Team:UC Davis/Project

From 2011.igem.org

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<h1>Overview</h1>
<h1>Overview</h1>
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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, and to prototype this protocol by creating mutant libraries of the LacI, TetR and λ cI repressible promoters. We also tested our protocol on GFP to visually its ability to create functional protein mutants.<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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As of October 2011, we have a functioning mutant library generation and screening process, and a selection of well-characterized promoter mutants, including seven LacI promoter mutants and seven λ cI promoter mutants. We also have nine TetR 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.
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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 id="whyMutants">Why Make Mutant Libraries?</h2>
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<h2>Project Selection</h2>
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When designing novel genetic circuits, synthetic biologists are limited to using parts that are already available or that they can manufacture themselves. The creation of new parts can be incredibly time-consuming, since they must be extracted from existing natural genes and converted to a DNA part standard (like <a href="http://biobricks.org/" target:"_blank">BioBricks</a>) or modified from existing parts.<br><br>
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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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The <a href="http://www.partsregistry.org" target="_blank">parts registry</a> contains many useful parts, but in cases where fine control over the function of a part is required for a genetic circuit to function properly, the existing selection may not offer enough variation in expression strength, chemical response, or other characteristics.<br><br>
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Fine-tuning the control of a part can be achieved by random mutation of the bases in the DNA sequence of a part to change some characteristic -- say, transcription initiation, fluorescence intensity, or protein binding affinity -- whilst maintaining the basic functionality of the part. This is generally done in such away that many mutants are created at once which are then screened for their activity levels.<br><br>
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This allows the creation of large numbers of mutants which are viable (that is, maintaining the core function of the part) in a relatively short period of time. By performing this process on popular parts, one team can produce not only parts that are of a suitable activity for their circuits, but also parts that might fall outside of their usage range but be useful to other teams. Submitting these mutant libraries to the <a href="http://www.partsregistry.org" target="_blank">registry</a> allows other scientists access to a broader range of parts with a broader activity range and familiar functionality, preventing wasted man-hours and reagents.
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<h2>Make a Part Family</h2>
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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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<h2 id="whyPromoters">Why mutate repressible promoters?</h2>
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<h2>Promoter Mutants</h2>
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R0010, R0040 and R0051 (the LacI, TetR, and λ cI repressible promoters) are among the most commonly used promoters in the <a href="http://www.partsregistry.org" target="_blank">registry</a>. They are useful because they allow control control over gene expression that can be regulated using their corresponding repressor proteins, C0012, C0040 and C0051. This permits the construction of complex circuits that take advantage of the ability to switch, oscillate, or otherwise control the expression of genes by combining these parts in different ways.<br><br>  
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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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J23101, another extremely popular promoter, is a member of a constitutive promoter mutant library based on the wild-type J23119. This family offers mutants with similar functionality at different expression activity levels, making it an ideal choice for designing circuits where fine control over gene expression is beneficial.
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The creation of promoter mutant libraries for R0010, R0040 and R0051 makes the same kind of fine expression control possible within repressible systems. Extensive characterization of these parts allows synthetic biologists to pick and  choose mutants with an ideal level of repressor binding affinity or overall promoter activity, which is vital for the efficient design of complex circuits.<br><br>
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Because promoters are integral to the function of all genetic circuits, it is especially important that they be available in a broad selection and that their activity is well-documented and predictable.
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<h2 id="GFP">Why Mutate GFP?</h2>
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<h2>KO3D Plotting Library</h2>
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For starters, GFP looks really cool, and a plate full of mutant GFP transformants looks even cooler.<br><br>
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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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Unlike the promoter activity of our LacI promoter mutants, the fluorescence activity and color of our GFP mutants is a result of the structure of the translated folded protein. We wanted to quickly and easily asses our mutagenic PCR protocol's ability to produce mutants of protein-coding genes that retain some level of the activity of the original protein. Because GFP expression is visible to the naked eye (and even more visible under long-wave UV light exposure from a handheld lamp), mutations to the GFP gene that produce alterations in protein structure that produce changes in color or fluorescence level can be quickly assessed. <br><br>
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<h2 id="libraries">Generating Mutant Libraries with Error-Prone PCR</h2>
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The process by which we produce our mutant libraries is both fast and simple. We select a BioBrick part and prepare an error-prone PCR reaction using a small amount of template, standard VF2 and VR primers, the <a href=https://2011.igem.org/Team:UC_Davis/Protocols#ER-PCR>error-prone PCR mix</a> listed on our <a href="https://2011.igem.org/Team:UC_Davis/Protocols">protocols</a> page and Taq polymerase enzyme. The products of this reaction may be diluted and run through another round of PCR to introduce more mutations.<br><br>
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Mutant parts are then ligated directly into a screening construct like those listed below to help assess part activity. For example, mutant promoters can be placed in front of GFP to estimate their strength by relative fluorescence, whereas repressors can be paired with their corresponding promoter in front of a reporter. Transformation of these ligation products into competent DH5-α cells yields plates with many colonies, many containing mutants of the selected BioBrick part.<br><br>
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Colonies with the desired level of reporter expression are transferred to replica plates, grown up in liquid culture, and assayed in a 96-well plate reader. Consecutive screens are performed to narrow down the number of potential mutants so that they may be run in triplicate in our plate reader, using Octave scripts to select mutants with the desired range of activity.<br><br>
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Once a final group of mutants has been picked, they are carefully characterized for their activity. You can read more about the characterization process for our promoter mutants <a>here</a>.
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<h1 id ="promotermut">Promoter and Repressor Mutants</h1>
 
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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 our mutagenic PCR process on parts R0010, R0040 and R0051 (the LacI, TetR and c1 Lambda promoters) using multiple 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 promoter / wild-type repressor pairs to IPTG at various repressor concentration levels.
 
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<h2 id="construct">General Mutant Screening Constructs</h2>
 
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The specific order in which the parts are depicted below allows the user to swap in any promoter/repressor or promoter/activator pair using our regulatory characterization plasmid, K611018.<br><br>
 
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<center><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.<br><br> 
 
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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 nearly identical to its promoter-based counterpart. The user simply ligates the wild-type promoter to the 5' end and a mutant repressor or activator to the 3' end.
 
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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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