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Team:UC Davis/PromoterFamilies
From 2011.igem.org
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| - | <h1>Promoter Families</h1> | + | <h1>Repressible Promoter Families</h1> |
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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. | 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. | ||
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| - | <h1>LacI</h1> | + | <h1 id="LacI">LacI</h1> |
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<p><iframe src="http://player.vimeo.com/video/27753866?title=0&byline=0&portrait=0" width="400" height="300" frameborder="0" align="left" style="margin-right:15px"></iframe>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. </p> | <p><iframe src="http://player.vimeo.com/video/27753866?title=0&byline=0&portrait=0" width="400" height="300" frameborder="0" align="left" style="margin-right:15px"></iframe>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. </p> | ||
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| - | <h2 id=" | + | <h2 id="LacIdnaseq">DNA Sequences</h2> |
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<img src="https://static.igem.org/mediawiki/2011/9/9f/UCD_R10mut_seq_REDMUTS_09242011.png"> | <img src="https://static.igem.org/mediawiki/2011/9/9f/UCD_R10mut_seq_REDMUTS_09242011.png"> | ||
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| - | <h1>TetR</h1> | + | <h1 id="TetR">TetR</h1> |
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| - | E. coli | + | 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. |
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<h1>Construct</h1> | <h1>Construct</h1> | ||
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| + | <h1 id="c1">λ cI</h1> | ||
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| + | <a href="http://vimeo.com/29301077"><iframe src="http://player.vimeo.com/video/29301077?title=0&byline=0&portrait=0" align="left" style="margin-right:15px" width="400" height="300" frameborder="0" webkitAllowFullScreen allowFullScreen></iframe></a> | ||
| + | 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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| + | </center> | ||
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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"> | ||
| + | </center> | ||
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</div> | </div> | ||
Latest revision as of 18:49, 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.
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
