Team:Harvard BioDesign

From 2014.igem.org

(Difference between revisions)
 
(32 intermediate revisions not shown)
Line 5: Line 5:
<!--main content -->
<!--main content -->
<table width="80%" align="center">
<table width="80%" align="center">
-
 
<!--welcome box -->
<!--welcome box -->
Line 26: Line 25:
<div align= "center">
<div align= "center">
-
<a href="https://2014.igem.org/Team:Harvard_BioDesign"style="><img src= "https://static.igem.org/mediawiki/2014/6/66/HarvardBioDesignLogo.jpg" width= "240px" height="200px"/>
+
<a href="https://2014.igem.org/Team:Harvard_BioDesign"><img src= "https://static.igem.org/mediawiki/2014/6/66/HarvardBioDesignLogo.jpg" width= "240px" height="200px"/></a>
</div>
</div>
Line 35: Line 34:
<table  width="100%">
<table  width="100%">
-
<tr heigth="15px"></tr>
+
<tr height="15px"></tr>
-
<tr heigth="75px">  
+
<tr height="75px">  
-
<td style="border:1px solid yellow;" align="center" height ="45px" onMouseOver="this.bgColor=yellow" onMouseOut="this.bgColor=yellow" bgColor=yellow>   
+
<td style="border:1px solid crimson;" align="center" width = "60px" height ="45px" onMouseOver="this.bgColor=color:#660000" onMouseOut="this.bgColor=color:#660000" bgColor=color:#660000>   
<a href="https://2014.igem.org/Team:Harvard_BioDesign"style="color:#000000">Home </a> </td>
<a href="https://2014.igem.org/Team:Harvard_BioDesign"style="color:#000000">Home </a> </td>
-
<td style="border:1px solid yellow;" align="center" height ="45px" onMouseOver="this.bgColor=yellow'" onMouseOut="this.bgColor=yellow'" bgColor=yellow>  
+
<td style="border:1px solid crimson;" align="center" width = "60px" height ="45px" onMouseOver="this.bgColor=color:#660000'" onMouseOut="this.bgColor=color:#660000" bgColor=color:#660000>  
<a href="https://2014.igem.org/Team:Harvard_BioDesign/Team"style="color:#000000"> Team </a> </td>
<a href="https://2014.igem.org/Team:Harvard_BioDesign/Team"style="color:#000000"> Team </a> </td>
-
<td style="border:1px solid yellow;" align="center" height ="45px" onMouseOver="this.bgColor=yellow'" onMouseOut="this.bgColor=yellow'" bgColor=yellow>
+
<td style="border:1px solid crimson" align="center" width = "60px" height ="45px" onMouseOver="this.bgColor=color:#660000'" onMouseOut="this.bgColor=color:#660000'" bgColor=color:#660000>   
-
<a href="https://igem.org/Team.cgi?year=2014&team_name=Harvard_BioDesign"style="color:#000000"> Official Team Profile </a></td>
+
-
 
+
-
<td style="border:1px solid yellow" align="center"  height ="45px" onMouseOver="this.bgColor=yellow'" onMouseOut="this.bgColor=yellow'" bgColor=yellow>   
+
<a href="https://2014.igem.org/Team:Harvard_BioDesign/Project"style="color:#000000"> Project</a></td>
<a href="https://2014.igem.org/Team:Harvard_BioDesign/Project"style="color:#000000"> Project</a></td>
-
<td style="border:1px solid yellow;" align="center"  height ="45px" onMouseOver="this.bgColor=yellow'" onMouseOut="this.bgColor=yellow'" bgColor=yellow>  
+
<td style="border:1px solid crimson;" align="center"  height ="45px" onMouseOver="this.bgColor=color:#660000'" onMouseOut="this.bgColor=color:#660000'" bgColor=color:#660000>  
<a href="https://2014.igem.org/Team:Harvard_BioDesign/Parts"style="color:#000000"> Parts</a></td>
<a href="https://2014.igem.org/Team:Harvard_BioDesign/Parts"style="color:#000000"> Parts</a></td>
-
<td style="border:1px solid yellow;" align="center" height ="45px" onMouseOver="this.bgColor=yellow'" onMouseOut="this.bgColor=yellow'" bgColor=yellow>
+
<td style="border:1px solid crimson;" align="center" height ="45px" onMouseOver="this.bgColor=color:#660000'" onMouseOut="this.bgColor=color:#660000'" bgColor=color:#660000>   
-
<a href="https://2014.igem.org/Team:Harvard_BioDesign/Modeling"style="color:#000000"> Modeling</a></td>
+
-
 
