Team:GeorgiaTech/Notebook

From 2014.igem.org

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<h1 id="May">May</h1>
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<h1 id="Notebook">Notebook</h1>
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<div class="bio">
<div class="bio">
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<h3>May</h3>
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<h3 id="May">May</h3>
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<p>We must constantly consider the safety implications of our project and how it could affect the environment, as we alter the genomes of organisms to produce novel functions. Understanding that our organism could create potentially harmful methanol and other hydrocarbons during methane remediation, it is envisioned that the bacteria will eventually be engineered with a kill-switch to prevent it's escape from industrial remediation of methane.
+
<h2>Project Design</h2>
-
<h2>Laboratory Safety</h2>
+
<p>The month of May was largely focused on the selection and design of our Biobrick parts and devices. Two weeks were dedicated to the selection process for our team's 2014 iGEM project. Each acting team member identified a problem and proposed a solution via a genetically engineered organism; through discussion and popular vote, it was eventually determined that our team would work to express functional sMMO in E.coli for the purpose of methane remediation.</p>
 +
<p>At this time, the team also voted to continue characterization and expansion of the RBS insertion primers first designed by Georgia Tech's 2013 iGEM Team.</p>
 +
<p>May saw very little bench work besides training modules to develop wet lab skills and build our supply of biological resources.</p>
-
<p> Our project involves regular use of powerful UV lights to visualize DNA bands after gel electrophoresis. Antibiotics used in the lab to selectively grow bacteria may be toxic to humans in large doses, and a Bunsen burner may be used on the bench to maintain a sterile environment or to sterilize some metal equipment during procedures involving bacteria.</p>
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<div class="c-1">
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<h2>Chassis Organisms</h2>
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</div>
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<h3 id="June">June</h3>
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<table rules="all" style="text-align:center; border:1px solid #C0C0C0;"><th width="20%">Species</th><th width="20%">Strain</th><th width="20%">Risk Group</th><th width="40%">Disease Risk to Humans</th>
+
<h2>RBS and Promoter Primer Testing</h2>
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<tr><td><i>Escherichia coli</i></td><td>DH5α (K-12 Derivative), BL21-A1</td><td>1</td><td>non-pathogenic, non-colonizing strains, although they may cause irritation to the skin, eyes, lungs, and kidneys.</td>
+
<p>Starting in June the team began testing of newly designed RBS insertion primers as well as newly designed promoter insertion primers based upon the same concept. Initial tests with these primers ultimately failed. It was deemed that the protocols and techniques developed by the team were not adequate enough for job. However, by the end of the month the team had achieved successes in inserting both a high and a low efficiency RBS site in front of the mCherry reporter gene in pSB1C3.</p>
-
</table>
+
<h2>sMMO Project Planning</h2>
-
<p>Our team works with E. coli DH5α and BL21 on a daily basis as a tool to selectively grow plasmids and express recombinant proteins respectively. Both of these strains are Biosafety level 1 organisms and can be handled safely in our existing lab space, however precautions are still undertaken to limit user exposure and the release of bacteria and recombinant DNA in the lab.</p>
+
<p>No bench work was performed on the sMMO project in the month of June, instead the time was spent meticulously designing our selected sMMO genes for DNA synthesis in order to ensure optimized expression in E. coli and an easy to manipulate gene in the lab. Additionally, the high cost of synthetic DNA drove our team to spend a great deal of time fundraising and obtaining discounts from molecular biology product suppliers.</p>
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<h2>Recombinant Coding DNA</h2>
+
-
<p>Our recombinant DNA does not pose a threat to any humans, however it may cause environmental harm if genes for antibiotic resistance or increased gene expression escaped from the lab. Precautions are therefore undertaken to ensure that our recombinant DNA does not leave the lab space.</p>
 
<div class="c-1">
<div class="c-1">
</div>
</div>
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<h3 id="Lab">Our Lab</h3>
+
<h3 id="July">July</h3>
 +
 
