Team:MIT tconst

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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

    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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