Part:BBa_K4881029
AdhE with AmilCP reporter
The construct starts with a T7 promoter, followed by a ribosome binding site and the adhE gene. adhE encodes for fused acetaldehyde-CoAdehydrogenase and catalyzes the reaction of acetyl-CoA to ethanol in anaerobic fermentation conditions. Lastly, it has AmilCP as a reporter gene that will encode for blue chromoprotein as a visual indicator the bacteria took the plasmid. We made this construct based on the preliminary research as a way to enhance the fermentation process.
Usage and Biology
According to the paper “Metabolic engineering of Escherichia coli for production of mixed-acid fermentation end products”, the main genes involved in the fermentation pathway of E. coli are alcohol dehydrogenase, pyruvate formate lyase, and pyruvate kinase. The image below depicts a diagram of this pathway. The AdhE enzyme sequentially reduces acetyl-CoA to acetaldehyde and then to ethanol under anaerobic conditions. This gene plays a vital role in the fermentation pathway, which is why our team developed a device to express more of this enzyme in E. coli and maximize the amount of ethanol produced in our project.
Transformation
In the project carried out by the FDR-HB-Peru team, we refer to the plasmid with BBa part K4881029 as Construct 3, since it was the third and final step to convert PET into ethanol. Throughout our experimentation, we were able to obtain transformants containing this device on a kanamycin-resistant plasmid backbone. However, as can be seen in the image of the Petri dishes labeled C3 with the transformed cells, the colonies did not express the blue chromoprotein reporter gene, and the same thing happened with liquid culture. A noteworthy finding during our analysis of the transformed cells was the low optical density in the liquid culture and minimal colony counts in the Petri dishes. One plausible hypothesis we are exploring is the possibility that ethanol production could be influencing cell growth and expression. However, we still have to perform experiments and testing in order to assess this properly.
Conclusion
In conclusion, improving the performance of this device requires a strategic adjustment in the position of the blue chromoprotein reporter gene. The enhancement of the fermentation process and ethanol production caused by the overexpression of AdhE still remains to be tested.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1
Illegal XbaI site found at 3575
Illegal PstI site found at 203
Illegal PstI site found at 538
Illegal PstI site found at 2104
Illegal PstI site found at 2693
Illegal PstI site found at 3590 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1
Illegal PstI site found at 203
Illegal PstI site found at 538
Illegal PstI site found at 2104
Illegal PstI site found at 2693
Illegal PstI site found at 3590
Illegal NotI site found at 7
Illegal NotI site found at 3583 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1
Illegal BglII site found at 570 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1
Illegal XbaI site found at 3575
Illegal PstI site found at 203
Illegal PstI site found at 538
Illegal PstI site found at 2104
Illegal PstI site found at 2693
Illegal PstI site found at 3590 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1
Illegal XbaI site found at 3575
Illegal PstI site found at 203
Illegal PstI site found at 538
Illegal PstI site found at 2104
Illegal PstI site found at 2693
Illegal PstI site found at 3590 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1777
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