 +
<h2>RBS and Promoter Primer Function Verification</h2>
 +
<p>Come July, refinement of the techniques and protocols used for RBS and promoter insertion finally came to fruition. Breaking the protocol into separate steps for RBS insertion and promoter insertion as well as a re-designing of the original promoter primers led to the verification of three new RBS primers and two new promoter primers. Although the strength of these PCR inserted sites had not yet been checked by flow cytometry, sequencing showed that the sequences had been inserted as predicted.</p>
 +
<h2>sMMO Gene Arrival</h2>
 +
<p>The synthesized sMMO genes arrived during this month, and all efforts went towards inserting these genes into vectors (if they weren't already) and transforming E.coli with them for the purpose of creating safe frozen stocks.</p>
-
<p>Our lab space is equipped with all of the essentials to genetically engineer our bacteria, centrifuges, thermo-cyclers, micropipettes (with a hand carved pipette rack), and a host of loving grad students. Although many other iGEM teams around the world have obtained an entire room for their work, we are quite cozy with our own lab bench and rented "think space"(note: insert hover picture of PBL room). Past Georgia Tech iGEM teams have done right by us and collected a large selection of hand-me-down lab tools (from pipettes to incubators) as well as having saved up the money to purchase a Qiacube to automatically perform almost any DNA, RNA, or Protein purification offered by Qiagen. Beyond this, our team also has access to many of the other tools shared by other labs in the building, including Gel-docs, cold rooms for slow growth of bacteria, flow cytometers, and much much more.</p>
 
<div class="c-1">
<div class="c-1">
</div>
</div>
-
<h3 id="Protocol">Safety Protocol</h3>
+
<h3 id="August">August</h3>
-
<p>All members of our lab are required to wear close-toed shoes, long pants, and nitrile gloves when working in the lab, as well as goggles when working with volatile chemicals or UV light. Team members are required to receive proper instruction from either a graduate student or experienced team member when using a new instrument or technique, and may not perform lab work unless at least one graduate supervisor is present in the lab also. </p>
+
<h2>RBS and Promoter Relative Expression Analysis</h2>
 +
<p>Finally Flow cytometry characterization of the RBS and promoter primers was initiated. The cytometry provided information on the relative expression rates of combinations of various strength RBS and promoters. This knowledge aided in our selection of primers to use with the individual sMMO genes.</p> <p>It was noted that the value of these primers would be more tangible if they were also compared to an mCherry reporter with an RBS and promoter site synthesized through more traditional 3A assembly or ordered digestion and ligation.</p>
-
<p>Our lab avoids use of carcinogenic substances wherever possible, although if one must be used, then it must be done in a taped off area of the lab and properly cleaned both before and after use. Fume hoods are available for use when working with volatile chemicals, as well as a fire cabinet for proper storage of such chemicals.</p>
+
<h2>Generation of New Biobrick Parts</h2>
 +
<p>sMMO genes Z, B and C were inserted into Biobrick format over the month of August in vector pSB1C3.</p>
-
<p>All disposable lab materials that come into contact with biological lab specimens such as pipette tips, petri dishes, gloves, and other objects are placed in biohazard bags for proper disposal. All sharps and glass containers are also similar disposed of in appropriate sharps collection boxes. All reusable glass ware is autoclaved before washing if it has been in contact with live cultures and all tools and bench spaces are cleaned with ethanol or bleach after use with recombinant DNA or live cultures respectively. It is not expected that any hazardous chemicals or biological samples will be used in the lab this year, although EHS has implemented proper disposal methods for such materials.</p>
+
<div class="c-1">
 +
</div>
 +
<h3 id="September">September</h3>
 +
 
 +
<h2>Expression Vector Creation</h2>
 +
<p>Over the course of September, the real work of inserting RBS and Promoter sequences into the sMMO pSB1C3 plasmids began. In this time RBS sites were added to the sMMO Z, B, and C genes. Additionally the promoters were also added to sMMO Z and B plasmids thus completing our production of the sMMO Z and B expression vectors.</p>
 +
 
 +
<h2>Generation of New Biobrick Parts</h2>
 +
<p>sMMO gene Y was inserted into Biobrick format over the month of September in vector pSB1C3.</p>
 +
 
 +
<h2>GSU Collaboration</h2>
 +
<p>Mambalgin-1 was biobricked using 5' Extension PCR in the pSB1C3 vector as the first step in the effort to express Mambalgin protein in E. coli.</p>
 +
 
