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

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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  
 
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  
 
binding capabilities into the population we uses the <i>in vivo</i> mutagenesis capabilities of the error-prone polymerase&nbsp;I  
 
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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<a href="http://parts.igem.org/wiki/Part:BBa_K2082106">BBa_K2082106</a>.
 
<br>
 
<br>
 
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  
 
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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We characterized the error prone polymerase&nbsp;I by performing <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Mutation/Reversion">reversion assays</a>, thus quantifiying the mutation rate.  
 
We characterized the error prone polymerase&nbsp;I by performing <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Mutation/Reversion">reversion assays</a>, thus quantifiying the mutation rate.  
 
<figure class="figure">
 
<figure class="figure">
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  <img src="Pfad" class="figure-img">
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  <img src="https://static.igem.org/mediawiki/2016/f/f3/Bielefeld_CeBiTec_2016_10_15_mutation_PolRevI.png" class="figure-img">
 
  <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 to using wild type polymerase&nbsp;I (<a href="http://parts.igem.org/wiki/Part:BBa_K2082107">BBa_K2082107</a>).</b></figcaption>
 
  <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 to using wild type polymerase&nbsp;I (<a href="http://parts.igem.org/wiki/Part:BBa_K2082107">BBa_K2082107</a>).</b></figcaption>
 
</figure>
 
</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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Furthermore we analyzed error-prone polymerase&nbsp;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.  
 
<figure class="figure">
 
<figure class="figure">
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  <img src="Pfad" class="figure-img">
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  <img src="https://static.igem.org/mediawiki/2016/3/34/Bielefeld_CeBiTec_2016_10_19_mutation_Pol_mutation.png" 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(<a href="http://parts.igem.org/wiki/Part:BBa_K2082107">BBa_K2082107</a>).</b></figcaption>
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  <figcaption class="figure-caption"><b>Figure 2: Mutagensis rate 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>) as determined by <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Mutation/Sequencing">high-throughput sequencing</a>.</b></figcaption>
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</figure>
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<figure class="figure">
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  <img src="https://static.igem.org/mediawiki/2016/3/34/Bielefeld_CeBiTec_2016_10_19_mutation_Pol_mutation.png" class="figure-img">
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  <figcaption class="figure-caption"><b>Figure 3: 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>).</b></figcaption>
 
</figure>
 
</figure>
 
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.
 
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.

Revision as of 20:42, 19 October 2016



Best Basic Part

like Lego they said

Error Prone Polymerase I - BBa_K2082106

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 the error-prone polymerase I BBa_K2082106.
This enzymes replace the normal polymerase I under certain conditions (further information) 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 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.