Team:MIT toggle

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<dl id="nav">
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<dl id="specialnav">
<dt><b>Bacteria</b></dt>
<dt><b>Bacteria</b></dt>
<dd>
<dd>
<ul>
<ul>
                         <li><a href="https://2010.igem.org/Team:MIT_toggle">Overview</a></li>
                         <li><a href="https://2010.igem.org/Team:MIT_toggle">Overview</a></li>
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                        <li><a href="https://2010.igem.org/Team:MIT_tmodel">Modelling</a></li>
<li><a href="https://2010.igem.org/Team:MIT_tconst">Toggle Construction</a></li>
<li><a href="https://2010.igem.org/Team:MIT_tconst">Toggle Construction</a></li>
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<li><a href="#">Characterization</a></li>
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<li><a href="https://2010.igem.org/Team:MIT_composite">Characterization</a></li>
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<dl id="nav">
<dt><b>Phage</b></dt>
<dt><b>Phage</b></dt>
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<ul>
<ul>
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                         <li><a href="#">Overview</a></li>
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                         <li><a href="https://2010.igem.org/Team:MIT_mammalian">Overview</a></li>
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<li><a href="#">Standard and Design</a></li>
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<li><a href="https://2010.igem.org/Team:MIT_mammalian_Standard">New Mammalian Standard </a></li>
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<li><a href="#">Experiments</a></li>
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                        <li><a href="https://2010.igem.org/Team:MIT_mammalian_Circuit">Circuit Design</a></li>
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<li><a href="https://2010.igem.org/Team:MIT_mammalian_Mechanosensation"> Mechanosensation</a></li>
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<li><a href="https://2010.igem.org/Team:MIT_mammalian_Bone"> Bone Formation</a></li>
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<li><a href="https://2010.igem.org/Team:MIT_mammalian_Switch"> Synthetic Switch</a></li>
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<tr><td><div class="bodybaby">bacterial toggle</div></td>
<tr><td><div class="bodybaby">bacterial toggle</div></td>
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<tr><td><br>The Bacterial team focused on implementing and improving the Collins toggle. The ultimate goal is to connect our toggle to our phage module, so that exposing UV light in a pattern on a lawn of bacteria will result in phage production and polymerization in the exposed area. As an intermediate in our project, we have managed to create what is essentially an improved bacterial camera capable of instantaneous photography.<br><br>We implemented part of the <a href="http://www.nature.com/nature/journal/v403/n6767/abs/403339a0.html">Collins toggle</a> in our bacteria, improving upon previous iGEM bacterial cameras by shortening the exposure time from hours <a href="http://parts.mit.edu/wiki/index.php/UT_Austin_2005">(UT-Austin 2005)</a> to seconds. During our experiments, noticeable cell death in UV-exposed regions prompted development of an additional feature. Our bacterial circuit improves upon the Collins toggle in that the power of UV light required to switch the toggle is reduced, resulting in significantly more cells surviving the image capture process.<br><br></td>
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<tr><td><br>A genetic switch or toggle follows the general form of two proteins that inhibit the others' transcription. Because of this arrangement, the toggle has two stable states -- on or off -- and can switch between the states with certain inputs. This pseudo-binary, reversible setup implies a simple but powerful control over a genetic system.<br><br>
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<tr><td><a href="https://static.igem.org/mediawiki/2010/d/dc/Uvoverview.png" class="thickbox"><img src="https://static.igem.org/mediawiki/2010/d/dc/Uvoverview.png"></a></td>
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<center><img src="https://static.igem.org/mediawiki/2010/4/4a/Toggle.png"></center><br>
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The Bacterial team focused on implementing and improving the Collins toggle, pTSMa. pTSMa switches on if the cell is exposed to UV, and switches off with the addition of IPTG. The ultimate goal was to use the toggle to fine-tune our control over the growth of our polymerizing phage M13. We created a biobrick that fluoresced red when exposed to UV light, or when the toggle was turned "on" to represent where phage would be produced when our modules are connected. As an intermediate in our project, we have managed to create what is essentially an improved bacterial camera capable of instantaneous photography.<br><br>
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<a href="https://static.igem.org/mediawiki/2010/d/dc/Uvoverview.png" class="thickbox"><img src="https://static.igem.org/mediawiki/2010/d/dc/Uvoverview.png"></a><br><br>
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We implemented part of the <a href="http://www.pnas.org/content/101/22/8414.full">Collins toggle</a> in our bacteria, improving upon previous iGEM bacterial cameras by shortening the exposure time from hours <a href="https://2006.igem.org/UT_Austin_2005">(UT-Austin 2005)</a> to seconds. However, during our experiments, we noticed two problems.<hr><br><br>
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<b class="bolded">Problem 1: Cell Death in Exposed Areas</b><br>
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<img src="http://partsregistry.org/wiki/images/e/eb/Igem_uv_prelim.png"><br>
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<br><br>Noticeable cell death in UV-exposed regions was immediately noticed in our cell lawns. According to established protocol, cells were exposed to UV power 80 J/m^2 in order to switch the Collins toggle to the "on" state. When we performed a drip assay to deduce the effect of this exposure on cell viability, we got the following death curve.<br><center><img width=400px src="https://static.igem.org/mediawiki/2010/0/05/Celldeath.png"> </center><br><br>
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<b>Solution: Low Power Toggle</b> <br>While searching literature, we found evidence that with a single site-directed mutagenesis reaction, we could increase toggle sensitivity to UV exposure. In doing this, we created a Low Power Toggle. Our bacterial circuit improves upon the Collins toggle in that the power of UV light required to switch the toggle is reduced 8x, resulting in significantly more cells surviving the image capture process.<br><img src="https://static.igem.org/mediawiki/2010/d/d5/Powermodulation.png"><br><br><br>
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<b class="bolded">Problem 2: Leaky Expression</b><br>
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Secondly, we noticed that even in areas that were not induced by UV, a low amount red fluorescence appeared. Looking back at all of our parts, we found on the parts registry page for R0065 that it is characterized as "very leaky". Analyzing its features, we noticed a second -10 promoter region in the hybrid promoter.<br>
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<br><b>Solution: Less Leaky Promoter</b><br>By removing one of these -10 sequences, we were able to reduce the leakiness of our signal, and increase the binary response of our circuit.<br>
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<center><img src="https://static.igem.org/mediawiki/2010/a/af/Leakiness.png"></center><br>In this FACS scatter, the x-axis represents green fluorescence, and the y-axis represents red fluorescence with zero UV exposure. Theoretically, there should be no red fluorescence. The red fluorescence in part K415023 is controlled by the original R0065, and in K415069 is controlled by our less leaky promoter K415031. You can see that the original promoter is more leaky than our fixed promoter: 3% of K415069 fluoresce red, while 15% of K415031 cells fluoresce red.
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</td>
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<tr><td></td>
</table>
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Latest revision as of 00:56, 28 October 2010