+
-
<td style="border:1px solid yellow;" align="center" height ="45px" onMouseOver="this.bgColor=yellow'" onMouseOut="this.bgColor=yellow'" bgColor=yellow>   
+
<a href="https://2014.igem.org/Team:Harvard_BioDesign/Notebook"style="color:#000000"> Notebook</a></td>
<a href="https://2014.igem.org/Team:Harvard_BioDesign/Notebook"style="color:#000000"> Notebook</a></td>
-
<td style="border:1px solid yellow;" align="center"  height ="45px" onMouseOver="this.bgColor=yellow'" onMouseOut="this.bgColor=yellow'" bgColor=yellow>  
+
<td style="border:1px solid crimson;" align="center"  height ="45px" onMouseOver="this.bgColor=color:#660000'" onMouseOut="this.bgColor=color:#660000'" bgColor=color:#660000>  
<a href="https://2014.igem.org/Team:Harvard_BioDesign/Safety"style=" color:#000000"> Safety </a></td>
<a href="https://2014.igem.org/Team:Harvard_BioDesign/Safety"style=" color:#000000"> Safety </a></td>
-
<td style="border:1px solid yellow;" align="center"  height ="45px" onMouseOver="this.bgColor=yellow'" onMouseOut="this.bgColor=yellow" bgColor=yellow>  
+
<td style="border:1px solid crimson;" align="center"  height ="45px" onMouseOver="this.bgColor=color:#660000'" onMouseOut="this.bgColor=color:#660000" bgColor=color:#660000>  
<a href="https://2014.igem.org/Team:Harvard_BioDesign/Attributions"style="color:#000000"> Attributions </a></td>
<a href="https://2014.igem.org/Team:Harvard_BioDesign/Attributions"style="color:#000000"> Attributions </a></td>
-
 
<td align ="center"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="55px"></a> </td>
<td align ="center"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="55px"></a> </td>
</tr>
</tr>
-
</table>
+
 
Line 91: Line 83:
<!--requirements section -->
<!--requirements section -->
-
<tr><td colspan="3"> <h3> Requirements </h3></td></tr>
+
<tr><td colspan="3" width = "30%"> <h3> Currently </h3></td><td colspan="3" width = "30%"> <h3> Our System </h3></td><td colspan="3" width = "30%"> <h3> Our Vision </h3></td></tr>
<tr>
<tr>
-
<td width="90%"  valign="top">  
+
<td colspan="3" width="30%"  valign="top">  
-
 
