Team:MIT tconst

From 2010.igem.org

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Revision as of 15:59, 25 October 2010

Bacteria

Phage

Mammalian

the bacterial uv toggle

In the beginning, there was a UV Toggle (Collins, 2000).

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. We planned for the toggle to create patterned fluorescence and phage polymerization in response to exposing the cells to UV light.

1 First Circuit
    1.1 Collins Toggle Validation
    1.2 First Biobrick
    1.3 Improved Bacterial Camera
2 AHL Propagation
    2.1 Construction
    2.2 Movie
3 Low Power Toggle
    3.1 Construction
    3.2 Improved Toggle

toggle by parts

toggle abstraction

toggle black-boxed

First Circuit

Collins Toggle Validation
We first wanted to make sure the parts we were working with were functional and reliable. As we wished to use the pTSMa plasmid as part of our circuit, we tested the original Collins toggle (pTSMa co-transformed with pCIRa).

Our recreation of the Collins toggle experiment validated pTSMa as a functional bistable toggle plasmid.

According to the paper, the area exposed to UV light should turn bright fluorescent green, and the rest of the cells should remain non-fluorescent. We recreated this experiment and our results demonstrate that the Collins toggle was working as it is described in the paper.

The basic Collins Toggle plasmids pTSMa and pCIRa. We used pTSMa in our own experiments as a bistable toggle.

Our First Biobrick
With our newly verified (borrowed) plasmid, we got to work on building our own response to UV induction. Using the same promoter used in pCIRa (the PcI-OR promoter that is induced by AHL/LuxR complex and inhibited by cI), we created a biobrick that would respond to UV induction by producing mCherry fluorescent protein when co-transformed with pTSMa. Our part would also constitutively produce LuxR, the protein that binds AHL and becomes a transcription factor that encourages the activity of the PcI-OR promoter.

Our first working part with a readout was K415006.

The first picture of mCherry fluorescence induced by 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.

Improved Bacterial Camera
As an intermediate in our project, we had created a bacterial camera with the ability to take and store images, reproduce the image on command (when AHL is added to the medium), and reset the entire plate to a blank slate whenever we pleased. All these abilities were a result of the bistable toggle we implemented in our design. In the context of past iGEM bacterial cameras, our toggle-based camera is a great improvement. UT-Austin created the first bacterial camera in 2005, exposing their cells to light for 12-15 hours to produce an image. Our toggle can snap and store an image in a matter of seconds.

AHL Propagation

We wanted our cells to be self-inducing and our signal to be self-propagating in addition to the circuit being sensitive and specific to user inputs. To do this, we added a part that would generate one of the necessary chemicals for transcription of the mCherry fluorescent protein.

Construction

We dubbed this part K415023, a part that now (if AHL and LuxR were available within the cell) fluoresced green or red in vivo depending on the state of the co-transformed toggle construct pTSMa. In addition, it would produce AHL under the same conditions as mCherry. This part can thus propagate and amplify a signal from a starting point of a droplet of AHL or even from leaky expression of AHL.

Movie
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.

Low Power Toggle

E.Coli population density as a function of UV exposure. Measured by drip assay.

According to established protocol, we were switching the state of our toggle (pTSMa from Collins 2003) with 80 J/m^2 UV. We noticed however, that significant cell death accompanied this exposure, likely due to fatal mutations. Searching through literature, we happened upon evidence of a super-sensitive cI (the lambda repressor from the pTSMa) that would be much more easily cleaved in UV-exposed cells. Luckily, this change from wildtype cI to the sensitive cI took a single SDM round, and we produced pLPTa, the Low Power Toggle.

LPT Construction

Through a single point mutation by SDM, the MIT iGEM team was able to change pTSMa into pLPTa, a toggle that requires much less UV power to switch the toggle between states.

@@ Line 90: / Line 90: @@
 <b class="bolded" id="improved">Improved Bacterial Camera</b><br>
-&nbsp;&nbsp;&nbsp;&nbsp;As an intermediate in our project, we had created a bacterial camera with the ability to take and store images, reproduce the image on command (when AHL is added to the medium), and reset the entire plate to a blank slate whenever we pleased. All these abilities were a result of the bistable toggle we implemented in our design. In the context of past iGEM bacterial cameras, our toggle-based camera is a great improvement. UT-Austin created the first bacterial camera in 2005, exposing their cells to light for 12-15 hours to produce an image. Our toggle can snap and store an image in a matter of seconds. <br><br>
+&nbsp;&nbsp;&nbsp;&nbsp;As an intermediate in our project, we had created a bacterial camera with the ability to take and store images, reproduce the image on command (when AHL is added to the medium), and reset the entire plate to a blank slate whenever we pleased. All these abilities were a result of the bistable toggle we implemented in our design. In the context of past iGEM bacterial cameras, our toggle-based camera is a great improvement. UT-Austin created the first bacterial camera in 2005, exposing their cells to light for 12-15 hours to produce an image. Our toggle can snap and store an image in a matter of seconds.
-&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>
-<br>
-&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
-<div class="sidep" style="width: 300px;">
-<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>
-&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.
 <br><br>
@@ Line 108: / Line 101: @@
 <b class="bolded" id="ahlmovie">Movie</b><br>
+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="620" height="372"><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="620" height="372"></embed></object><br><br>
@@ Line 113: / Line 107: @@
   <div class="sidep" style="float:right; width:200px;"><a href="https://static.igem.org/mediawiki/2010/0/05/Celldeath.png" class="thickbox"><img width=200px src="https://static.igem.org/mediawiki/2010/0/05/Celldeath.png"></a>E.Coli population density as a function of UV exposure. Measured by drip assay.</div>According to established protocol, we were switching the state of our toggle (pTSMa from Collins 2003) with 80 J/m^2 UV. We noticed however, that significant cell death accompanied this exposure, likely due to fatal mutations. Searching through literature, we happened upon evidence of a super-sensitive cI (the lambda repressor from the pTSMa) that would be much more easily cleaved in UV-exposed cells. Luckily, this change from wildtype cI to the sensitive cI took a single SDM round, and we produced pLPTa, the Low Power Toggle.<br><br><b class="bolded" id="lptconst">LPT Construction</b><br>
 <div class="sidep" style="float:left; width:400px;"><a href="https://static.igem.org/mediawiki/2010/c/cb/Sdm.png" class="thickbox"><img src="https://static.igem.org/mediawiki/2010/c/cb/Sdm.png" width=400px></a>Through a single point mutation by SDM, the MIT iGEM team was able to change pTSMa into pLPTa, a toggle that requires much less UV power to switch the toggle between states.</div>
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