Team:UChicago

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<h1 >WELCOME TO iGEM 2014! </h1>
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<p>Your team has been approved and you are ready to start the iGEM season!
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<br>On this page you can document your project, introduce your team members, document your progress <br> and share your iGEM experience with the rest of the world! </p>
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<p style="color:#E7E7E7"> <a href="https://2014.igem.org/wiki/index.php?title=Team:UChicago&action=edit"style="color:#FFFFFF"> Click here  to edit this page!</a> </p>
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<a href="https://2014.igem.org/Team:UChicago"style="color:#000000">Home </a> </td>
 
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<a href="https://2014.igem.org/Team:UChicago/Team"style="color:#000000"> Team </a> </td>
 
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<a href="https://igem.org/Team.cgi?year=2014&team_name=UChicago"style="color:#000000"> Official Team Profile </a></td>
 
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<a href="https://2014.igem.org/Team:UChicago/Project"style="color:#000000"> Project</a></td>
 
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<a href="https://2014.igem.org/Team:UChicago/Attributions"style="color:#000000"> Attributions </a></td>
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<tr><td colspan="3"> <h3> Requirements </h3></td></tr>
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<p> Please be sure to keep these links, your audience will want to find your: </p>
<p> Please be sure to keep these links, your audience will want to find your: </p>
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<!-- Links to other team pages -->
 