Bacteria
bacterial toggle

A genetic switch or toggle follows the general form of two proteins that inhibit the others' transcription. Because of this arrangement, the toggle has two stable states -- on or off -- and can switch between the states with certain inputs. This pseudo-binary, reversible setup implies a simple but powerful control over a genetic system.


The Bacterial team focused on implementing and improving the Collins toggle, pTSMa. pTSMa switches on if the cell is exposed to UV, and switches off with the addition of IPTG. The ultimate goal was to use the toggle to fine-tune our control over the growth of our polymerizing phage M13. We created a biobrick that fluoresced red when exposed to UV light, or when the toggle was turned "on" to represent where phage would be produced when our modules are connected. As an intermediate in our project, we have managed to create what is essentially an improved bacterial camera capable of instantaneous photography.



We implemented part of the Collins toggle in our bacteria, improving upon previous iGEM bacterial cameras by shortening the exposure time from hours (UT-Austin 2005) to seconds. However, during our experiments, we noticed two problems.


Problem 1: Cell Death in Exposed Areas



Noticeable cell death in UV-exposed regions was immediately noticed in our cell lawns. According to established protocol, cells were exposed to UV power 80 J/m^2 in order to switch the Collins toggle to the "on" state. When we performed a drip assay to deduce the effect of this exposure on cell viability, we got the following death curve.


Solution: Low Power Toggle
While searching literature, we found evidence that with a single site-directed mutagenesis reaction, we could increase toggle sensitivity to UV exposure. In doing this, we created a Low Power Toggle. Our bacterial circuit improves upon the Collins toggle in that the power of UV light required to switch the toggle is reduced 8x, resulting in significantly more cells surviving the image capture process.



Problem 2: Leaky Expression
Secondly, we noticed that even in areas that were not induced by UV, a low amount red fluorescence appeared. Looking back at all of our parts, we found on the parts registry page for R0065 that it is characterized as "very leaky". Analyzing its features, we noticed a second -10 promoter region in the hybrid promoter.

Solution: Less Leaky Promoter
By removing one of these -10 sequences, we were able to reduce the leakiness of our signal, and increase the binary response of our circuit.

In this FACS scatter, the x-axis represents green fluorescence, and the y-axis represents red fluorescence with zero UV exposure. Theoretically, there should be no red fluorescence. The red fluorescence in part K415023 is controlled by the original R0065, and in K415069 is controlled by our less leaky promoter K415031. You can see that the original promoter is more leaky than our fixed promoter: 3% of K415069 fluoresce red, while 15% of K415031 cells fluoresce red.

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