western Blot and Coomassie stain on both the pre-induction samples from above and the purified protein (namely, the<br />
first elution from the batch purification).<br />
</p><br />
<p><br />
The Western Blot used mouse monoclonal antibodies against the N-terminal HIS-tag as primary antibodies and anti-mouse secondary antibodies:<br />
</p><br />
<br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/5/54/Western_Blot_Melb.png" height="400" width="460" border="0"/><br />
</p><br />
<p><br />
The corresponding Coomassie stain is show below:<br />
</p><br />
<br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f0/Purified_CS.png" height="365" width="486" border="0"/><br />
</p><br />
<p><br />
There are several interesting aspects of the Western Blot. Firstly, there was a strong band between 25 and 32 kDa in most lanes. However, it<br />
appears in all of the pre-induction controls, suggesting non-specific binding of the anti-HIS antibodies.<br />
</p><br />
<p><br />
Secondly, we observed a band in most of the lanes around 17 kDa (with the exception being the USP protein in SHuffle cells). As these bands were not<br />
present in the pre-induction controls, we suspected they were a result of the induction.<br />
</p><br />
<h3><br />
Mass spectroscopy<br />
</h3><br />
<p><br />
In order to assess the identity of the bands, we performed an in-gel tryptic digestion on select bands. We digested bands in the Coomassie stain which<br />
corresponded to the HIS-tagged bands in the Western Blot. This was followed by mass spec analysis (LC MS/MS). We focused our analysis on the Magainin 1<br />
Star Peptide bands and the Linear Magainin 1 band. Our USP peptide was, by design, highly rich in basic residues, greatly reducing the likelihood that<br />
tryptic fragments would be detected by the mass spec.<br />
</p><br />
<p><br />
We found that the Linear Magainin 1 peptide appeared to be present in the approximately 17 kDa band, with the following detected tryptic fragments in the<br />
sequence below (bold; basic residues underlined):<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/1/1b/Tryptic_digest_linear_mag1.PNG" height="63" width="601" border="0"/><br />
</p><br />
<p><br />
Note that spaces have been added in the sequence to emphasise distinct domains in the protein (in this case, the fusion protein versus the Magainin 1<br />
peptide). Together with the fact that the protein runs close to the expected molecular weight, this seems to provide good evidence that the protein is<br />
being expressed.<br />
</p><br />
<p><br />
We then examined the bands corresponding to the Magainin 1 Star Peptide.<br />
</p><br />
<p><br />
Again, we digested bands in the Coomassie stained gel corresponding to the prominent HIS-tagged bands on the <em>Western Blot</em> (near 17 kDa).<br />
</p><br />
<p><br />
The mass spec coverage for the Magainin 1 Star Peptide expressed in SHuffle cells was as follows:<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/6/63/Tryptic_digest_Sample_A_Star_Mag_Shuffle.PNG" height="90" width="601" border="0"/><br />
</p><br />
<p><br />
The coverage for the same peptide expressed in BL21(DE3) was found to be:<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/d/db/Tryptic_digest_Sample_A_Star_Mag_BL21%28DE3%29.PNG" height="85" width="602" border="0"/><br />
</p><br />
<p><br />
Again, we can see that tryptic peptides from the expressed protein appear to present in the gel band under analysis.<br />
</p><br />
<p><br />
There is a relatively lower sequence coverage. This could be a limitation of our procedure: for example, improper destaining during the in gel digestion<br />
procedure can inhibit tryptic digestion.<br />
</p><br />
<p><br />
However, even if the digestion was complete, the mass spec will only detect fragments which ionize well. The Magainin 1 Star peptide consists of four<br />
repeats of the Magainin 1 sequence (corresponding to the arms of the star). If the two tryptic fragments within Magainin 1 do not ionize well, then indeed<br />
the entire peptide would not be read.<br />
</p><br />
<p><br />
Finally, it is possible that the protein has been cleaved or degraded. This would account for the slightly lower-than-expected mass on the SDS page gel.<br />
</p><br />
<p><br />
Nonetheless, there is the distinct possibility that the peptide fragments are there but not detected. Replication of the in-gel mass spec would be useful<br />
in clarifying this point, or provided the purity of the sample is acceptable, it may be possible to run an intact mass spec.<br />
</p><br />
<h2><br />
Conclusions<br />
</h2><br />
<p><br />
We designed several novel star peptides based on several rational design criteria. Further, we began the optimisation process required to express these peptides in <em>E. coli</em>.<br />
</p><br />
<p><br />
To this end, we constructed an expression system based on the SUMO protein and standard BioBrick parts. The function of this system was confirmed, as it<br />
appears it does produce HIS-tagged SUMO fusion protein in <em>E. coli</em>, as expected.<br />
</p><br />
<p><br />
Although it appears the expression system works, there was some uncertainty about particular peptides, namely the Magainin 1 Star Peptide and USP Peptide.<br />
A Westerm Blot suggested that at least part of the peptides are present. However, additional sequence coverage in a mass spec analysis and intact mass spec<br />
would help us determine the precise identity of our products.<br />
</p><br />
<p><br />
We have shown that the SUMO fusion can be expressed in our BioBrick. Given the success of SUMO in the general scientific community, we hope our BioBrick<br />
will encourage further use of the fusion domain within the iGEM community. In addition, we hope that our ideas and concepts will inspire future<br />
iGEM efforts to explore the applications of synthetic biology for material science. We continue to believe that bacteria have great promise for the <em>in vivo</em><br />