<ul>
<ul>
<li><a href="https://2014.igem.org/Team:UChicago">Home</a> </li>
<li><a href="https://2014.igem.org/Team:UChicago">Home</a> </li>
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<li><a href="https://2014.igem.org/Team:UChicago/Safety">Safety</a> </li>
<li><a href="https://2014.igem.org/Team:UChicago/Safety">Safety</a> </li>
<li><a href="https://2014.igem.org/Team:UChicago/Attributions">Attributions</a> </li>
<li><a href="https://2014.igem.org/Team:UChicago/Attributions">Attributions</a> </li>
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<p>There are a few wiki requirements teams must follow:</p>
<p>There are a few wiki requirements teams must follow:</p>
<ul>
<ul>
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<li>All pages, images and files must be hosted on the <a href ="https://2014.igem.org/Special:Upload">  2014.igem.org server</a>. </li>
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<li>All pages must be created under the team’s name space.</li>
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<li>The <a href="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png"> iGEM logo </a> should be placed on the upper part of every page and should link to <a href="https://2014.igem.org/Main_Page">2014.igem.org</a>.</li>
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<li>As part of your documentation, keep the links from the menu to the left. </li>
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<li>The <a href="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png"> iGEM logo </a> should be placed on the upper part of every page and should link to <a href="https://2014.igem.org/Main_Page">2014.igem.org</a>.</li>
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<p>Visit the <a href="https://2014.igem.org/Wiki_How-To"> Wiki How To page </a> for a complete list of requirements, tips and other useful information. </p>
<p>Visit the <a href="https://2014.igem.org/Wiki_How-To"> Wiki How To page </a> for a complete list of requirements, tips and other useful information. </p>
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<h3>Tips</h3>
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<p>This wiki will be your team’s first interaction with the rest of the world, so here are a few tips to help you get started: </p>
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<h1>UChicago iGEM 2014</h1>
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<h2>2014: A Sequence Space Odyssey: Diversifying Mutators to Optimize Directed Evolution</h2>
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<div>
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<h3>Project Abstract</h3>
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<p>
 +
Directed evolution is a fundamental technique in bioengineering organisms to express proteins of novel function and to mass produce industrially relevant biomolecules. Generally, directed evolution simulates an algorithmic process in which the entire sequence space is searched for an optimal genotype by increasing the natural mutation rate to artificially speed the process of selective evolution. Current methods of in vivo directed evolution typically rely on physical or chemical mutagens to accomplish stochastic genomic or plasmid mutagenesis. However, the extremely high and uncontrolled mutation rate often accumulates deleterious mutations nonspecific to the process of interest. This can result in a toxic effect to the organism, leading to suboptimal evolved levels of production.
 +
</p><p>
 +
A novel directed evolution system, termed feedback-regulated evolution of phenotype (FREP), incorporates a dynamic mutation rate to overcome the existing problems of directed evolution by mimicking the plasticity of the mutation rate in natural evolution. This is achieved through dynamic control of a mutator element that is negatively regulated by the desired end product. In this feedback scheme, as more of the desired biomolecule is produced, the rate of mutation decreases and eventually approaches zero, allowing evolution and maintenance of a high level of production while minimizing the accumulation of toxic, nonspecific mutations.
 +
</p><p>
 +
Additionally, many directed evolution systems that incorporate mutator genes often rely on the strongest known mutator, MutD. However, the use of MutD alone in these systems is problematic because like all other individual mutator genes, it may only catalyze certain types of base pair substitutions reflective of its “mutational bias”, limiting the search of sequence space. Diversifying the mutator genes to eliminate mutational bias should increase the efficiency by which the sequence space can be searched. Ultimately, the addition of multiple mutators should lead to higher overall levels of evolved production. Despite a very few number of past studies to incorporate multiple mutators, these efforts have failed to show an increased efficiency of evolution. They fail to encompass a systematic screen of all mutators identified in literature, which would be required for determination of the ideal combination of mutators. In general, there is a surprising lack of studies on the use of different combinations of mutators in directed evolution.
 +
</p><p>
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The 2014 UChicago iGEM team has two main goals. The first goal is to implement and optimize FREP in E. coli by evolving mutants with elevated production of a given biomolecule, specifically evolving tyrosine production as a relatively simple pathway to demonstrate proof of concept. The mutator element will be controlled by the tyrosine-sensitive repressible promoter ParoF, suppressing the mutation rate as the tyrosine production of the cell increases. The mutator gene is linked to a fluorescent reporter gene which allows for convenient screening of desired phenotype, as bacteria with low levels of fluorescence should also have the highest levels of production due to the negative feedback loop.
 +
</p><p>
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The second goal is to enable a deeper exploration in the search space by combining multiple mutators with different biases to increase the initial mutation rate and speed the accumulation of beneficial mutations. We have identified previously characterized mutator genes that function in a diversity of pathways, including polymerases, proofreading enzymes, methylases and topoisomerases. We will characterize the mutation rates of these mutator genes in their active dominant negative forms individually as well as in various combinations, aiming to determine the ideal combination for directed evolution. We hope to introduce this ideal combination of mutators into the FREP system and demonstrate increased levels of evolved tyrosine production compared to the single mutator. If successful, our project should significantly improve the process of directed evolution of any biomolecule of industrial importance.
 +
</p>
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</div>
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<div>
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<h2>Project Summary</h2>
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<h4>What are mutators genes?</h4>
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<p>
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Mutators genes are defective copies of genes that can induce an elevated spontaneous mutation rate for all other genes in the organism. They increases genomic instability and hence are often employed as a tool for mutagenesis.
 +
</p>
 +
<h4>How do mutator genes work?</h4>
 +
<p>
 +
Mutator genes often confers defects in the DNA repair or replication pathways. For example, defects in polymerases, proofreading enzymes or recombinases can lead to inaccurate DNA replication, leading to significantly increased spontaneous mutation rate. Each mutator gene product has its mutational bias due to the inherent differences in function and nature of mutation. The mutT gene product, for instance, is a G-A mismatch repair mutator. The major type of mutation caused, hence its mutational bias, is AT to CG transversions.
 +
</p>
 +
<h4>Our Project</h4>
 +
<p>
 +
Keasling et. al has used the most potent mutator gene, mutD, in implementing FREP. However, the use of only one mutator can limit the search space due to low initial mutation rates and mutational biases. Our team has therefore chosen to investigate the following 11 well-characterized dominant negative mutators:
 +
</p>
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<table class="bortable">
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<th>Mutator</th>
