Difference between revisions of "Team:Bielefeld-CeBiTec/Basic Part"

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Best Basic Part
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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&nbsp;I <a href=http://parts.igem.org/wiki/Part:BBa_K2082106>BBa_K2082106</a>.
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This enzymes replace the normal polymerase&nbsp;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>.
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Characterization
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<h1 style="margin-bottom: 0px; text-align:left">Best Basic Part</h1>
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We characterized the error prone polymerase&nbsp;I per performing <a href=https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Mutation/Reversion>reversion assays</a>, thus quantifiying the mutation rate.  
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<h2 style="color:#ffffff; text-align:left">like Lego they said</h2>
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Figure: Mutation rate when using the error prone polymerase&nbsp;I (<a href=http://parts.igem.org/wiki/Part:BBa_K2082106>BBa_K2082106</a>) in comparison to using wild type polymerase&nbsp;I (<a href=http://parts.igem.org/wiki/Part:BBa_K2082107>BBa_K2082107</a>).
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Furthermore we analyzed error-prone polymerase&nbsp;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&nbsp;I (<a href=http://parts.igem.org/wiki/Part:BBa_K2082106>BBa_K2082106</a>) in comparision to wild type polymerase&nbsp;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&nbsp;I by working out all necessary information to use this BioBrick in further directed evolution applications.
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<div class="container text_header"><h3>Error Prone Polymerase I - <a href="http://parts.igem.org/wiki/Part:BBa_K2082106">BBa_K2082106</a></h3></div>
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<div class="container text"> <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 uses the <i>in vivo</i> mutagenesis capabilities of the error-prone polymerase&nbsp;I  
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<a href="http://parts.igem.org/wiki/Part:BBa_K2082106">BBa_K2082106</a>.
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<br>
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This enzymes replace the normal polymerase&nbsp;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 that 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/wiki/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>.
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</div>
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<div class="container text_header"><h4>Characterization</h4></div>
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<div class="container text">
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We characterized the error prone polymerase&nbsp;I per performing <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Mutation/Reversion">reversion assays</a>, thus quantifiying the mutation rate.  
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  <img src="Pfad" class="figure-img">
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  <figcaption class="figure-caption"><b>Figure 1: Increase in mutation rate when using the error prone polymerase&nbsp;I (<a href="http://parts.igem.org/wiki/Part:BBa_K2082106">BBa_K2082106</a>) in comparison the using wild type polymerase&nbsp;I (<a href="http://parts.igem.org/wiki/Part:BBa_K2082107">BBa_K2082107</a>).</b></figcaption>
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</figure>
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Furthermore we analyzed error-prone polymerase&nbsp;I <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Mutation/Sequencing">mutation spectrum</a> using Illumina sequencing.  
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<figure class="figure">
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  <img src="Pfad" class="figure-img">
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  <figcaption class="figure-caption"><b>Figure 2: Mutatagenic spectrum of error prone polymerase&nbsp;I (<a href="http://parts.igem.org/wiki/Part:BBa_K2082106">BBa_K2082106</a>) in comparision to wild type polymerase&nbsp;I.</b></figcaption>
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</figure>
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In combination we benchmarked the error-prone polymerase&nbsp;I by working out all necessary information to use this BioBrick in further directed evolution applications.
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</div>
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Revision as of 10:56, 19 October 2016

Best Basic Part Error-prone polymerase I as best in vivo mutator  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 in vivo mutagenesis capabilities of an error-prone polymerase I <a href=http://parts.igem.org/wiki/Part:BBa_K2082106>BBa_K2082106</a>. 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 in vivo mutagenesis approach circumvents the main problems restraining in vivo mutagenesis: the unintentional mutagenesis of essential proteins of a bacteria. 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 in vivo. Characterization 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>). 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. 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>). In combination we benchmarked the error-prone polymerase I by working out all necessary information to use this BioBrick in further directed evolution applications.