construction of unique biomaterials, and we look forward to seeing how the synthetic biology community will develop in this area.<br />
</p><br />
<br />
<br />
<br />
<h2>References</h2><br />
<p>ADER, C., FREY, S., MAAS, W., SCHMIDT, H. B., GÖRLICH, D. &amp; BALDUS, M. 2010. Amyloid-like interactions within nucleoporin FG hydrogels. <em>Proceedings of the National Academy of Sciences,</em> 107<strong>,</strong> 6281-6285.</p><br />
<p><br />
BANEYX, F. 1999. Recombinant protein expression in Escherichia coli. <em>Current Opinion in Biotechnology,</em> 10<strong>,</strong> 411-421.</p><br />
<p><br />
BOMMARIUS, B., JENSSEN, H., ELLIOTT, M., KINDRACHUK, J., PASUPULETI, M., GIEREN, H., JAEGER, K. E., HANCOCK, R. E. W. &amp; KALMAN, D. 2010. Cost-effective expression and purification of antimicrobial and host defense peptides in Escherichia coli. <em>Peptides,</em> 31<strong>,</strong> 1957-1965.</p><br />
<p><br />
BROGDEN, K. A. 2005. Antimicrobial peptides: Pore formers or metabolic inhibitors in bacteria? <em>Nature Reviews Microbiology,</em> 3<strong>,</strong> 238-250.</p><br />
<p><br />
CHEN, X., ZHU, F., CAO, Y. &amp; QIAO, S. 2009. Novel expression vector for secretion of cecropin AD in Bacillus subtilis with enhanced antimicrobial activity. <em>Antimicrobial agents and chemotherapy,</em> 53<strong>,</strong> 3683-3689.</p><br />
<p><br />
GLINEL, K., JONAS, A. M., JOUENNE, T., LEPRINCE, J., GALAS, L. & HUCK, W. T. 2008. Antibacterial and antifouling polymer brushes incorporating antimicrobial peptide. <em>Bioconjug Chem,</em> 20<strong>,</strong> 71-77.</p><br />
<p><br />
HUMBLOT, V., YALA, J.-F., THEBAULT, P., BOUKERMA, K., HÉQUET, A., BERJEAUD, J.-M. & PRADIER, C.-M. 2009. The antibacterial activity of Magainin I immobilized onto mixed thiols self-assembled monolayers. <em>Biomaterials,</em> 30<strong>,</strong> 3503-3512.</p><br />
<p><br />
DAWSON, P. E., MUIR, T. W., CLARK-LEWIS, I. &amp; KENT, S. 1994. Synthesis of proteins by native chemical ligation. <em>Science,</em> 266<strong>,</strong> 776-779.</p><br />
<p><br />
KADOKURA, H. &amp; BECKWITH, J. 2009. Detecting folding intermediates of a protein as it passes through the bacterial translocation channel. <em>Cell,</em> 138<strong>,</strong> 1164-1173.</p><br />
<p><br />
KADOKURA, H., KATZEN, F. &amp; BECKWITH, J. 2003. Protein disulfide bond formation in prokaryotes. <em>Annual review of biochemistry,</em> 72<strong>,</strong> 111-135.</p><br />
<p><br />
LI, Y. 2011. Recombinant production of antimicrobial peptides in&lt; i&gt; Escherichia coli&lt;/i&gt;: A review. <em>Protein Expression and Purification,</em> 80<strong>,</strong> 260-267.</p><br />
<p><br />
LOBSTEIN, J., EMRICH, C. A., JEANS, C., FAULKNER, M., RIGGS, P. &amp; BERKMEN, M. 2012. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. <em>Microb Cell Fact,</em> 11<strong>,</strong> 56-56.</p><br />
<p><br />
STEINER, D., FORRER, P., STUMPP, M. T. &amp; PLUCKTHUN, A. 2006. Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display. <em>Nat Biotech,</em> 24<strong>,</strong> 823-831.</p><br />
<p><br />
SULISTIO, A., GURR, P. A., BLENCOWE, A. &amp; QIAO, G. G. 2012. Peptide-Based Star Polymers: The Rising Star in Functional Polymers. <em>Australian Journal of Chemistry,</em> 65<strong>,</strong> 978-984.</p><br />
<p><br />
SULISTIO, A., LOWENTHAL, J., BLENCOWE, A., BONGIOVANNI, M. N., ONG, L., GRAS, S. L., ZHANG, X. &amp; QIAO, G. G. 2011. Folic Acid Conjugated Amino Acid-Based Star Polymers for Active Targeting of Cancer Cells. <em>Biomacromolecules,</em> 12<strong>,</strong> 3469-3477.</p><br />
<p><br />
WIRADHARMA, N., LIU, S. Q. &amp; YANG, Y. Y. 2012. Branched and 4-Arm Starlike α-Helical Peptide Structures with Enhanced Antimicrobial Potency and Selectivity. <em>Small,</em> 8<strong>,</strong> 362-366.</p><br />
<p><br />
YU, Z., WANG, Q., MA, Q. &amp; ZHANG, R. 2013. Secretory expression of lacticin Q fused with SUMO in Bacillus subtilis. <em>Protein Expression and Purification,</em> 89<strong>,</strong> 51-55.</p><br />
<p><br />
ZASLOFF, M. 1987. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. <em>Proceedings of the National Academy of Sciences,</em> 84<strong>,</strong> 5449-5453.</p><br />
<p>&nbsp;</p><br />
<br />
<A NAME="Oxford"></A><br />
<h1>Collaboration with Oxford</h1><br />
<p>To strengthen the sense of iGEM community, we contacted <a target="_blank" href="https://2014.igem.org/Team:Oxford">University of Oxford iGEM team</a> to discuss the possibility of collaboration between our teams. The Oxford project aims to develop a system that can dispose of the carcinogenic, hazardous solvent dichloromethane (DCM). To do this, Oxford team has proposed the use of the DcmA enzyme. This enzyme degrades DCM to form a toxic intermediate which in turn is converted by another enzyme, FdhA, into a neutral molecule. Schematically this can be presented in the following way:</p><br />
<br />
<p><br />
Reaction 1: DCM +&nbsp;DcmA <strong>toxic intermediate</strong><br><br />
Reaction 2: <strong>toxic intermediate</strong> + FdhA neutral product.</p><br />
<p>The problem for their system lies in bringing the two enzymes together to ensure efficient reaction kinetics. An idea that we discussed with Oxford was to use the star peptide platform to link the two enzymes together, in a structure similar to the following: </p><br />
<img src="https://static.igem.org/mediawiki/2014/2/2e/Melbourne_Collab_1.png" width="500" height="325" alt=""/><br />
<p>We worked with Oxford to study this system from a theoretical standpoint. Our team studied the 3D structure of both enzymes involved to confirm that the enzyme could in theory be attached to a linker in this manner, while Oxford team did stochastic modelling to determine how reaction rate changes when the linker length, labeled D on the diagram, changes. </p><br />
<p><br />
As a first check, it was necessary to determine whether anchoring these enzymes to our star would be sterically possible. We studied the structures of FdhA and DcmA determined by crystallography using data from the protein data bank. As no crystallographic data was available in the databank for DcmA, GST was examined as a proxy, as GST is structurally homologous to DcmA. </p><br />