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<p>We are currently working on providing teams with some easy to use design templates.
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<th>Gene Function/Gene Product</th>
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<br> In the meantime you can also view other team wikis for inspiration! Here are some very good examples</p>
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<th>Mutational Bias/Preferred Mutations</th>
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<li> <a href="https://2013.igem.org/Team:SDU-Denmark/"> 2013 SDU Denmark </a> </li>
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<li> <a href="https://2013.igem.org/Team:SYSU-China">2013 SYSU China</a> </li>
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<li> <a href="https://2013.igem.org/Team:Shenzhen_BGIC_ATCG"> 2013 Shenxhen BGIG ATCG </a></li>
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<li> <a href="https://2013.igem.org/Team:Colombia_Uniandes">2013 Colombia Unianades </a></li>
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<li> <a href="https://2013.igem.org/Team:Lethbridge">2013 Lethbridge</a></li>
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</ul>
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<p>For a full wiki list, you can visit <a href="https://igem.org/Team_Wikis?year=2013">iGEM 2013 web sites </a> and <a href="https://igem.org/Team_Wikis?year=2012">iGEM 2012 web sites</a>  lists. </p>
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<td> </td>
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<p>This wiki will be your team’s first interaction with the rest of the world, so here are a few tips to help you get started: </p>
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<ul>
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<li>State your accomplishments! Tell people what you have achieved from the start. </li>
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<li>Be clear about what you are doing and what you plan to do.</li>
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<li>You have a global audience! Consider the different backgrounds that your users come from.</li>
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<li>Make sure information is easy to find; nothing should be more than 3 clicks away.  </li>
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<li>Avoid using very small fonts and low contrast colors; information should be easy to read.  </li>
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<li>Start documenting your project as early as possible; don’t leave anything to the last minute before the Wiki Freeze. For a complete list of deadlines visit the <a href="">iGEM 2013 calendar</a> </li>
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<li>Have lots of fun! </li>
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<tr>
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<td>Dam</td>
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<td>DNA methyltransferase that methylates A in -GATC-</td>
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<td>Transversions</td></tr>
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<tr><td>DinB</td>
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<td>Error-prone DNA polymerase (pol IV)</td>
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<td>Frameshift and base substitution</td></tr>
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<tr><td>DnaE</td>
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<td>Alpha subunit of DNA polymerase III holoenzyme</td>
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<td>Transversions</td></tr>
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<tr><td>EmrR</td>
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<td>Transcriptional repressor of microcin biosynthesis genes</td>
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<td>AT->GC transversions, frameshift, base substitutions</td></tr>
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<tr><td>MutD</td>
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<td>Epsilon subunit of DNA polymerase III</td>
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<td>Affects all bases through Transitions, Transversions, Frameshifts</td></tr>
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<tr><td>MutH</td>
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<td>Part of the multimeric methyl-directed DNA mismatch repair protein (MutSHL)</td>
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<td>Transitions, Frameshifts</td></tr>
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<tr><td>MutL</td>
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<td>Part of the multimeric methyl-directed DNA mismatch repair protein (MutSHL)</td>
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<td>Transitions, Frameshifts</td></tr>
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<tr><td>MutS</td>
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<td>Part of the multimeric methyl-directed DNA mismatch repair protein (MutSHL)</td>
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<td>Transitions, Frameshifts</td></tr>
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<tr><td>MutT</td>
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<td>Nucleoside triphosphatase</td>
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<td>AT to CG Transversions</td></tr>
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<tr><td>MutY</td>
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<td>Adenine DNA glycosylase</td>
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<td>GC to TA Transversions</td></tr>
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<tr><td>ParC</td>
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<td>Alpha subunit of DNA topoisomerase IV</td>
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<td>Nicks DNA</td></tr>
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<tr><td>UmuD’C</td>
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<td>Subunits of DNA polumerase IV</td>
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<td>Hypersensitivity to UV light damage</td></tr>
</table>
</table>
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<p>
 +
To further increase the search space, we have planned to use the following combinations of mutator genes:
 +
</p>
 +
<table class="bortable">
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<tr>
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<th>Combinations</th>
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<th>Reasoning</th>
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</tr>
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<tr><td>MutS + MutH + MutL</td>
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<td>Belong to same multimeric protein</td></tr>
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<tr><td>MutS + MutY + MutH</td>
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<td>Mutants have been shown to increase mismatches between 8oxo-G and A</td></tr>
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<tr><td>MutD + MutS + MutT</td>
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<td>The E. coli strain XL1-Red contains mutations in these genes</td></tr>
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<tr><td>MutY + EmrR</td>
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<td>Different biases</td></tr>
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<tr><td>Dam + MutH</td>
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<td>Different biases</td></tr>
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<tr><td>Dam + MutS</td>
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<td>Different biases</td></tr>
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<tr><td>MutD + MutH</td>
 +
<td>Different biases</td></tr>
 +
<table>
 +
<p>
 +
In our project, we only focused on point mutators to verify our hypothesis that a combination of mutators with different biases can increase the initial mutation rate and facilitate the search for the global optimum. If our hypothesis is proven correct, we will move onto more complex and larger-scale mutational mechanisms such as transposition and recombination to investigate their effects on the diversification of the search space.
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<html><!--
==Fun Extras==
==Fun Extras==
See a fluctuation analysis program in action!:
See a fluctuation analysis program in action!:
-
[[Team:UChicago/Fluctuation Analysis|]]
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[[Team:UChicago/Fluctuation Analysis|UChicago/Fluctuation Analysis]]
 +
--></html>

