Part:BBa_K4881028

fucO and aldA with AmilGFP Reporter

PgyrA promoter is a constitutive promoter, which according to the paper “Improving ethylene glycol utilization in Escherichia coli fermentation”, worked better than an inducible promoter (Panda et al., 2021). It is followed by a ribosome binding site, then gene fucO, a Mutase ribosome binding site, and then the aldA gene. fucO encodes for the production of lactaldehyde reductase, which catalyzes the breakdown of ethylene glycol (EG) into glycolaldehyde. aldA encodes for the production of aldehyde dehydrogenase A, which catalyzes the breakdown of glycolaldehyde into glycolate. The product, glycolate, can enter the glycolysis pathway to serve as a source of energy for the cell. In between the fucO and aldA genes, there is a Mutase RBS to enhance enzyme production. Finally, before the double terminator, we decided to include a reporter gene that would express yellow chromoprotein as a visual indicator that the bacteria took the plasmid.

Usage and Biology

This part was initially used for the project PlastE.co from the team FDR-HB-Peru in the season 2023. This device was the insert of a kanamycin-resistant plasmid that was transformed into competent E. coli cells. This device was our team's second step in turning PET plastic into ethanol. The lactaldehyde reductase produced by the gene fucO and aldehyde dehydrogenase A from the gene aldA will help to break down ethylene glycol into glycolate. That way, it can naturally enter the glycolate degradation pathway to become 2-phospho-D-glycerate, and 2-phospho-D-glycerate will enter glycolysis to turn into pyruvate. Finally, pyruvate will have the possibility to enter fermentation in the right anaerobic conditions.

Transformation

In the PET bioassay carried out by the FDR-HB-Peru team, we refer to the plasmid with part BBa K4881028 as Construct 2, since it was the second step of our project. We were able to obtain transformants that contained this device in a kanamycin-resistant plasmid backbone. However, as can be seen in the image of the liquid culture of transformed cells, the cells were not expressing the reporter gene of the yellow chromoprotein.

Conclusion

In conclusion, improving the performance of this device requires a strategic adjustment in the position of the yellow chromoprotein reporter gene. The expression of proteins lactaldehyde reductase and aldehyde dehydrogenase A still has to be tested.