+
Natural cellular mechanisms are co-opted by synthetic biologists for use in biological sensors. Typical reporters such as GFP are only detectable inside living cells and used to gather information in the laboratory environment.  
-
<div class="global indent">
+
<br/>
-
    <!--content-->
+
<br/>
-
    <div class="container">
+
<br/>
-
        <div class="row">
+
<br/>
-
            <div class="col-lg-12">
+
<img src = "https://static.igem.org/mediawiki/2014/6/6a/Past.jpg" width =100% height =350px/>
-
                <h2 class="indent">Project description</h2>
+
-
                <p><strong>Overall Project Summary</strong></p>
+
-
                <p><em>E. coli</em>, along with many other gram-negative bacteria, produce beta-amyloid proteins called curli which form the basis of their biofilms. These amyloid structures are highly resistant to degradation and can survive extreme pH and temperature changes. Such robust features of curli proteins make them a great medium for the stable encoding of information. Additionally, curli fibers are assembled extracellularly throughout the lifetime of the cells, so information stored in curli can be read well after cells have died.<br>
+
-
                              We designed a sensing system in <em>E. coli</em> to store information about the cells&rsquo; environment by linking curli proteins to half of a strong affinity tag pairing. The two peptides that compose an affinity tag pair bind only to each other, allowing information to be stored until both peptides are present. Using a variety of affinity tags, we placed each one under the control of a different inducible promoter. The ratio of different affinity-tagged curli produced by the cells is determined by the relative concentrations of the inducers present in the environment. Next, we created fusion chromoproteins with the orthogonal affinity tags. By applying a standard mixture of affinity-tagged chromoproteins, we identify the ratio of bound chromoproteins via an RYB color system. This ratio corresponds to the information encoded by the <em>E. coli</em>. This work has potential applications in the fields of cryptography, information storage, as well as the formation of a new reporting toolbox for synthetic biology.</p>
+
-
                <p><strong>Project Details</strong><br>
+
-
                  Our system is composed of the following components:</p>
+
-
                <p>CsgA variants - We have engineered four versions of the CsgA protein with four different affinity domains: three coiled coil SynZip domains, and one covalent bond-based SpyTag domain. The affinity domains are fused to the endogenous CsgA with a 24-amino acid linker sequence, to ensure that the domains are available for binding as the CsgA polymerizes into a curli fiber.</p>
+
-
                <p>Chromoprotein constructs - to demonstrate the feasibility of our concept, we used chromoproteins fused to binding domains reciprocal to those fused to the CsgA variants. These are three SynZip domains, and one SpyCatcher domain, which spontaneously forms a covalent bond with the SpyTag domain when the two come close enough to one another in solution.</p>
+
-
                <p>Sensing promoters – thus far we have coupled expression of the CsgA variants to two inducuble promoters: LacZ and … In the future we hope to include promoters induced by more interesting environmental conditions, like temperature or atmospheric chemical concentrations (i.e. carbon monoxide), so that the composition of the curli fibers produced by the bacteria will reflect such environmental conditions.</p>
+
-
                <p><strong>Materials and Methods</strong><br>
+
-
                  Genetic constructs were created by PCR cloning reactions, Gibson assembly reactions, and related protocols. CsgA production was monitored via Congo Red assays, and chromoprotein-CsgA association was determined by culturing both transgenic proteins together for 24 hours, subjecting the solution to SDS gel electrophoresis, and observing the resultant protein bands for conjugation between the SpyTag and SpyCatcher-associated proteins.</p>
+
-
                <p><strong>The Experiments</strong></p>
+
-
                <ul>
+
-
                  <li>Congo Red assays</li>
+
-
                  <li>Gel shift pictures</li>
+
-
                </ul>
+
-
                <p><strong>Results</strong><br>
+
-
                  According to the results we have obtained thus far, modifying native CsgA to include our selected binding domain reduces the total amount of curli that is ultimately produced by <em>E. Coli</em> (according to Congo Red analysis), by anywhere from 20-50%. However, the curli that is produced is stained by Congo Red, indicating that it has chemical similarity to the curli produced by wild-type cells.</p>
+
-
                <p>Gel shift goes here.</p>
+
-
                <p><strong>Analysis</strong><br>
+
-
                  The reduction in curli production that was observed after we added binding domains to CsgA is not too great to make our system unworkable. Various methods can be used to counteract this problem, the simplest of which is to simply use a greater initial number of cells such that the desired or required amount of curli is ultimately produced. In the future, we may attempt to determine whether using linker sequences with different lengths, compositions, and 3-dimensional structures can affect the amount of curli that is ultimately produced – it is possible that the shape of the modified CsgA subunit is less optimal for the self-assembly of curli that is an important feature of our system.</p>
+
-
                <p><strong>Conclusion</strong><br>
+
-
                  The results of our Congo Red analysis indicate that it is possible to produce significant amounts of curli with <em>E. coli</em> engineered to express mutant variants of CsgA with binding domains that allow our data processing and storage system to function. Small adjustments will be made to optimize curli production and self-assembly, but we have shown that it is possible to modify CsgA in ways required for our system to function without limiting curli production to an unacceptable degree.</p>
+
-
<h5 class="indent">&nbsp;</h5>
+
-
            </div>
+
-
        </div>
+
-
    </div>
+
 +
</td>
 +
<td colspan="3"width="30%"  valign="top">
 +
Our system encodes sensing information into the amyloid protein curli within bacterial biofilms. Unlike common reporter proteins, curli fibers are highly resistant to degradation and can survive extreme pH and temperature changes extracellularly. Applying chromoproteins engineered to bind specifically to corresponding curli units, information is easily displayed.
 +
<br/>
 +
<br/>
 +
<img src = "https://static.igem.org/mediawiki/2014/5/55/Idea.jpg" width =100% height = 350px/>
 +
</td>
 +
<td colspan="3"width="30%"  valign="top">
 +
Biological sensing can be applied outside of the laboratory settings with information that is robust and can be easily read in a biomaterial with functional potential.
 +
<br/>
 +
<br/>
 +
<br/>
 +
<br/>
 +
<br/>
 +
<img src = "https://static.igem.org/mediawiki/2014/5/5b/Future.jpg" width =100% height =350px/>
</td>
</td>
</tr>
</tr>
-
 
