Team:Tsinghua-E/Part1

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==[[File:4_1s.png‎|68px|left]] '''Part 1: THU-E Mutation Part''' ==
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<h2>[[File:4_1s.png‎|68px|left]] '''Part 1: THU-E Mutation Part''' </h2>
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A plasmid  used for the construction of high-diversity library in vivo ingenome level. In  this vector, highly error-prone <em>dnaQ</em> mutant, <em>mutD</em><html><a href="#_ENREF_1" title="Lou, 2012 #499">1</a></html>was cloned downstream  of <em>araBAD</em> promoter to control the  mutation rate of the target genome by the concentration of <em>araBAD</em> promoter’s inducer, L-arabinose, in a strict manner.<em>E. Coli</em> JM109 carrying different vectors  of pBAD_B0030-<em>mutD</em>-<em>sfGFP</em>, pBAD_B0032-<em>mutD</em>-<em>sfGFP</em> and pBAD_SDA_RBS-<em>mutD-sfGFP</em>(this RBS sequence was derived  from the RBS sequence upstream of sfGFP in original AraC_pBAD_CI_OR222-sfGFP  vector<html><a href="#_ENREF_2" title="Lou, 2012 #499">2</a></html>)were  constructed. By detecting the induced fluorescence intensity, we found that pBAD_B0030-<em>mutD</em>-<em>sfGFP</em>,  andpBAD_SDA_RBS-<em>mutD- sfGFP</em>have  relatively higher <em>mutD</em> expression. The increaseof mutation rate induced  by our mutation part was measured by quantifying the reversion of rifampinresistance  caused by mutation in genome.pBAD_SDA_RBS-<em>mutD-  sfGFP</em>could increase the genome mutation rate up to 10 times compared with  negative control with 1g/L induction concentration of L-arabinose. <br />
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A plasmid  used for the construction of high-diversity library in vivo ingenome level. In  this vector, highly error-prone <em>dnaQ</em> mutant, <em>mutD</em><html><a href="#_ENREF_1" title="Lou, 2012 #499">[1]</a></html>was cloned downstream  of <em>araBAD</em> promoter to control the  mutation rate of the target genome by the concentration of <em>araBAD</em> promoter’s inducer, L-arabinose, in a strict manner.<em>E. Coli</em> JM109 carrying different vectors  of pBAD_B0030-<em>mutD</em>-<em>sfGFP</em>, pBAD_B0032-<em>mutD</em>-<em>sfGFP</em> and pBAD_SDA_RBS-<em>mutD-sfGFP</em>(this RBS sequence was derived  from the RBS sequence upstream of sfGFP in original AraC_pBAD_CI_OR222-sfGFP  vector<html><a href="#_ENREF_2" title="Lou, 2012 #499">[2]</a></html>)were  constructed. By detecting the induced fluorescence intensity, we found that pBAD_B0030-<em>mutD</em>-<em>sfGFP</em>,  andpBAD_SDA_RBS-<em>mutD- sfGFP</em>have  relatively higher <em>mutD</em> expression. The increaseof mutation rate induced  by our mutation part was measured by quantifying the reversion of rifampinresistance  caused by mutation in genome.pBAD_SDA_RBS-<em>mutD-  sfGFP</em>could increase the genome mutation rate up to 10 times compared with  negative control with 1g/L induction concentration of L-arabinose. <br />
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<strong>1 Schaaper, R. M. MECHANISMS OF MUTAGENESIS IN THE ESCHERICHIA-COLI  MUTATOR MUTD5 - ROLE OF DNA MISMATCH REPAIR. <em>Proc. Natl. Acad. Sci. U. S. A.</em> 85, 8126-8130,doi:10.1073/pnas.85.21.8126  (1988).</strong><br />
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<p><font style="font-size: 12px">[1] Schaaper, R. M. Mechanisms of mutagenesis in the Escherichia-Coli mutator mutd5 - role of DNA mismatch repair. <em>Proc. Natl. Acad. Sci. U. S. A.</em> 85, 8126-8130,doi:10.1073/pnas.85.21.8126  (1988).</font></p>
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  <strong>2 Lou, C. B., Stanton, B., Chen, Y. J., Munsky, B. &amp;  Voigt, C. A. Ribozyme-based insulator parts buffer synthetic circuits from  genetic context. <em>Nature Biotechnology</em> 30, 1137-+, doi:10.1038/nbt.2401 (2012).</strong><strong> </strong></p>
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<p><font style="font-size: 12px">[2] Lou, C. B., Stanton, B., Chen, Y. J., Munsky, B. &amp;  Voigt, C. A. Ribozyme-based insulator parts buffer synthetic circuits from  genetic context. <em>Nature Biotechnology</em> 30, 1137-+, doi:10.1038/nbt.2401 (2012).</font></p>
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Latest revision as of 03:07, 28 September 2013

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4 1s.png
Part 1: THU-E Mutation Part

Plasmid1.png

A plasmid used for the construction of high-diversity library in vivo ingenome level. In this vector, highly error-prone dnaQ mutant, mutD[1]was cloned downstream of araBAD promoter to control the mutation rate of the target genome by the concentration of araBAD promoter’s inducer, L-arabinose, in a strict manner.E. Coli JM109 carrying different vectors of pBAD_B0030-mutD-sfGFP, pBAD_B0032-mutD-sfGFP and pBAD_SDA_RBS-mutD-sfGFP(this RBS sequence was derived from the RBS sequence upstream of sfGFP in original AraC_pBAD_CI_OR222-sfGFP vector[2])were constructed. By detecting the induced fluorescence intensity, we found that pBAD_B0030-mutD-sfGFP, andpBAD_SDA_RBS-mutD- sfGFPhave relatively higher mutD expression. The increaseof mutation rate induced by our mutation part was measured by quantifying the reversion of rifampinresistance caused by mutation in genome.pBAD_SDA_RBS-mutD- sfGFPcould increase the genome mutation rate up to 10 times compared with negative control with 1g/L induction concentration of L-arabinose.



Figure.1 Conception illustration of the working mechanism of mutD
Figure.2 rifampicin reversion mutants caused by mutD expression and the counts by agar plate



[1] Schaaper, R. M. Mechanisms of mutagenesis in the Escherichia-Coli mutator mutd5 - role of DNA mismatch repair. Proc. Natl. Acad. Sci. U. S. A. 85, 8126-8130,doi:10.1073/pnas.85.21.8126 (1988).

[2] Lou, C. B., Stanton, B., Chen, Y. J., Munsky, B. & Voigt, C. A. Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nature Biotechnology 30, 1137-+, doi:10.1038/nbt.2401 (2012).