<p><br />
The crystalographically resolved structure of FdhA enzyme (Tanaka et al., 2002) from the protein data bank revealed the following image:</p><br />
<img src="https://static.igem.org/mediawiki/2014/d/dc/Melbourne_Collab_2.png" width="700" height="355" alt=""/><br />
<p>The crystalographically resolved structure of GST was as follows (Chang et al., 2014):</p><br />
<img src="https://static.igem.org/mediawiki/2014/e/e9/Melbourne_Collab_3.png" width="800" height="361" alt=""/><br />
<p>It can be seen that the amino- and carboxy-terminals are located away from the active site. This suggests that both enzymes, DcmA and FdhA, might be linked together via linkers that are used in our star peptide. That is, the active site will not be sterically hindered by attachment to the star peptide. That said, it is difficult to predict how anchoring the protein to the star will affect its tertiary structure. Further, it is difficult to predict how limiting the rotational degrees of freedom will affect enzyme activity.</p><br />
<p>The Oxford team modeled this system and found that, indeed, the star peptide as an enzyme linker was favourable to enzyme kinetics. Their work can be found here: <a target="_blank" href="https://2014.igem.org/Team:Oxford/alternatives_to_microcompartments#show2" target="_blank">https://2014.igem.org/Team:Oxford/alternatives_to_microcompartments#show2</a></p><br />
</html></div>Slowehttp://2014.igem.org/Team:Melbourne/ProjectTeam:Melbourne/Project2014-10-18T00:48:24Z<p>Slowe: </p>
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<h1>Project and results</h1><br />
<br />
<p>Read about our experimental work below, or jump to our theoretical collaboration with the <A HREF="#Oxford">University of Oxford</A><br />
<br />
<h2>Introduction and theory</h2><br />
<p>Synthetic peptide chemists have long produced peptide-based materials <em>in vitro</em>. Star-shaped peptides are a promising type of biomaterial being explored in the field of nanomedicine (Sulistio et al., 2012). Star peptides can have several biomedical uses, such as acting as drug delivery vehicles (Sulistio et al., 2011) or linkers for other biomacromolecules. Star peptides generally take the form of several linear peptide arms linked together in a central core. One way of linking these linear peptide arms together is to used covalent bonds such as those in disulfides. Typically, disulfide bonds are formed synthetically by taking several linear arms and treating them with an oxidant <em>in vitro</em>. Here, we introduce a new approach to forming star peptides using <em>E. coli</em> and synthetic biology. Thus, we aimed to show how the peptides synthesis and disulfide bond forming machinery of <em>E. coli</em> can be used to form disulfide linked star peptides and key star peptide precursors.</p><br />
<p>&nbsp;</p><br />
<h2>Synthesis approach</h2><br />
<p><em>E. coli</em> naturally possesses the capacity to form disulfide bonds. In native strains, disulfide bonds are naturally formed by an array of enzymes which are part of the Dsb family (e.g. DsbA and DsbC) (Kadokura et al., 2003, Kadokura and Beckwith, 2009). Normally, these enzymes are found in the oxidizing periplasm of the cell. However, several new strains of <em>E. coli</em> have recently been engineered which contain an oxidizing cytoplasm conducive to disulfide bond formation. One example of this is the SHuffle cell line (Lobstein et al., 2012). The cell line contains mutations to key enzymes responsible for the reducing nature of the cytoplasm, namely thioredoxin reductase (<em>trxB</em>) and glutathione reductase (<em>gor</em>). Furthermore, the Shuffle cell line over expresses the disulfide bond isomerase DsbC to the cytoplasm. Together, these mutations allow SHuffle to fold disulfide-bonded proteins in the cytoplasm at a higher success rate compared to non-mutants. We aimed to take advantage of the disulfide bond forming capabilities of this strain of <em>E. coli</em> to synthesize star peptides in cells. As shown in the figure below, the synthesis steps may proceed as follows:</p><br />
<p>&nbsp;</p><br />
<ol><br />
<li> Express a short peptide containing two cysteine residues either to the <em>E. coli</em> periplasm or the cytoplasm of a <em>trxB gor </em>mutant.</li><br />
<li><em>E. coli</em> disulfide bond forming enzymes fold the peptide into a hairpin loop structure.<br />
</li><br />
<li>Cut the loop at a protease recognition site engineered into the peptide. This may be done by extracting the folded peptide from the cell and treating it with the protease in vitro. Alternatively, the protease may be co-expressed in the cell to allow for in vivo cleavage.<br />
</li><br />
</ol><br />
<p>&nbsp;</p><br />
<table align="center"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9d/Project_concept-v2.png" width="480" height="600" alt=""/></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<p>This synthesis approach has several benefits over purely <em>in vitro</em> approaches. Firstly, the exact peptide sequence can be precisely programmed into <em>E. coli</em> using recombinant DNA synthesis. Secondly, by performing the disulfide bond formation in cells and optionally the proteolytic cleavage, several synthesis steps which would need to be performed <em>in vitro</em> are eliminated. From a scale up perspective, this would eliminate entire unit operations which would otherwise be required to produce this product. Given these benefits, in the current study, we aimed to express a star peptide precursor to the cytoplasm of SHuffle cells, which would later be extracted and externally digested with the star-forming protease. In order to achieve this, we first designed several star peptides which might be amenable to this synthetic strategy. These peptides are described below.</p><br />
<p>&nbsp;</p><br />
<h2>Rationally designed peptides</h2><br />
