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Error-prone polymerase I as best in vivo mutator
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Integral to our project is the directed evolution of our Evobodies. To continuously introduce new Evobody variants with potential binding capabilities into the population we uses the <i>in vivo</i> mutagenesis capabilities of an error-prone polymerase I <a href=http://parts.igem.org/wiki/Part:BBa_K2082106>BBa_K2082106</a>.
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This enzymes replace the normal polymerase I under certain conditions (see here) and introduces a high amount of mutations inside our Evobody coding sequence. In that the mutations are more frequently inside our Evobody sequence and rarely inside other genomic proteins this <i>in vivo</i> mutagenesis approach circumvents the main problems restraining <i>in vivo</i> mutagenesis: the unintentional mutagenesis of essential proteins of a bacteria.
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By adding <a href=http://parts.igem.org/wiki/Part:BBa_K2082106>BBa_K2082106</a> to the iGEM parstreg and characterizing it we hope to give coming iGEM teams the possibility to easily optimize their proteins by means of directed evolution directly <i>in vivo</i>.
We characterized the error prone polymerase I per performing <a href=https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Mutation/Reversion>reversion assays</a>, thus quantifiying the mutation rate.
Figure: Mutation rate when using the error prone polymerase I (<a href=http://parts.igem.org/wiki/Part:BBa_K2082106>BBa_K2082106</a>) in comparison to using wild type polymerase I (<a href=http://parts.igem.org/wiki/Part:BBa_K2082107>BBa_K2082107</a>).
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<h2 style="color:#ffffff; text-align:left">like Lego they said</h2>
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Furthermore we analyzed error-prone polymerase I <a href=https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Mutation/Sequencing>mutation spectrum</a> using Illumina sequencing.
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Figure: Mutatagenic spectrum of error prone polymerase I (<a href=http://parts.igem.org/wiki/Part:BBa_K2082106>BBa_K2082106</a>) in comparision to wild type polymerase I(<a href=http://parts.igem.org/wiki/Part:BBa_K2082107>BBa_K2082107</a>).
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In combination we benchmarked the error-prone polymerase I by working out all necessary information to use this BioBrick in further directed evolution applications.
<div class="container text_header"><h3>Error Prone Polymerase I - <a href="http://parts.igem.org/Part:BBa_K2082106">BBa_K2082106</a></h3></div>
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<div class="container text">
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Integral to our project is the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Project/Mutation">directed evolution</a> of our Evobodies. To continuously introduce new Evobody variants with potential
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binding capabilities into the population we used the <i>in vivo</i> mutagenesis capabilities of the error-prone polymerase I
This enzyme replaces the normal polymerase I under certain conditions (<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Project/Mutation/EpPolI">further information</a>) and introduces a high amount of mutations
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inside our Evobody coding sequence. In because the mutations are more frequently inside our Evobody sequence and rarely inside other
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genomic proteins this <i>in vivo</i> mutagenesis approach circumvents the main problems restraining <i>in vivo</i> mutagenesis: the
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unintentional mutagenesis of essential proteins of a bacteria.
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<br>
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By adding <a href="http://parts.igem.org/Part:BBa_K2082106">BBa_K2082106</a> to the iGEM parstreg and characterizing it we hope
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to give coming iGEM teams the possibility to easily optimize their proteins by means of directed evolution <i>in vivo</i>.
We characterized the error prone polymerase I by performing <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Mutation/Reversion">reversion assays</a>, thus quantifiying the mutation rate.
<figcaption class="figure-caption"><b>Figure 1: Increase in mutation rate when using the error prone polymerase I (<a href="http://parts.igem.org/Part:BBa_K2082106">BBa_K2082106</a>) in comparison to using wild type polymerase I (<a href="http://parts.igem.org/Part:BBa_K2082107">BBa_K2082107</a>).</b></figcaption>
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</figure>
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Furthermore we analyzed error-prone polymerase I by <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Mutation/Sequencing">high-throughput sequencing</a> obtaining the mutation rate as well as the mutation spectrum.
<figcaption class="figure-caption"><b>Figure 2: Mutagensis rate of error prone polymerase I (<a href="http://parts.igem.org/Part:BBa_K2082106">BBa_K2082106</a>) in comparision to wild type polymerase I(<a href="http://parts.igem.org/Part:BBa_K2082107">BBa_K2082107</a>) as determined by <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Mutation/Sequencing">high-throughput sequencing</a>.</b></figcaption>
<figcaption class="figure-caption"><b>Figure 3: Mutatagenic spectrum of error prone polymerase I (<a href="http://parts.igem.org/Part:BBa_K2082106">BBa_K2082106</a>) in comparision to wild type polymerase I(<a href="http://parts.igem.org/Part:BBa_K2082107">BBa_K2082107</a>).</b></figcaption>
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</figure>
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In combination we benchmarked the error-prone polymerase I by working out all necessary information to use this BioBrick in further directed evolution applications.
Integral to our project is the directed evolution of our Evobodies. To continuously introduce new Evobody variants with potential
binding capabilities into the population we used the in vivo mutagenesis capabilities of the error-prone polymerase I
BBa_K2082106.
This enzyme replaces the normal polymerase I under certain conditions (further information) and introduces a high amount of mutations
inside our Evobody coding sequence. In because the mutations are more frequently inside our Evobody sequence and rarely inside other
genomic proteins this in vivo mutagenesis approach circumvents the main problems restraining in vivo mutagenesis: the
unintentional mutagenesis of essential proteins of a bacteria.
By adding BBa_K2082106 to the iGEM parstreg and characterizing it we hope
to give coming iGEM teams the possibility to easily optimize their proteins by means of directed evolution in vivo.
Characterization
We characterized the error prone polymerase I by performing reversion assays, thus quantifiying the mutation rate.
Figure 1: Increase in mutation rate when using the error prone polymerase I (BBa_K2082106) in comparison to using wild type polymerase I (BBa_K2082107).
Furthermore we analyzed error-prone polymerase I by high-throughput sequencing obtaining the mutation rate as well as the mutation spectrum.
Figure 2: Mutagensis rate of error prone polymerase I (BBa_K2082106) in comparision to wild type polymerase I(BBa_K2082107) as determined by high-throughput sequencing.Figure 3: Mutatagenic spectrum of error prone polymerase I (BBa_K2082106) in comparision to wild type polymerase I(BBa_K2082107).
In combination we benchmarked the error-prone polymerase I by working out all necessary information to use this BioBrick in further directed evolution applications.
