Team:MIT toggle

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<div class="bodybaby" style="font-size: 12px;">toggle by parts</div><a href="https://static.igem.org/mediawiki/2010/c/cc/UV_Circuit1.jpg" class="thickbox" title="Bacterial UV Toggle for controlling phage polymerization"><img src="https://static.igem.org/mediawiki/2010/c/cc/UV_Circuit1.jpg" width=215px></a>
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<div class="bodybaby" style="font-size: 12px;">toggle abstraction</div><a href="https://static.igem.org/mediawiki/2010/4/43/Abstractcircuit.png" class="thickbox" title="Abstracted bacterial toggle circuit to logic gates."><img src="https://static.igem.org/mediawiki/2010/4/43/Abstractcircuit.png" width=215px></a><br><br><br>
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<div style="width:250px; margin: 10px; position: relative; top: -4px; left:-11px; display: block; float:right; padding: 7px; background-color: white;">
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<div class="bodybaby" style="font-size: 12px;">toggle black-boxed</div><a href="https://static.igem.org/mediawiki/2010/1/18/Circuit.png" class="thickbox" title="Black-boxed bacterial toggle circuit. Also shown are the phage linker constructs."><img src="https://static.igem.org/mediawiki/2010/1/18/Circuit.png" width=215px></a>
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<dl id="specialnav">
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<dt><b>Bacteria</b></dt>
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<dd>
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<ul>
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                        <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>
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<li><a href="https://2010.igem.org/Team:MIT_tconst">Toggle Construction</a></li>
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<li><a href="https://2010.igem.org/Team:MIT_composite">Characterization</a></li>
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</ul>
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<dl id="nav">
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<dt><b>Phage</b></dt>
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<dd>
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<ul>
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<li><a href="https://2010.igem.org/Team:MIT_phage">Introduction</a></li>
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<li><a href="https://2010.igem.org/Team:MIT_phage_background">Background</a></li>
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<li><a href="https://2010.igem.org/Team:MIT_phage_design">Design</a></li>
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<li><a href="https://2010.igem.org/Team:MIT_phage_construction">Construction</a></li>
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<li><a href="https://2010.igem.org/Team:MIT_phage_results">Results</a></li>
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<li><a href="https://2010.igem.org/Team:MIT_phage_context">Context</a></li>
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</ul>
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</dd>
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<dt><b>Mammalian</b></dt>
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<dd>
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<ul>
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                        <li><a href="https://2010.igem.org/Team:MIT_mammalian">Overview</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="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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</dd>
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<div id="unique" style="padding:5px; font-size: 14px; border: 1px solid black; margin:5px;">
 
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<table width=70%><tr><td><div class="bodybaby">bacterial biobrick construction</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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<table width=650px style="background-color: white; margin-top:5px; padding: 10px;">
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<tr><td><img src="https://static.igem.org/mediawiki/2010/d/dc/Uvoverview.png"></td>
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<tr><td><div class="bodybaby">bacterial toggle</div></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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<center><img src="https://static.igem.org/mediawiki/2010/4/4a/Toggle.png"></center><br>
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<tr><td><div class="bodybaby">the bacterial uv toggle</div></td>
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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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<tr><td><br>In the beginning, there was a UV Toggle
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(<a href="http://www.nature.com/nature/journal/v403/n6767/abs/403339a0.html">Collins, 2000</a>). <br> <br>&nbsp;&nbsp;&nbsp;&nbsp;The 2010 MIT iGEM team saw that it was good, and decided to implement the Collins toggle in E.coli to create cells with bistable phenotypes.
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<div class="sidep"><a href="https://static.igem.org/mediawiki/2010/8/80/First.png" class="thickbox" title="The first patterned image created by exposing masked cells to UV light. The cells were made by co-transforming the Collins toggle plasmid pTSMa with our composite biobrick K415006."><img height=100px src="https://static.igem.org/mediawiki/2010/8/80/First.png"></a><br>The first picture of mCherry fluorescence induced by UV light.</div>
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We planned for the toggle to create patterned fluorescence and phage polymerization in response to exposing the cells to UV light. <br>&nbsp;&nbsp;&nbsp;&nbsp;Our first construction resulted in a composite biobrick K415006 that, once co-transformed with pTSMa from the Collins toggle, induced mCherry fluorescence in cells that were exposed to UV light. After much fine-tuning of the power of the UV exposure, the concentrations of AHL and IPTG, and mask cutting, a pattern of fluorescence finally emerged -- the first image.<br>
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<br> <br>
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&nbsp;&nbsp;&nbsp;&nbsp;Then we decided to make the signal amplify itself. We added a gene onto our composite biobrick that would produce additional AHL if induced by UV light. The new composite biobrick was dubbed K415022. In order to view the propagation, we recorded a movie beginning from the moment the cells were induced with UV. In addition to viewing the progression of the fluorescence, we were able to record how long it took for a true pattern of fluorescence to emerge in our cell lawn.<br><object width="300" height="180" style="display: inline; margin: 0px 16%;"><param name="movie" value="http://www.youtube.com/v/6KHabnSzCJ8?fs=1&amp;hl=en_US"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/6KHabnSzCJ8?fs=1&amp;hl=en_US" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="400" height="240"></embed></object>
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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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<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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&nbsp;&nbsp;&nbsp;&nbsp;After further inspection of the movie, we realized it might not be an accurate representation of the fluorescent propagation because we noticed a circle of cell death where the UV exposure had killed some of the cells in the lawn. We then decided to make pLPTa, a low power toggle that
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<div class="sidep" style="width: 300px;">
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<b class="bolded">Problem 1: Cell Death in Exposed Areas</b><br>
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<a href="https://static.igem.org/mediawiki/2010/e/eb/Igem_uv_prelim.png" class="thickbox" title="Power Modulation using the Low Power Toggle and cells that fluoresce red with UV induction."><img src="https://static.igem.org/mediawiki/2010/e/eb/Igem_uv_prelim.png" width=300px></a><br>Here we see cells controlled by the Low Power Toggle. The cells fluoresce red with UV induction, but at higher UV levels cell death can be seen in the green field.</div> still provided bistability, but required less UV power to induce a toggle switch. <br>
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<img src="http://partsregistry.org/wiki/images/e/eb/Igem_uv_prelim.png"><br>
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&nbsp;&nbsp;&nbsp;&nbsp;By site-directed mutagenesis, we changed the lambda repressor (cI) gene in the Collins pTSMa to a cI that is more sensitive to cleavage by Rec-A, the enzyme activated by UV light exposure. Thus, we modified the Collins toggle to minimize cell death in the UV-exposed regions while still maintaining its switch-like behavior.
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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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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.

Retrieved from "http://2010.igem.org/Team:MIT_toggle"