<p>There are two approaches to functionalising star peptides. In the first, the identity of the arms can be chosen to be bioactive peptide molecules. This is the simplest approach to producing a biologically-relevant star. In the second, the arms can be functionalised by ligating molecules to them at any point. We used both of these approaches to design two separate peptides.</p><br />
<h3>Magainin 1 star and linear peptides</h3><br />
<p>Our first strategy was to make a star peptide using antimicrobial peptides(AMPs) as building blocks. AMPs are small, approximately 50 residue peptides secreted by some bacterial and eukaryotic cells which selectively kill microbial cells. It is thought that AMPs work by forming pores in the membrane of prokaryotic cells (Brogden, 2005). AMPs have been recombinantly expressed in a number of organisms, including <em>E. coli</em> (for a review, see Li, 2011) and <em>B. Subtilis</em> (Chen et al., 2009, Yu et al., 2013).</p><br />
<p>Our concept was to design a star peptide with antimicrobial peptide arms. Wiradharma et al. (2012) first showed that placing linear antimicrobial peptides in a star configuration could lead to enhanced antimicrobial activity and decreased hemolytic activity. Although it is unclear why this is the case, it may be due to the ability of neighbouring antimicrobial peptide arms to interact with each other to synergistically rupture the membrane.<br><br />
While Wiradharma used a synthetic peptide sequence, we designed a peptide using the naturally occurring AMP, Magainin 1 (Zasloff, 1987). Magainin 1 peptides will be placed to the ends of each star arm.</p><br />
<br />
<p>Magainin 1 was chosen because the Magainins are one of the major classes of antimicrobial peptides, being well studied and characterised. In addition, we were concerned that tethering the antimicrobial peptide to the star peptide might interfere with its antimicrobial activity. Magainin 1, however, has previously been tethered to surfaces, where it has imparted the surfaces with microbicidal properties (Glinel et al., 2008; Humblot et al., 2009). We surmised that if Magainin 1 maintained its activity while anchored to surfaces, it may also maintain its activity while anchored to a star peptide.</p><br />
<p>The sequence for Magainin 1 is: GIGKFLHSAGKFGKAFVGEIMKS.</p><br />
<p>When attached to a star peptide it will have the following structure:</p><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/MAGAININ_1_peptide.png" width="720" height="300" alt=""/><br />
<p>There are several design elements to note:</p><br />
<ul><br />
<li><br />
The antimicrobial peptide star will be expressed with a SUMO fusion protein. This is because without the fusion, it is likely that the peptide would be toxic to the host cell.</li><br />
<li>The peptide includes a Factor X cutting site between the two cysteines for eventual proteolytic cleavage and formation of the star peptide.</li><br />
</ul><br />
<p>In addition to the star peptide Magainin 1, we synthesised a gene for a linear Magainin 1 peptide as well. This is identical to the construct above, except that there is only one Magainin 1 peptide attached to the SUMO fusion.</p><br />
<br />
<h3>Unstructured Peptide (USP) Construct</h3><br />
<p>In the second approach, we designed a peptide which can be functionalised using chemical approaches. This peptide was designed to have flexible, unstructured arms and was termed the USP construct. Unlike the Magainin 1 star, the arms of this peptide serve not as active peptides themselves, but as inert structural linkers.</p><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/USPI_Peptide.png" width="720" height="216" alt=""/><br />
<p>The arms were designed with the following elements in mind:</p><br />
<ul><br />
<li><br />
<b>Lack of structure.</b> The arms were designed with a bioinspired approach, using the FxFG motif of nucleoporins (where x is a variable amino acid residue). Such segments naturally repeat in nucleoporins and are thought to lead to disorder/lack of stable secondary structure. Nucleoporins are found in mammalian cells, serving as flexible brushes around nuclear pores (Ader et al., 2010).<br />
</li><br />
<li><b>Water-soluble.</b> The arms were designed with several charged amino acids to improve solubility.<br />
</li><br />
<li><b>Designed to form a disulfide bond.</b> Although it is difficult to rationally ensure that the disulfide bond will form between two cysteines in our peptide, we incorporated a beta turn between the two cysteines which may encourage the peptide to fold at the apex of the hairpin loop. This may bring the cysteines into closer proximity, providing more probable bond formation.</li></ul><br />
<br />
<p>The ultimate utility of this peptide lies in its ability to be functionalised with other biomacromolecules. For example, the technique of Native Chemical Ligation(NCL) can be used to join peptides, proteins, and other ligands to the arms (Dawson et al., 1994). The idea of attaching enzymes to the star peptide was explored by the University of Oxford iGEM 2014 team in a collaborative effort between our two teams (See Supplementary Project Work at the end of this page). </p><br />
<br />
<p>&nbsp;</p><br />
<h2>Expression system</h2><br />
<p>In order to successfully express our constructs, we designed our protein expression vectors to include a fusion protein. The fusion protein was necessary for two reasons. First, some of our constructs are very small (e.g. the non-star Magainin 1), and expression levels of very small peptides can be difficult without a fusion partner. Second, two of our constructs code for antimicrobial peptides. Without a fusion partner, it is likely that these genes would be toxic to their hosts upon induction.</p><br />
<p>To find a suitable expression system, we looked towards the Registry of Standard Parts. We used the SUMO protein expression system designed by TU Delft 2014 (for example, see<a target="_blank" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1022101">http://parts.igem.org/wiki/index.php?title=Part:BBa_K1022101</a>). This system essentially consists of a N-terminal HIS-tag followed by the SUMO protein (also known as UlpI).</p><br />
