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Team:Bielefeld-CeBiTec/Results/Pathway
From 2014.igem.org
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 by 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). In this approach we were able to amplify pSB1C3 as backbone, so no recloning was necessary.
Additionally we wanted to combine our part K1465302 with the adhA from Lactococcus lactis. This alcoholdehydrogenase was identified as the best enzyme for the last step in the 2-keto-acid pathway (Atsumi2008, Atsumi2010). This pathway is responsible for the isobutanol production.
We wanted to add the adhA to pSB1C3_alsS_ilvC_ilvD_kivD by BioBricck Assembly, but adhA was not available as BioBrick, so we designed it. How we did this you can read in the Results of adhA. We did a BioBrick Sufffix Insertion and the result was the pSB1C3_alsS_ilvC_ilvD_kivD_adhA construct which is the part K1465303.
Constructs with promoter
To characterize our BioBricks, we wanted to add promoter to them. We chose the ptac promoter (BBa_K731500) for characterizations during cultivations and the stronger T7 promoter (BBa_I719005) for SDS pages.
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
