Latest revision as of 02:19, 18 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.

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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+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.
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+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.
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+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.
+<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.
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Team:Oxford/directed evolution

From 2014.igem.org

Latest revision as of 02:19, 18 October 2014

Introduction: directed evolution