<p><br />
The strategy of expressing toxic AMPs using SUMO has been successfully reported in the literature (Bommarius et al., 2010). We surmise that the SUMO protein could inhibit the antimicrobial activity of single, linear peptides, and that it may also inhibit the activity of our star antimicrobial peptide.</p><br />
<p><br />
SUMO as a fusion protein also has the benefit of leaving no residues at the C-terminal end of the cleavage site. This means that upon cleavage with the SUMO protease, the native protein can be recovered. In our case, this means that one of the arms of the star can be designed without the need to take into account the addition of any amino acid residues left behind by the protease.<br><br />
We used the SUMO peptide sequence reported by TU Delft. However, our construct contained the following unique features:</p><br />
<ol><br />
<li> Standardisation of the biobrick by substituting the T7 promoter and RBS (the origins of which are both not specified in the Delft documentation) with the standard T7 promoter and RBS BioBrick<a target="_blank" href="http://parts.igem.org/Part:BBa_K525998">BBa_K525998</a>. In addition to supporting the principle of standardization, using the well-characterized promoter BioBrick should help assure expression levels.<br><br />
</li><br />
<li>Biobrick BBa_K1022101 lacks a terminator sequence (this was presumably because the part was meant to be integrated into a larger genetic construct with a terminator). A terminator from the registry of standard parts was added (specifically, the wild type terminator from T7 bacteriophage,<a target="_blank" href="http://parts.igem.org/Part:BBa_K731721">BBa_K731721</a>).<br />
</li><br />
<li>The original biobrick BBa_K1022101 codes for three amino acid residues before the HIS-tag (ASM), which appeared to be redundant. Correspondence with the 2013 TU Delft team suggested that these residues were unnecessary and appear to be cleaved within the cell as part of the cells post-translational modifications. However, their presence complicates the addition of additional tags at the N-terminus of the protein (e.g. periplasmic export tags), and therefore were not included.<br><br />
</li><br />
<li>The SUMO sequence was codon optimised for E. coli during synthesis. As the Delft documentation did not specify whether the gene was codon optimal, codon optomisation was undertaken to potentially improve expression levels. The linear Magainin 1 construct, however, was not codon optimised in the SUMO region in order to provide a control condition.</li><br />
</ol><br />
<p>To summarise, the protein expression devices used in our project to the following form:</p><br />
<img src="https://static.igem.org/mediawiki/2014/d/d0/Gene_circuit_export.png" width="720" height="91" alt=""/></td><br />
<p>The protein coding region consisted of a 6x-HIS tag followed by the SUMO protein and the relevant star or linear peptide.</p><br />
<p>&nbsp;</p><br />
<h2>Plasmid preparation: Cloning and acquisition of the genetic constructs</h2><br />
<h3>Magainin 1 Star Peptide</h3><br />
<p>This construct was synthesised in the standard shipping plasmid from Life Technologies- plasmid pMK. We had originally planned to express our protein to the periplasm of E. coli, and therefore had included in the synthesis a periplasmic export tag, the TorTss signal sequence (Steiner et al., 2006).</p><br />
<p><br />
Cytoplasmic and periplasmic expression may both allow for disulfide bond formation in the cell. We decided, however, to focus on cytoplasmic expression. There are distinct advantages to cytoplasmic expression (e.g. the absence of several periplasmic proteases and potentially higher expression levels (Baneyx, 1999)).</p><br />
<p><br />
Another reason the cytoplasm was chosen was to allow us to test the effects of an oxidising versus reducing intracellular environment on disulfide bond formation. We planned to express the construct in both SHuffle T7 cells (oxidising cytoplasm) and BL21(DE3) (reducing cytoplasm) to probe whether there was a difference in disulfide bond formation. Therefore, we needed to remove the periplasmic export tag from the gene.</p><br />
<p><br />
To do this, we use the following cloning strategy (noting that the top row represents the gene initially synthesised in plasmid pMK):</p><br />
<img src="https://static.igem.org/mediawiki/2014/2/2c/Melbourne_cloning_strategy_diagram.jpg" width="900" height="437" alt=""/><br />
<p>The gene was inserted into plasmid pSB1C3 containing the T7 promoter and ribosome binding site, BBa_K525998. It was inserted after the promoter and before the BioBrick suffix. This was accomplished by digesting the destination vector with SpeI and PstI. At the same time, PCR was used to amplify the segment of the gene containing the SUMO fusion and the Magainin 1 Star peptide, adding XbaI and keeping the PstI site existing in the gene.</p><br />
<p><br />
Digestion of the PCR product then allowed for ligation of the insert and destination vector using the PstI sites and XbaI and SpeI (XbaI and SpeI have compatible sticky ends). Note that after the ligation, there will be a scar in the gene where the XbaI and the SpeI sites were ligated.</p><br />
<p><br />
The ligation appeared to be successful. The ligation mixture was transformed to DH5α competent cells and plated onto chloramphenicol-containing agar plates.</p><br />
<p><br />
Plasmid from 5 colonies (C1, C2, C3, C4, and C5) was cultured, extracted, and then digested with EcoRI and PstI.</p><br />
<p><br />
As evident in the figure below, at least 4 of the colonies appeared to contain the insert. The empty, linearised pSB1C3 backbone ran at approximately the correct size (2070 bps), as did the insert (740 bps).</p><br />
<img src="https://static.igem.org/mediawiki/2014/9/95/Magainin_1_subcloning.png" width="353" height="400" alt=""/><br />
