Team:UC Davis/Project

From 2011.igem.org

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<h1>Overview</h1>
<h1>Overview</h1>
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The <a href="http://www.partsregistry.org" target="_blank">parts registry</a> contains many useful parts, many of which could be made more useful if they were available in a range of activity levels and characteristics. Because of this, we set out to develop a rugged, easy-to-use mutagenic PCR protocol for the rapid production of mutant libraries of any <a href="http://biobricks.org/" target:"_blank">BioBrick</a> part using standard primers.<br><br>
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We set out to develop a quick, easy process for the expansion of basic parts into a part families. Our method employs a suped-up <a href="https://2011.igem.org/Team:UC_Davis/Protocols#ER-PCR">mutagenic PCR protocol</a> that uses standard VF2 and VR primers and materials most iGEM teams already have on hand. We chose to prototype this process by creating a part family from the LacI promoter R0010, and to mutate GFP to visually assess our ability to create functional protein mutants.<br><br>
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To test our protocol's ability to produce functional protein mutants, we ran E0240 (GFP) through our error-prone PCR procedure and performed visual inspections of the resulting colonies for their variation from wild-type GFP.
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To show that our protocol can also be used to diversify the application of promoters and other regulatory gene sequences, we performed the same procedure on parts R0010, R0040 and R0051 (the LacI, TetR and c1 Lambda promoters), changing only the number of successive rounds of error-prone PCR. We screened these mutants for promoter activity compared to wild type, and characterized their response to changes in repressor concentration. We also characterized the response of wild-type and mutant LacI to IPTG at various repressor concentration levels.
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As of November 2011, we have a functioning part family generation process and seven <a href="https://2011.igem.org/Team:UC_Davis/Data_LacI">well-characterized</a> LacI promoter mutants and eight GFP mutants (two of which have been lovingly named Orange-Mutated Green Fluorescent Protein or [OMGfp] 1 and 2) which await further characterization.<br><br>
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We have also begun the process of generating part families from the TetR (R0040) and Lambda c1 (R0051) promoters, which are currently being selected for screening.
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<h2>General Construct</h2>
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<h2>Project Selection</h2>
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Why would we want to make mutants? What's so special about repressible promoters? Read about how we came to decide on this project idea <a href="https://2011.igem.org/Team:UC_Davis/Project_Selection">here.</a><br><br>
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<img src="https://static.igem.org/mediawiki/2011/2/21/UCD_Gen_pMut_construct.png"></center><br><br>
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We designed this construct for characterizing promoter mutants.  When pBAD is induced with arabinose, the repressor of choice is transcribed leading to decreased levels of the reporter, GFP.  The specific order in which the parts are depicted allows the user to swap in any promoter/repressor or promoter/activator pair using our<a href="http://partsregistry.org/Part:BBa_K611018"> regulatory characterization plasmid.</a> 
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<center><img src="https://static.igem.org/mediawiki/2011/1/12/UCD_Gen_rMut_construct.png"><br><br></center>
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The mutant repressor characterization construct is identical to the promoter mutant construct with the exception that the user must put the wildtype promoter on the 5' end and a mutant repressor/activator on the 3' end. 
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<h1>LacI</h1>
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<h2>Make a Part Family</h2>
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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. Read more about it
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Want to make your own mutant library? Curious as to how we assayed our promoter mutants, or how we selected variants that had well-spaced activity levels? Read about our library generation process <a href="https://2011.igem.org/Team:UC_Davis/PartFamilies">here</a>. <br>
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<a href="https://2011.igem.org/Team:UC_Davis/LacI" style="color:#bb66ff;">here.</a>
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<h1>Tet</h1>
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<h2>Promoter Mutants</h2>
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E. coli have evolved a resistance to the tetracycline antibiotic which is found in the tetracycline operon. This operon is regulated by TetR, the tetracycline repressor, which is a dimeric protein that binds the tetracycline repressible promoter. We mutated both of these. Read more about it <a href="https://2011.igem.org/Team:UC_Davis/TetR" style="color:#bb66ff;">here.</a>  
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We chose to mutate repressible promoters because they are useful in designing complex circuits and are relatively easy to screen for changes in activity level. You can view detailed information on our <a href="https://2011.igem.org/Team:UC_Davis/PromoterFamilies#LacI">LacI</a>, <a href="https://2011.igem.org/Team:UC_Davis/PromoterFamilies#TetR">TetR</a> and <a href="https://2011.igem.org/Team:UC_Davis/PromoterFamilies#c1">λ cI</a> part families on their respective pages.<br><br>
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<h1>cI Lambda</h1>
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<h2>KO3D Plotting Library</h2>
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This repressor originates from Lambda phage. Having either lytic or lysogenic life cycles, this bacteriophage infects it's E. coli host with double stranded DNA. cI binds at OR1, OR2 and OR3 sites with preference given to the OR1 site. For more information on how we mutated this repressor and it's associated promoter, click <a href="https://2011.igem.org/Team:UC_Davis/cilambda" style="color:#bb66ff;" > here.</a>
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When researching ways to present <a href="https://2011.igem.org/Team:UC_Davis/Data_LacI">LacI characterization data</a> clearly on this website, we realized there were no simple, cross-platform javascript libraries for interactive 3D data plotting. To rectify this, we coded our own. Read more about it <a href="https://2011.igem.org/Team:UC_Davis/KO3D">here</a>.
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Latest revision as of 20:15, 23 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.

Overview

We set out to develop a quick, easy process for the expansion of basic parts into a part families. Our method employs a suped-up mutagenic PCR protocol that uses standard VF2 and VR primers and materials most iGEM teams already have on hand. We chose to prototype this process by creating a part family from the LacI promoter R0010, and to mutate GFP to visually assess our ability to create functional protein mutants.

As of November 2011, we have a functioning part family generation process and seven well-characterized LacI promoter mutants and eight GFP mutants (two of which have been lovingly named Orange-Mutated Green Fluorescent Protein or [OMGfp] 1 and 2) which await further characterization.

We have also begun the process of generating part families from the TetR (R0040) and Lambda c1 (R0051) promoters, which are currently being selected for screening.

Project Selection

Why would we want to make mutants? What's so special about repressible promoters? Read about how we came to decide on this project idea here.

Make a Part Family

Want to make your own mutant library? Curious as to how we assayed our promoter mutants, or how we selected variants that had well-spaced activity levels? Read about our library generation process here.

Promoter Mutants

We chose to mutate repressible promoters because they are useful in designing complex circuits and are relatively easy to screen for changes in activity level. You can view detailed information on our LacI, TetR and λ cI part families on their respective pages.

KO3D Plotting Library

When researching ways to present LacI characterization data clearly on this website, we realized there were no simple, cross-platform javascript libraries for interactive 3D data plotting. To rectify this, we coded our own. Read more about it here.

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