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Team:Bielefeld-CeBiTec/Results/AdhA
From 2014.igem.org
Module III - Isobutanol production
Alcohol dehydrogenase (AdhA)
We found out, that the AdhA from Lactococcus lactis is the best alcoholdehydrogenase in literature (Atsumi et al., 2010). For that reason we want to increase the production of isobutanol by putting the adhA gene behind our producing pathway. The adhA from L. Lactis was not available as a BioBrick so we designed a new part which contains the coding sequence of the adhA gene from L. Lactis (BBa_K1465301). You can find our approach in the following sections.
Cloning
For the isolation of the adhA gene from L. lactis we first had to isolate the DNA of it. Afterwards we tried to amplify the adhA gene with the primer rev_pSB1C3_adhA and fw_adhA_pSB1C3. Via the primer the ahdA genes was combined with the RBS BBa_B0034. Together with the amplified backbone we performed a Gibson Assembly. All verifications showed that our cloning was successful but our backbone was pSB1K3 and not pSB1C3. For that reason we performed a successful recloning in pSB1C3 and our part BBa_K1465301 was ready for usage.
For the characterization of our BioBrick BBa_K1465301 we performed a BioBrick Suffix Assembly with the pSB1A2_T7
(BBa_ and our part. We wanted the new created part in pSB1C3 too, so a successful recloning in pSB1C3 was done. The new part can now be found as the BioBrick BBa_K1465304.
Additionally we performed a BioBrick Prefix Assembly with the pSB1C3_ptac
(BBa_ and our part. The successfully created part can now also be found as the BioBrick BBa_K1465305
Expression
Cultivation
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