<p>N.B. MW marker is the 100 bp Ladder from Axygen. * indicates the colony picked for sequencing and eventual transformation to the expression cell lines.</p><br />
<p>The DNA from Colony C2 was confirmed using Sanger sequencing at the Australian Genome Research Facility. This DNA appears in the registry of standard parts as BioBrick <a target="_blank" href="http://parts.igem.org/Part:BBa_K1394000">BBa_K1394000</a>.</p><br />
<p><br />
For the USP peptide and the linear Magainin 1 peptide, the genes were synthesised by GenScript and delivered in pSB1C3. The expression vectors had identical gene regulatory elements to that used for the Magainin 1 Star peptide. They only differed in the codon optimisation used, and they also lacked the assembly scar described above.</p><br />
<br />
<h2><br />
Protein expression and characterisation<br />
</h2><br />
<p><br />
After acquiring our genes, the project moved to protein expression and purification. We expressed both star peptides (Magainin 1 Star Peptide and the USP I<br />
peptide) in both the SHuffle T7 and BL21(DE3) cell lines, both sourced from New England Biolabs. As the Linear Magainin 1 peptide does not have any special disulfide bonding<br />
requirements, we expressed it in BL21(DE3).<br />
</p><br />
<p><br />
The plasmids were transformed to the expression cells, and a single colony was cultured and induced overnight at 17 °C. A whole cell sample both before<br />
IPTG induction (-IPTG) and after the induction period (+IPTG) were boiled in SDS-PAGE sample buffer and loaded on a 15% tris-glycine gel. The whole cell<br />
Coomassie stained gel is shown below alongside the NEB P7712S molecular weight marker.<br />
</p><br />
<br><br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/2/24/Whole_cell_CS.png" height="330" width="439"/><br />
</p><br />
<br><br />
<p><br />
From theoretical prediction of the peptide masses, we would expect the following distribution of molecular weights:<br />
</p><br />
<br><br />
<table border="1" cellpadding="0" cellspacing="0" align="center"><br />
<tbody><br />
<tr><br />
<td width="156"><br />
<p align="center"><br />
Magainin 1 Star Peptide (Mag1 Star)<br />
</p><br />
</td><br />
<td width="145"><br />
<p align="center"><br />
USP 1<br />
</p><br />
</td><br />
<td width="156"><br />
<p align="center"><br />
Linear Magainin 1 (Linear Mag1)<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="156"><br />
<p align="center"><br />
22.15 kDa<br />
</p><br />
</td><br />
<td width="145"><br />
<p align="center"><br />
29.3 kDa<br />
</p><br />
</td><br />
<td width="156"><br />
<p align="center"><br />
14.32 kDa<br />
</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<br><br />
<p><br />
We looked for bands corresponding to overexpression of the proteins. We observed a thick band, post IPTG around 32 kDa in the BL21(DE3) cells carrying the<br />
USP plasmid. However, we saw this same band appearing in the Linear Magainin 1 lane but the Linear Magainin 1 protein should have a much lower molecular<br />
weight than what was observed. Therefore, we did not assume that we had overexpression overexpression of the USP 1 protein.<br />
</p><br />
<p><br />
We also noted bands in all post-IPTG lanes around 17 kDa. This would be roughly consistent with both the Linear Magainin 1 and Magainin 1 Star Peptide,<br />
noting that small proteins may not run at their expected molecular weight. Again, the analysis is complicated by the fact that the band also appears in the<br />
lane corresponding to the larger molecular weight protein, USP I. Given this ambiguity, we decided to purify all the protein in the sample using Ni-NTA<br />
purification.<br />
</p><br />
<h3><br />
Purification and further characterisation<br />
</h3><br />
<p><br />
A small-scale purification was carried out according to our <a href="https://static.igem.org/mediawiki/2014/e/e1/MELBOURNE_D1V1_Column_Purification_of_His-Tagged_Proteins.pdf">protocol</a>. The cells were lysed with iodoacetamide, an alkylating agent, added to the lysis buffer. Iodoacetamide blocks all free cysteines on proteins with a short alkyl group. This was added<br />
because we wanted to determine if a disulphide bond had formed inside the cell. If we did not block free cysteines, then any bond that had formed could be<br />
attributed to spontaneous/air oxidation of disulfides outside the cell.<br />
</p><br />
<p><br />
After purification, we ran a concurrent Western Blot and Coomassie stain on both the pre-induction samples from above and the purified protein (namely, the<br />
first elution from the batch purification).<br />
</p><br />
<p><br />
The Western Blot used mouse monoclonal antibodies against the N-terminal HIS-tag as primary antibodies and anti-mouse secondary antibodies:<br />
</p><br />
<br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/5/54/Western_Blot_Melb.png" height="400" width="460" border="0"/><br />
</p><br />
<p><br />
The corresponding Coomassie stain is show below:<br />
</p><br />
<br />
<p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f0/Purified_CS.png" height="365" width="486" border="0"/><br />
</p><br />
<p><br />
There are several interesting aspects of the Western Blot. Firstly, there was a strong band between 25 and 32 kDa in most lanes. However, it<br />
appears in all of the pre-induction controls, suggesting non-specific binding of the anti-HIS antibodies.<br />
</p><br />
<p><br />
Secondly, we observed a band in most of the lanes around 17 kDa (with the exception being the USP protein in SHuffle cells). As these bands were not<br />
present in the pre-induction controls, we suspected they were a result of the induction.<br />
</p><br />
<h3><br />
Mass spectroscopy<br />
</h3><br />
<p><br />
In order to assess the identity of the bands, we performed an in-gel tryptic digestion on select bands. We digested bands in the Coomassie stain which<br />
corresponded to the HIS-tagged bands in the Western Blot. This was followed by mass spec analysis (LC MS/MS). We focused our analysis on the Magainin 1<br />