-
 
-
<tr> <td colspan="3"  height="15px"> </td></tr>
 
-
<tr><td bgColor=yellow colspan="3" height="1"> </tr>
 
-
 
-
 
-
<!--tips  -->
 
-
<tr><td colspan="3" > <h3> Tips  </h3></td></tr>
 
-
 
-
<tr>
 
-
<td width="45%" valign="top">
 
-
<p>We are currently working on providing teams with some easy to use design templates.
 
-
<br> In the meantime you can also view other team wikis for inspiration! Here are some very good examples</p>
 
-
 
-
<ul>
 
-
<li> <a href="https://2013.igem.org/Team:SDU-Denmark/"> 2013 SDU Denmark </a> </li>
 
-
<li> <a href="https://2013.igem.org/Team:SYSU-China">2013 SYSU China</a> </li>
 
-
<li> <a href="https://2013.igem.org/Team:Shenzhen_BGIC_ATCG"> 2013 Shenxhen BGIG ATCG </a></li>
 
-
<li> <a href="https://2013.igem.org/Team:Colombia_Uniandes">2013 Colombia Unianades </a></li>
 
-
<li> <a href="https://2013.igem.org/Team:Lethbridge">2013 Lethbridge</a></li>
 
-
</ul>
 
-
 
-
<p>For a full wiki list, you can visit <a href="https://igem.org/Team_Wikis?year=2013">iGEM 2013 web sites </a> and <a href="https://igem.org/Team_Wikis?year=2012">iGEM 2012 web sites</a>  lists. </p>
 
-
</td>
 
<td> </td>
<td> </td>
-
<td width="45%">
 
-
 
-
<p>This wiki will be your team’s first interaction with the rest of the world, so here are a few tips to help you get started: </p>
 
-
 
-
<ul>
 
-
<li>State your accomplishments! Tell people what you have achieved from the start. </li>
 
-
<li>Be clear about what you are doing and what you plan to do.</li>
 
-
<li>You have a global audience! Consider the different backgrounds that your users come from.</li>
 
-
<li>Make sure information is easy to find; nothing should be more than 3 clicks away.  </li>
 
-
<li>Avoid using very small fonts and low contrast colors; information should be easy to read.  </li>
 
-
<li>Start documenting your project as early as possible; don’t leave anything to the last minute before the Wiki Freeze. For a complete list of deadlines visit the <a href="">iGEM 2013 calendar</a> </li>
 
-
<li>Have lots of fun! </li>
 
-
</ul>
 
-
</br>
 
-
</td>
 
-
</tr>
 
</table>
</table>
-
 
+
</html>
-
<a href="https://2014.igem.org/wiki/index.php?title=Team:Harvard_BioDesign&action=edit"style="color:#C610F4"> Click here  to edit this page!</a>
+

Latest revision as of 01:43, 18 October 2014


Home Team Project Parts Notebook Safety Attributions

Currently

Our System

Our Vision

Natural cellular mechanisms are co-opted by synthetic biologists for use in biological sensors. Typical reporters such as GFP are only detectable inside living cells and used to gather information in the laboratory environment.



Our system encodes sensing information into the amyloid protein curli within bacterial biofilms. Unlike common reporter proteins, curli fibers are highly resistant to degradation and can survive extreme pH and temperature changes extracellularly. Applying chromoproteins engineered to bind specifically to corresponding curli units, information is easily displayed.

Biological sensing can be applied outside of the laboratory settings with information that is robust and can be easily read in a biomaterial with functional potential.