Latest revision as of 03:33, 18 October 2014

Uchi banner.jpg

UChicago iGEM 2014

2014: A Sequence Space Odyssey: Diversifying Mutators to Optimize Directed Evolution

Project Abstract

Directed evolution is a fundamental technique in bioengineering organisms to express proteins of novel function and to mass produce industrially relevant biomolecules. Generally, directed evolution simulates an algorithmic process in which the entire sequence space is searched for an optimal genotype by increasing the natural mutation rate to artificially speed the process of selective evolution. Current methods of in vivo directed evolution typically rely on physical or chemical mutagens to accomplish stochastic genomic or plasmid mutagenesis. However, the extremely high and uncontrolled mutation rate often accumulates deleterious mutations nonspecific to the process of interest. This can result in a toxic effect to the organism, leading to suboptimal evolved levels of production.

A novel directed evolution system, termed feedback-regulated evolution of phenotype (FREP), incorporates a dynamic mutation rate to overcome the existing problems of directed evolution by mimicking the plasticity of the mutation rate in natural evolution. This is achieved through dynamic control of a mutator element that is negatively regulated by the desired end product. In this feedback scheme, as more of the desired biomolecule is produced, the rate of mutation decreases and eventually approaches zero, allowing evolution and maintenance of a high level of production while minimizing the accumulation of toxic, nonspecific mutations.

Additionally, many directed evolution systems that incorporate mutator genes often rely on the strongest known mutator, MutD. However, the use of MutD alone in these systems is problematic because like all other individual mutator genes, it may only catalyze certain types of base pair substitutions reflective of its “mutational bias”, limiting the search of sequence space. Diversifying the mutator genes to eliminate mutational bias should increase the efficiency by which the sequence space can be searched. Ultimately, the addition of multiple mutators should lead to higher overall levels of evolved production. Despite a very few number of past studies to incorporate multiple mutators, these efforts have failed to show an increased efficiency of evolution. They fail to encompass a systematic screen of all mutators identified in literature, which would be required for determination of the ideal combination of mutators. In general, there is a surprising lack of studies on the use of different combinations of mutators in directed evolution.