Star Peptide bands and the Linear Magainin 1 band. Our USP peptide was, by design, highly rich in basic residues, greatly reducing the likelihood that<br />
tryptic fragments would be detected by the mass spec.<br />
</p><br />
<p><br />
We found that the Linear Magainin 1 peptide appeared to be present in the approximately 17 kDa band, with the following detected tryptic fragments in the<br />
sequence below (bold; basic residues underlined):<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/1/1b/Tryptic_digest_linear_mag1.PNG" height="63" width="601" border="0"/><br />
</p><br />
<p><br />
Note that spaces have been added in the sequence to emphasise distinct domains in the protein (in this case, the fusion protein versus the Magainin 1<br />
peptide). Together with the fact that the protein runs close to the expected molecular weight, this seems to provide good evidence that the protein is<br />
being expressed.<br />
</p><br />
<p><br />
We then examined the bands corresponding to the Magainin 1 Star Peptide.<br />
</p><br />
<p><br />
Again, we digested bands in the Coomassie stained gel corresponding to the prominent HIS-tagged bands on the <em>Western Blot</em> (near 17 kDa).<br />
</p><br />
<p><br />
The mass spec coverage for the Magainin 1 Star Peptide expressed in SHuffle cells was as follows:<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/6/63/Tryptic_digest_Sample_A_Star_Mag_Shuffle.PNG" height="90" width="601" border="0"/><br />
</p><br />
<p><br />
The coverage for the same peptide expressed in BL21(DE3) was found to be:<br />
</p><br />
<br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/d/db/Tryptic_digest_Sample_A_Star_Mag_BL21%28DE3%29.PNG" height="85" width="602" border="0"/><br />
</p><br />
<p><br />
Again, we can see that tryptic peptides from the expressed protein appear to present in the gel band under analysis.<br />
</p><br />
<p><br />
There is a relatively lower sequence coverage. This could be a limitation of our procedure: for example, improper destaining during the in gel digestion<br />
procedure can inhibit tryptic digestion.<br />
</p><br />
<p><br />
However, even if the digestion was complete, the mass spec will only detect fragments which ionize well. The Magainin 1 Star peptide consists of four<br />
repeats of the Magainin 1 sequence (corresponding to the arms of the star). If the two tryptic fragments within Magainin 1 do not ionize well, then indeed<br />
the entire peptide would not be read.<br />
</p><br />
<p><br />
Finally, it is possible that the protein has been cleaved or degraded. This would account for the slightly lower-than-expected mass on the SDS page gel.<br />
</p><br />
<p><br />
Nonetheless, there is the distinct possibility that the peptide fragments are there but not detected. Replication of the in-gel mass spec would be useful<br />
in clarifying this point, or provided the purity of the sample is acceptable, it may be possible to run an intact mass spec.<br />
</p><br />
<h2><br />
Conclusions<br />
</h2><br />
<p><br />
We designed several novel star peptides based on several rational design criteria. Further, we began the process of generating these peptides in <em>E. coli</em>.<br />
</p><br />
<p><br />
To this end, we constructed an expression system based on the SUMO protein and standard BioBrick parts. The function of this system was confirmed, as it<br />
appears it does produce HIS-tagged SUMO fusion protein in <em>E. coli</em>, as expected.<br />
</p><br />
<p><br />
Although it appears the expression system works, there was some uncertainty about particular peptides, namely the Magainin 1 Star Peptide and USP Peptide.<br />
A Westerm Blot suggested that at least part of the peptides are present. However, additional sequence coverage in a mass spec analysis and intact mass spec<br />
would help us determine the precise identity of our products.<br />
</p><br />
<p><br />
We have shown that the SUMO fusion can be expressed in our BioBrick. Given the success of SUMO in the general scientific community, we hope our BioBrick<br />
will encourage further use of the fusion domain within the iGEM community. In addition, we hope that our ideas and concepts will inspire future<br />
iGEM efforts to explore the applications of synthetic biology for material science. We continue to believe that bacteria have great promise for the <em>in vivo</em><br />
construction of unique biomaterials, and we look forward to seeing how the synthetic biology community will develop in this area.<br />
</p><br />
<br />
<br />
<br />
<h2>References</h2><br />
<p>ADER, C., FREY, S., MAAS, W., SCHMIDT, H. B., GÖRLICH, D. &amp; BALDUS, M. 2010. Amyloid-like interactions within nucleoporin FG hydrogels. <em>Proceedings of the National Academy of Sciences,</em> 107<strong>,</strong> 6281-6285.</p><br />
<p><br />
BANEYX, F. 1999. Recombinant protein expression in Escherichia coli. <em>Current Opinion in Biotechnology,</em> 10<strong>,</strong> 411-421.</p><br />
<p><br />
BOMMARIUS, B., JENSSEN, H., ELLIOTT, M., KINDRACHUK, J., PASUPULETI, M., GIEREN, H., JAEGER, K. E., HANCOCK, R. E. W. &amp; KALMAN, D. 2010. Cost-effective expression and purification of antimicrobial and host defense peptides in Escherichia coli. <em>Peptides,</em> 31<strong>,</strong> 1957-1965.</p><br />
<p><br />
BROGDEN, K. A. 2005. Antimicrobial peptides: Pore formers or metabolic inhibitors in bacteria? <em>Nature Reviews Microbiology,</em> 3<strong>,</strong> 238-250.</p><br />
<p><br />
CHEN, X., ZHU, F., CAO, Y. &amp; QIAO, S. 2009. Novel expression vector for secretion of cecropin AD in Bacillus subtilis with enhanced antimicrobial activity. <em>Antimicrobial agents and chemotherapy,</em> 53<strong>,</strong> 3683-3689.</p><br />
<p><br />
GLINEL, K., JONAS, A. M., JOUENNE, T., LEPRINCE, J., GALAS, L. & HUCK, W. T. 2008. Antibacterial and antifouling polymer brushes incorporating antimicrobial peptide. <em>Bioconjug Chem,</em> 20<strong>,</strong> 71-77.</p><br />
<p><br />
