Revision as of 22:10, 11 October 2014

Directed evolution

Introduction: directed evolution

The next step in improving the genes and gene products we are working with is to use directed evolution to enhance protein function and/or stability. Because of the limited time we had available for lab work, we weren’t able to fully pursue this. Nevertheless, the information below lays out our plans for potential future work.

Evolution occurs by natural processes such as point mutations, insertions, and deletions, as well as larger rearrangements and duplications of part of the genome. All of these can change properties of both translated and untranslated regions. Directed evolution is a technique used in protein engineering that accelerates this natural process with the aims of improving the function of a protein of interest.

For our project DCMation, we wanted to use random mutagenesis on dcmA, the enzyme that breaks down DCM. The thought behind this was to increase the enzyme’s activity, catalytic efficiency, and/or stability, thus accelerating the rate of DCM turnover in our system. Sadly, we never got around to doing this … :(

The plan was to use hypermutagenic PCR, which is a method that relies on inaccurate polymerisation reactions to introduce point mutations. Taq polymerase would be ideal for this purpose, since it lacks proofreading ability and is therefore prone to errors. With suitable conditions, this can lead to overall mutation frequencies of 10% per amplification (JP Vartanian, 1996). A screen would then have been used to determine which colony’s purified and mutated dcmA was more efficient at turning over DCM compared to the wild-type enzyme.

Oxford iGEM 2014

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-<div style="background-color:white; opacity:0.9; Height:75px; width:100%;margin-top:5px:margin-bottom:5px;font-size:65px;font-family:Helvetica;padding-top:5px; color:#00000; font-weight: 450;"><br><center><font style="opacity:0.7">Codon optimisation</font></center></div>
+<div style="background-color:white; opacity:0.9; Height:75px; width:100%;margin-top:5px:margin-bottom:5px;font-size:65px;font-family:Helvetica;padding-top:5px; color:#00000; font-weight: 450;"><br><center><font style="opacity:0.7">Directed evolution</font></center></div>
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-<h1>Introduction: codon optimisation</h1>
+<h1>Introduction: directed evolution</h1>
-In our quest to optimise the process of DCM breakdown, the first step was to move the genes we were interested in into well-characterised host strains. The source bacterium Methylobacterium extorquens DM4 proved very difficult to grow in the lab, and took its leisurely time once it did decide to grow. It's also not a particularly well-studied bacterium, unlike E. coli and P. putida, which we decided to work with. This section explains the concept of codon optimisation, why we used it, and how we did so. <br><br>
+The next step in improving the genes and gene products we are working with is to use directed evolution to enhance protein function and/or stability. Because of the limited time we had available for lab work, we weren’t able to fully pursue this. Nevertheless, the information below lays out our plans for potential future work.
+<br><br>
 </div>
-<div class="white_news_block">
-Cells use codons, which are composed of three consecutive nucleotides, to read the genetic code and translate it into proteins. This genetic code can not only vary between species, but it is also degenerate, meaning that several codons may specify a particular amino acid. Different species have unique codon usage biases, implying that they prefer particular codons over other codons that define the same amino acid. An excerpt of a codon usage frequency table is given for the three strains we worked with to highlight different codon biases.
+<div class="white_news_block">
+Evolution occurs by natural processes such as point mutations, insertions, and deletions, as well as larger rearrangements and duplications of part of the genome. All of these can change properties of both translated and untranslated regions. Directed evolution is a technique used in protein engineering that accelerates this natural process with the aims of improving the function of a protein of interest.
 <br><br>
-<img src="https://static.igem.org/mediawiki/2014/0/0d/Oxford_codon_optimisation.png" style="float:left;position:relative; width:80%;margin-left:10%;margin-right:10%" />
+For our project DCMation, we wanted to use random mutagenesis on dcmA, the enzyme that breaks down DCM. The thought behind this was to increase the enzyme’s activity, catalytic efficiency, and/or stability, thus accelerating the rate of DCM turnover in our system. Sadly, we never got around to doing this … :(
 <br><br>
-Since we are expressing the dcmA enzyme from M. extorquens in E. coli and P. putida, we used an online tool to optimise the sequence of dcmA so that it can be translated by our host strains. To ensure that dcmA is not over-produced and therefore a strain on the metabolic capacity of the cell, codon usage was only optimised to ~70%.<br><br>
+The plan was to use hypermutagenic PCR, which is a method that relies on inaccurate polymerisation reactions to introduce point mutations. Taq polymerase would be ideal for this purpose, since it lacks proofreading ability and is therefore prone to errors. With suitable conditions, this can lead to overall mutation frequencies of 10% per amplification (JP Vartanian, 1996). A screen would then have been used to determine which colony’s purified and mutated dcmA was more efficient at turning over DCM compared to the wild-type enzyme.
 </div>

Team:Oxford/directed evolution1

From 2014.igem.org

Revision as of 22:10, 11 October 2014

Introduction: directed evolution