The 2014 UChicago iGEM team has two main goals. The first goal is to implement and optimize FREP in E. coli by evolving mutants with elevated production of a given biomolecule, specifically evolving tyrosine production as a relatively simple pathway to demonstrate proof of concept. The mutator element will be controlled by the tyrosine-sensitive repressible promoter ParoF, suppressing the mutation rate as the tyrosine production of the cell increases. The mutator gene is linked to a fluorescent reporter gene which allows for convenient screening of desired phenotype, as bacteria with low levels of fluorescence should also have the highest levels of production due to the negative feedback loop.

The second goal is to enable a deeper exploration in the search space by combining multiple mutators with different biases to increase the initial mutation rate and speed the accumulation of beneficial mutations. We have identified previously characterized mutator genes that function in a diversity of pathways, including polymerases, proofreading enzymes, methylases and topoisomerases. We will characterize the mutation rates of these mutator genes in their active dominant negative forms individually as well as in various combinations, aiming to determine the ideal combination for directed evolution. We hope to introduce this ideal combination of mutators into the FREP system and demonstrate increased levels of evolved tyrosine production compared to the single mutator. If successful, our project should significantly improve the process of directed evolution of any biomolecule of industrial importance.

Project Summary

What are mutators genes?

Mutators genes are defective copies of genes that can induce an elevated spontaneous mutation rate for all other genes in the organism. They increases genomic instability and hence are often employed as a tool for mutagenesis.

How do mutator genes work?

Mutator genes often confers defects in the DNA repair or replication pathways. For example, defects in polymerases, proofreading enzymes or recombinases can lead to inaccurate DNA replication, leading to significantly increased spontaneous mutation rate. Each mutator gene product has its mutational bias due to the inherent differences in function and nature of mutation. The mutT gene product, for instance, is a G-A mismatch repair mutator. The major type of mutation caused, hence its mutational bias, is AT to CG transversions.

Our Project

Keasling et. al has used the most potent mutator gene, mutD, in implementing FREP. However, the use of only one mutator can limit the search space due to low initial mutation rates and mutational biases. Our team has therefore chosen to investigate the following 11 well-characterized dominant negative mutators:

Mutator Gene Function/Gene Product Mutational Bias/Preferred Mutations
Dam DNA methyltransferase that methylates A in -GATC- Transversions
DinB Error-prone DNA polymerase (pol IV) Frameshift and base substitution
DnaE Alpha subunit of DNA polymerase III holoenzyme Transversions
EmrR Transcriptional repressor of microcin biosynthesis genes AT->GC transversions, frameshift, base substitutions
MutD Epsilon subunit of DNA polymerase III Affects all bases through Transitions, Transversions, Frameshifts
MutH Part of the multimeric methyl-directed DNA mismatch repair protein (MutSHL) Transitions, Frameshifts
MutL Part of the multimeric methyl-directed DNA mismatch repair protein (MutSHL) Transitions, Frameshifts
MutS Part of the multimeric methyl-directed DNA mismatch repair protein (MutSHL) Transitions, Frameshifts
MutT Nucleoside triphosphatase AT to CG Transversions
MutY Adenine DNA glycosylase GC to TA Transversions
ParC Alpha subunit of DNA topoisomerase IV Nicks DNA
UmuD’C Subunits of DNA polumerase IV Hypersensitivity to UV light damage

To further increase the search space, we have planned to use the following combinations of mutator genes:

Combinations Reasoning
MutS + MutH + MutL Belong to same multimeric protein
MutS + MutY + MutH Mutants have been shown to increase mismatches between 8oxo-G and A
MutD + MutS + MutT The E. coli strain XL1-Red contains mutations in these genes
MutY + EmrR Different biases
Dam + MutH Different biases
Dam + MutS Different biases
MutD + MutH Different biases

In our project, we only focused on point mutators to verify our hypothesis that a combination of mutators with different biases can increase the initial mutation rate and facilitate the search for the global optimum. If our hypothesis is proven correct, we will move onto more complex and larger-scale mutational mechanisms such as transposition and recombination to investigate their effects on the diversification of the search space.