HUMBLOT, V., YALA, J.-F., THEBAULT, P., BOUKERMA, K., HÉQUET, A., BERJEAUD, J.-M. & PRADIER, C.-M. 2009. The antibacterial activity of Magainin I immobilized onto mixed thiols self-assembled monolayers. <em>Biomaterials,</em> 30<strong>,</strong> 3503-3512.</p><br />
<p><br />
DAWSON, P. E., MUIR, T. W., CLARK-LEWIS, I. &amp; KENT, S. 1994. Synthesis of proteins by native chemical ligation. <em>Science,</em> 266<strong>,</strong> 776-779.</p><br />
<p><br />
KADOKURA, H. &amp; BECKWITH, J. 2009. Detecting folding intermediates of a protein as it passes through the bacterial translocation channel. <em>Cell,</em> 138<strong>,</strong> 1164-1173.</p><br />
<p><br />
KADOKURA, H., KATZEN, F. &amp; BECKWITH, J. 2003. Protein disulfide bond formation in prokaryotes. <em>Annual review of biochemistry,</em> 72<strong>,</strong> 111-135.</p><br />
<p><br />
LI, Y. 2011. Recombinant production of antimicrobial peptides in&lt; i&gt; Escherichia coli&lt;/i&gt;: A review. <em>Protein Expression and Purification,</em> 80<strong>,</strong> 260-267.</p><br />
<p><br />
LOBSTEIN, J., EMRICH, C. A., JEANS, C., FAULKNER, M., RIGGS, P. &amp; BERKMEN, M. 2012. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. <em>Microb Cell Fact,</em> 11<strong>,</strong> 56-56.</p><br />
<p><br />
STEINER, D., FORRER, P., STUMPP, M. T. &amp; PLUCKTHUN, A. 2006. Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display. <em>Nat Biotech,</em> 24<strong>,</strong> 823-831.</p><br />
<p><br />
SULISTIO, A., GURR, P. A., BLENCOWE, A. &amp; QIAO, G. G. 2012. Peptide-Based Star Polymers: The Rising Star in Functional Polymers. <em>Australian Journal of Chemistry,</em> 65<strong>,</strong> 978-984.</p><br />
<p><br />
SULISTIO, A., LOWENTHAL, J., BLENCOWE, A., BONGIOVANNI, M. N., ONG, L., GRAS, S. L., ZHANG, X. &amp; QIAO, G. G. 2011. Folic Acid Conjugated Amino Acid-Based Star Polymers for Active Targeting of Cancer Cells. <em>Biomacromolecules,</em> 12<strong>,</strong> 3469-3477.</p><br />
<p><br />
WIRADHARMA, N., LIU, S. Q. &amp; YANG, Y. Y. 2012. Branched and 4-Arm Starlike α-Helical Peptide Structures with Enhanced Antimicrobial Potency and Selectivity. <em>Small,</em> 8<strong>,</strong> 362-366.</p><br />
<p><br />
YU, Z., WANG, Q., MA, Q. &amp; ZHANG, R. 2013. Secretory expression of lacticin Q fused with SUMO in Bacillus subtilis. <em>Protein Expression and Purification,</em> 89<strong>,</strong> 51-55.</p><br />
<p><br />
ZASLOFF, M. 1987. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. <em>Proceedings of the National Academy of Sciences,</em> 84<strong>,</strong> 5449-5453.</p><br />
<p>&nbsp;</p><br />
<br />
<A NAME="Oxford"></A><br />
<h1>Collaboration with Oxford</h1><br />
<p>To strengthen the sense of iGEM community, we contacted <a target="_blank" href="https://2014.igem.org/Team:Oxford">University of Oxford iGEM team</a> to discuss the possibility of collaboration between our teams. The Oxford project aims to develop a system that can dispose of the carcinogenic, hazardous solvent dichloromethane (DCM). To do this, Oxford team has proposed the use of the DcmA enzyme. This enzyme degrades DCM to form a toxic intermediate which in turn is converted by another enzyme, FdhA, into a neutral molecule. Schematically this can be presented in the following way:</p><br />
<br />
<p><br />
Reaction 1: DCM +&nbsp;DcmA <strong>toxic intermediate</strong><br><br />
Reaction 2: <strong>toxic intermediate</strong> + FdhA neutral product.</p><br />
<p>The problem for their system lies in bringing the two enzymes together to ensure efficient reaction kinetics. An idea that we discussed with Oxford was to use the star peptide platform to link the two enzymes together, in a structure similar to the following: </p><br />
<img src="https://static.igem.org/mediawiki/2014/2/2e/Melbourne_Collab_1.png" width="500" height="325" alt=""/><br />
<p>We worked with Oxford to study this system from a theoretical standpoint. Our team studied the 3D structure of both enzymes involved to confirm that the enzyme could in theory be attached to a linker in this manner, while Oxford team did stochastic modelling to determine how reaction rate changes when the linker length, labeled D on the diagram, changes. </p><br />
<p><br />
As a first check, it was necessary to determine whether anchoring these enzymes to our star would be sterically possible. We studied the structures of FdhA and DcmA determined by crystallography using data from the protein data bank. As no crystallographic data was available in the databank for DcmA, GST was examined as a proxy, as GST is structurally homologous to DcmA. </p><br />
<p><br />
The crystalographically resolved structure of FdhA enzyme (Tanaka et al., 2002) from the protein data bank revealed the following image:</p><br />
<img src="https://static.igem.org/mediawiki/2014/d/dc/Melbourne_Collab_2.png" width="700" height="355" alt=""/><br />
<p>The crystalographically resolved structure of GST was as follows (Chang et al., 2014):</p><br />
<img src="https://static.igem.org/mediawiki/2014/e/e9/Melbourne_Collab_3.png" width="800" height="361" alt=""/><br />
<p>It can be seen that the amino- and carboxy-terminals are located away from the active site. This suggests that both enzymes, DcmA and FdhA, might be linked together via linkers that are used in our star peptide. That is, the active site will not be sterically hindered by attachment to the star peptide. That said, it is difficult to predict how anchoring the protein to the star will affect its tertiary structure. Further, it is difficult to predict how limiting the rotational degrees of freedom will affect enzyme activity.</p><br />
<p>The Oxford team modeled this system and found that, indeed, the star peptide as an enzyme linker was favourable to enzyme kinetics. Their work can be found here: <a target="_blank" href="https://2014.igem.org/Team:Oxford/alternatives_to_microcompartments#show2" target="_blank">https://2014.igem.org/Team:Oxford/alternatives_to_microcompartments#show2</a></p><br />
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