Part:BBa_K4407014
Plac-dps-aceE，Coexpression of dps and aceE in response to lactose
This part is responsible for co-expressing dps and aceE proteins in response to lactose under the control of a Plac promotor and a terminator. It is composed of BBa_K4119006 (Plac), BBa_K4407001 (dps), BBa_K4407002 (aceE), and BBa_K3585002 (terminator). dps protects the bacteria from oxidative damage. It protects DNA against oxidative damage by binding with Fe2+ to prevent it from reacting with H2O2 (Fenton’s reaction). 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 construct facultative anaerobic Clostridium tyrobutyricum (C. tyrobutyricum). This device combines the function of the two proteins, enhancing viability in aerobic environment by protecting DNA and substituting the main metabolic enzyme PFOR which is inactivated under aerobic condition with PDH in the presence of oxygen.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 368
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 368
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 368
Illegal BglII site found at 428
Illegal BglII site found at 2942
Illegal BglII site found at 3350
Illegal BamHI site found at 1902 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 368
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 368
Illegal NgoMIV site found at 3972
Illegal AgeI site found at 2683 - 1000COMPATIBLE WITH RFC[1000]
Results
The Plac promoter fragment and linearized pMTL-dps-aceE vector were ligated by the Gibson assembly method. 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-Plac-dps-aceE by agarose gel electrophoresis and gene sequencing (Figure 1). The promotor vector was also confirmed to be Plac promoter with the same method, as shown in figure 2.
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