Team:MIT tconst

From 2010.igem.org

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<b class="bolded" id="biobrick">Our First Biobrick</b><br>
<b class="bolded" id="biobrick">Our First Biobrick</b><br>
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&nbsp;&nbsp;&nbsp;&nbsp;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.<br><div class="sidep" style="width: 450px; float: left;"><a href="https://static.igem.org/mediawiki/igem.org/e/e3/K415006.png" class="thickbox" title="Our first working part with a readout was K415006."><img width=450px src="https://static.igem.org/mediawiki/igem.org/e/e3/K415006.png"></a><br>Our first working part with a readout was K415006.</div>
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&nbsp;&nbsp;&nbsp;&nbsp;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.<br><div class="sidep" style="width: 450px; float: left;"><a href="https://static.igem.org/mediawiki/igem.org/e/e3/K415006.png" class="thickbox" title="Our first working part with a readout was K415006."><img width=450px src="https://static.igem.org/mediawiki/igem.org/e/e3/K415006.png"></a><br>Our first working part with a readout was K415006.</div><br><br><br><br>
<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<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> 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>
<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<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> 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>

Revision as of 23:18, 18 October 2010



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 Edge Detection
    2.1 Construction
3 AHL Propogation
    3.1 Construction
    3.2 Movie
4 Low Power Toggle
    4.1 Construction
    4.2 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.





    Our first construction resulted in a composite biobrick K415006 that, once co-transformed with pTSMa from the Collins toggle, induced mCherry

The first picture of mCherry fluorescence induced by UV light.
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.


    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.

    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

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.
still provided bistability, but required less UV power to induce a toggle switch.
    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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