Team:Bielefeld-CeBiTec/Results/Pathway

From 2014.igem.org

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   <h4>Coding sequences</h4>
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<p>We started with the <b>pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></b> construct which is the part <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1465302" target="_blank"> K1465302</a>. Therefor we used the CDS of the NCTU team Formosa 2011/2012 BioBricks <i>alsS</i> (<a href="http://parts.igem.org/Part:BBa_K539627" target="_blank">BBa_K539627</a>), <i>ilvC</i> (<a href="http://parts.igem.org/Part:BBa_K539621" target="_blank">BBa_K539621</a>), <i>
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<p>We started with the <b>pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></b> construct which is the part <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1465302" target="_blank"> K1465302</a>. Therefore we used the CDS of the NCTU team Formosa 2011/2012 BioBricks <i>alsS</i> (<a href="http://parts.igem.org/Part:BBa_K539627" target="_blank">BBa_K539627</a>), <i>ilvC</i> (<a href="http://parts.igem.org/Part:BBa_K539621" target="_blank">BBa_K539621</a>), <i>
ilvD</i> (<a href="http://parts.igem.org/Part:BBa_K539626" target="_blank">BBa_K539626</a>) and <i>kivD</i> (<a href="http://parts.igem.org/Part:BBa_K539742" target="_blank">BBa_K539742</a>)</li>. We used the various parts of the Parts Kit to combine them with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a>. For this we amplified the various CDS and combined them with a RBS ((<a href="http://parts.igem.org/Part:BBa_B0034" target="_blank">BBa_B0034</a>) by a PCR. <br>In the beginning it didn’t worked with pSB1C3 so we used pSB1K3. <br> When we had pSB1K3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i>, we wanted to reclone it in pSB1C3 and combine it with a promoter. This we wanted to do with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a>. Thereby we identified an illegal restriction side in the end of <i>alsS</i> because of the combination with the following RBS. This we removed by PCR amplification and Gibson with new primer .  
ilvD</i> (<a href="http://parts.igem.org/Part:BBa_K539626" target="_blank">BBa_K539626</a>) and <i>kivD</i> (<a href="http://parts.igem.org/Part:BBa_K539742" target="_blank">BBa_K539742</a>)</li>. We used the various parts of the Parts Kit to combine them with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a>. For this we amplified the various CDS and combined them with a RBS ((<a href="http://parts.igem.org/Part:BBa_B0034" target="_blank">BBa_B0034</a>) by a PCR. <br>In the beginning it didn’t worked with pSB1C3 so we used pSB1K3. <br> When we had pSB1K3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i>, we wanted to reclone it in pSB1C3 and combine it with a promoter. This we wanted to do with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a>. Thereby we identified an illegal restriction side in the end of <i>alsS</i> because of the combination with the following RBS. This we removed by PCR amplification and Gibson with new primer .  
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Revision as of 10:18, 17 October 2014


Module III - Isobutanol production

Cloning

Coding sequences

We started with the pSB1C3_alsS_ilvC_ilvD_kivD construct which is the part K1465302. Therefore we used the CDS of the NCTU team Formosa 2011/2012 BioBricks alsS (BBa_K539627), ilvC (BBa_K539621), ilvD (BBa_K539626) and kivD (BBa_K539742). We used the various parts of the Parts Kit to combine them with Gibson Assembly. For this we amplified the various CDS and combined them with a RBS ((BBa_B0034) by a PCR.
In the beginning it didn’t worked with pSB1C3 so we used pSB1K3.
When we had pSB1K3_alsS_ilvC_ilvD_kivD, we wanted to reclone it in pSB1C3 and combine it with a promoter. This we wanted to do with BioBrick Assembly. Thereby we identified an illegal restriction side in the end of alsS because of the combination with the following RBS. This we removed by PCR amplification and Gibson with new primer .

Constructs with promoter

Expression

Cultivation

Production

Conclusion

References
  • Atsumi S, Wu TY, Eckl EM, Hawkins SD, Buelter T, Liao JC. 2010. Engineering the isobutanol biosynthetic pathway in Escherichia coli by comparison three aldehyde reductase/alcohol dehydrogenase genes. In: Appl. Microbiol. Biotechnol 85, 651–657