Team:UC Davis/PromoterFamilies

From 2011.igem.org

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<h1>λ cI</h1>
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<a href="http://vimeo.com/29301077"><iframe src="http://player.vimeo.com/video/29301077?title=0&amp;byline=0&amp;portrait=0" align="left" style="margin-right:15px" width="400" height="300" frameborder="0" webkitAllowFullScreen allowFullScreen></iframe></a>
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This promoter/repressor pair originates from Lambda phage. Employing either a lytic or lysogenic life cycle, this bacteriophage infects its E. coli host with double stranded DNA. cI binds at OR1, OR2 and OR3 sites with preference given to the OR1 site.
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<h2>Construct</h2>
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<a href="http://partsregistry.org/Part:BBa_K611017"><img src="https://static.igem.org/mediawiki/2011/a/a5/UCD_R51mut_construct.png">
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<h2>Mutant Screening</h2>
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<a href="http://farm7.static.flickr.com/6014/6190729685_171b292c18_b.jpg"><img src="http://farm7.static.flickr.com/6014/6190729685_171b292c18_b.jpg" width="400" height="212"></a>
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We selected mutants by first visually picking colonies from transformation plates.  From there we used our plate reader to read the GFP fluorescence of each mutant on a 96 well screening plate.  In the graph above, the green bars represent mutant expression that is 1.5 standard deviations from the average wildtype fluorescence.  Many of our mutants show very low to no expression which indicates that the error-prone pcr which produced them was notably mutagenic. We found 9 suitable candidates to further characterize using the process outlined in the <a href="https://2011.igem.org/Team:UC_Davis/Data"> Data page.</a>
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Revision as of 18:37, 22 October 2011

Our Sponsors

Start a Family

Got a favorite BioBrick? Check our our process for expanding basic parts into part families.

Criteria

View our judging criteria for iGEM 2011 here.

Repressible Promoter Families

The activity of repressible promoters can be modulated through the expression repressor proteins, chemical induction, and other factors. We chose to expand the LacI, TetR and Lambda c1 BioBrick promoters into part families to offer synthetic biologists a broader selection of repressible promoters from which to build genetic circuits.

LacI

The lac repressor is responsible for regulating the metabolism of lactose. In the absence of lactose, LacI forms a tetramer with identical subunits which appears as two dimers. Each dimer binds in the major groove of the DNA binding region which subsequently blocks the RNA polymerase from binding. In nature, allolactose will bind the repressor leading to transcription of the lac operon. Using IPTG as an inducer has the same effect as allolactose.

To the left is a small render of the LacI tetramer bound to its operator.

Construct

Mutant Screening


The above graph shows our initial mutants. We picked 87 potential mutants from transformation plates and ran them in our plate reader to quantitatively measure fluorescence. The green bars represent variants that are at least 1.5 standard deviations from the average wildtype expression level.



After gathering this data, we picked 29 mutants which represented a good range of expression. From there we did more fluorescence tests to obtain a final 7 mutants. After choosing our mutants, we did more rigorous characterization as outlined on our Data page.

DNA Sequences



The sequences above show our 7 LacI mutants. There are between 1 and 7 mutations in each sequence as indicated by the red bases. All 7 sequences have mutations between bases 100 and 200 which contain the known locations of the CAP binding site(bases 88-127) and LacI binding site (bases 166-200). Read more about our mutants on their Parts Registry pages.

TetR

The Tet operon, found in E. coli, codes for resistance to the antibiotic tetracycline. This operon is regulated by the tetracycline repressor TetR, a dimeric protein which binds to and inhibits the TetR repressible promoter.


TetR monomer



Construct

λ cI

This promoter/repressor pair originates from Lambda phage. Employing either a lytic or lysogenic life cycle, this bacteriophage infects its E. coli host with double stranded DNA. cI binds at OR1, OR2 and OR3 sites with preference given to the OR1 site.

Construct

Mutant Screening

We selected mutants by first visually picking colonies from transformation plates. From there we used our plate reader to read the GFP fluorescence of each mutant on a 96 well screening plate. In the graph above, the green bars represent mutant expression that is 1.5 standard deviations from the average wildtype fluorescence. Many of our mutants show very low to no expression which indicates that the error-prone pcr which produced them was notably mutagenic. We found 9 suitable candidates to further characterize using the process outlined in the Data page.

Retrieved from "http://2011.igem.org/Team:UC_Davis/PromoterFamilies"