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Team:Bielefeld-CeBiTec/Results/Pathway
From 2014.igem.org
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<h4>Coding sequences</h4> | <h4>Coding sequences</h4> | ||
<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> | <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 parts of the Parts Kit to combine them | + | 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 parts of the Parts Kit to combine them by <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 did not 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 (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvC_alsS-new" target="_blank">rv_ilvC_alsS-new</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC-new" target="_blank">fw_alsS_ilvC-new</a>). |
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Revision as of 10:31, 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 parts of the Parts Kit to combine them by Gibson Assembly. For this we amplified the various CDS and combined them with a RBS (BBa_B0034) by a PCR.
In the beginning it did not 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 (rv_ilvC_alsS-new, fw_alsS_ilvC-new).
Constructs with promoter
Expression
Cultivation
Production
Conclusion
References
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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
