Part:BBa_K4407011
Pthl-aceE，Expression of aceE with Pthl promoter
This part is responsible for expressing the aceE protein under the control of a Plac promotor, a ribosome binding site (RBS) and a terminator. It is composed of BBa_K3443002 (Pthl), BBa_K103015 (RBS), BBa_K4407002 (aceE), and BBa_K3585002 (terminator). aceE protein is a component of the PDH complex and catalyzes the conversion of pyruvate to acetyl-CoA and CO2. It can function well in aerobic environment. In our project, we used this device to provide a new metabolic pathway for C. tyrobutyricum in aerobic environment.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1684
Illegal BglII site found at 2092
Illegal BamHI site found at 644 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2714
Illegal AgeI site found at 1425 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1956
Illegal SapI.rc site found at 2076
Results
1)Plasmid construction: pMTL-Pthl-aceE
Using the plasmid pMTL-Pthl-BS2 provided by NJTech_China team as the template, the linearized vector pMTL-Pthl was amplified. The fragment of aceE gene was amplified from the genome of Escherichia coli DH5α. Gibson assembly method was used to connect aceE gene fragment with the linearized pMTL-Pthl vector. Then, we ran a colony PCR for the transformed bacterial colony (E .coli JM109), inoculated the positive bacterial colonies, and extracted the plasmids, which were then confirmed to be pMTL-Pthl-aceE by gel electrophoresis and gene sequencing.
2) C. tyrobutyricum transformed with pMTL-Pthl-aceE
After the recombinant plasmid pMTL-Pthl-aceE was conjugated into C. tyrobutyricum using E. coli CA434 as the donor strain. The expression of aceE protein in the recombinant bacteria was confirmed by SDS-PAGE.
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