 +
<div class="c-1">
 +
</div>
 +
<h3 id="October">October</h3>
-
<p>Additionally, as required by Georgia Tech EHS, two copies of all MSDS sheets for chemicals stored or used in the lab were maintained, one for our reference and another for the lab manager. There are also mandated fire extinguishers, safety showers, eyewashes, and clearly marked emergency exits.</p>
+
<h2>GSU Collaboration</h2>
 +
<p>Biobricked Mambalgin-1 had regulatory RBS and Promoter sequences inserted via our insertion primer process. It has since been sent back to GSU and we have assisted in the characterization of Mambalgin-1 expression in E. coli.</p>
-
<p>Lastly, all team members are required to undergo EHS training in general biosafety and complete a right-to-know training module before being allowed to perform any lab work. These two sessions covered proper use, handling, and storage of hazardous substances as well as the labeling of such substances, emergency procedures, and general lab policies maintained at Georgia Tech. Although not necessary, most of the team also received training for handling blood and tissue samples and information on blood borne pathogens as part of a recommended training session.</p>
+
<p></p>
</div>
</div>

Latest revision as of 19:55, 17 October 2014

Notebook

May

Project Design

The month of May was largely focused on the selection and design of our Biobrick parts and devices. Two weeks were dedicated to the selection process for our team's 2014 iGEM project. Each acting team member identified a problem and proposed a solution via a genetically engineered organism; through discussion and popular vote, it was eventually determined that our team would work to express functional sMMO in E.coli for the purpose of methane remediation.

At this time, the team also voted to continue characterization and expansion of the RBS insertion primers first designed by Georgia Tech's 2013 iGEM Team.

May saw very little bench work besides training modules to develop wet lab skills and build our supply of biological resources.

June

RBS and Promoter Primer Testing

Starting in June the team began testing of newly designed RBS insertion primers as well as newly designed promoter insertion primers based upon the same concept. Initial tests with these primers ultimately failed. It was deemed that the protocols and techniques developed by the team were not adequate enough for job. However, by the end of the month the team had achieved successes in inserting both a high and a low efficiency RBS site in front of the mCherry reporter gene in pSB1C3.

sMMO Project Planning

No bench work was performed on the sMMO project in the month of June, instead the time was spent meticulously designing our selected sMMO genes for DNA synthesis in order to ensure optimized expression in E. coli and an easy to manipulate gene in the lab. Additionally, the high cost of synthetic DNA drove our team to spend a great deal of time fundraising and obtaining discounts from molecular biology product suppliers.

July

RBS and Promoter Primer Function Verification

Come July, refinement of the techniques and protocols used for RBS and promoter insertion finally came to fruition. Breaking the protocol into separate steps for RBS insertion and promoter insertion as well as a re-designing of the original promoter primers led to the verification of three new RBS primers and two new promoter primers. Although the strength of these PCR inserted sites had not yet been checked by flow cytometry, sequencing showed that the sequences had been inserted as predicted.

sMMO Gene Arrival

The synthesized sMMO genes arrived during this month, and all efforts went towards inserting these genes into vectors (if they weren't already) and transforming E.coli with them for the purpose of creating safe frozen stocks.

August

RBS and Promoter Relative Expression Analysis

Finally Flow cytometry characterization of the RBS and promoter primers was initiated. The cytometry provided information on the relative expression rates of combinations of various strength RBS and promoters. This knowledge aided in our selection of primers to use with the individual sMMO genes.

It was noted that the value of these primers would be more tangible if they were also compared to an mCherry reporter with an RBS and promoter site synthesized through more traditional 3A assembly or ordered digestion and ligation.

Generation of New Biobrick Parts

sMMO genes Z, B and C were inserted into Biobrick format over the month of August in vector pSB1C3.

September

Expression Vector Creation

Over the course of September, the real work of inserting RBS and Promoter sequences into the sMMO pSB1C3 plasmids began. In this time RBS sites were added to the sMMO Z, B, and C genes. Additionally the promoters were also added to sMMO Z and B plasmids thus completing our production of the sMMO Z and B expression vectors.

Generation of New Biobrick Parts

sMMO gene Y was inserted into Biobrick format over the month of September in vector pSB1C3.

GSU Collaboration

Mambalgin-1 was biobricked using 5' Extension PCR in the pSB1C3 vector as the first step in the effort to express Mambalgin protein in E. coli.

October

GSU Collaboration

Biobricked Mambalgin-1 had regulatory RBS and Promoter sequences inserted via our insertion primer process. It has since been sent back to GSU and we have assisted in the characterization of Mambalgin-1 expression in E. coli.

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