BBa K4881027 - Construct 1

The construct starts with a T7 promoter, followed by a ribosome binding site and MHETase linked to PETase. PETase encodes for the PETase enzyme, which catalyzes the breakdown of polyethylene terephthalate (PET) to monomeric mono-2-hydroxyethyl terephthalate (MHET). MHETase encodes for the production of the MHETase enzyme, which catalyzes the breakdown of MHET to ethylene glycol and terephthalic acid. These genes are joined together by a 12 amino acid linker containing glycine and serine. This combination seemed to obtain better results in expression according to the paper “Characterization and engineering of a two-enzyme system for plastics depolymerization”, where it was compared with an 8 amino acid linker and a 20 amino acid linker. Finally, before the double terminator, our team decided to add a reporter gene that would express pink chromoprotein as a visual indicator that the bacteria took the plasmid.

Usage and Biology

This device was the insert of an ampicillin-resistant plasmid that was transformed into competent E. coli cells. This device was our team's first step for turning PET plastic into ethanol, as both of these enzymes will work together to break down PET into ethylene glycol and terephthalic acid.

Transformation

Through our transformation we were able to obtain transformants containing this device on an ampicillin-resistant plasmid backbone. Demonstrating that the plasmid can be taken up by E. coli cells. However, as can be seen in the image of the liquid culture of transformed cells, the cells did not express the pink chromoprotein reporter gene.

Bioassay Results

For our 72-hour bioassay setup, we used 15 autoclaved Falcon tubes of 50mL, 250mL autoclaved liquid LB, 250mL autoclaved liquid LB + Amp, 250mL autoclaved liquid LB + Kan, 250mL autoclaved liquid LB + Amp+Kan, and 3 mL of liquid culture of each type of bacteria to use. The plastic samples came from a PET bottle that was pierced with an office hole puncher. Afterward, the PET circles were weighted in groups of 6 to be 0.056 ± 0.002 g. Then, they were treated in 70% ethanol for 15 minutes and washed with distilled water.

The liquid culture contained 500µL of overnight culture to 5mL of media placed on 50mL Falcon tubes for aeration. After that, we placed 6 PET circles inside the tubes. Then, 20 hours after the bioassay tubes were closed, we added 5mL of media. Finally, they were left until the end of the 72-hour period.

Results

In conclusion, improving the performance of this device requires a strategic adjustment in the position of the pink chromoprotein reporter gene. On the other hand, the results of our bioassays reveal a promising aspect: the plastic degradation of 5 to 7% in the tubes containing the E. coli cells with Construct 1. To obtain definitive information, it is imperative to replicate the bioassay using plastic samples with greater mass and surface area, supplemented with larger amounts of media and liquid cultures, all housed in expanded containers. Only through this comprehensive approach will we be able to reveal the true extent of plastic biodegradation facilitated by the device.

References:

Knott, B. C., Erickson, E., Allen, M. D., Gado, J. E., Graham, R., Kearns, F. L., Pardo, I., Topuzlu, E., Anderson, J. J., Austin, H. P., Dominick, G., Johnson, C. W., Rorrer, N. A., Szostkiewicz, C. J., Copié, V., Payne, C. M., Woodcock, H. L., Donohoe, B. S., Beckham, G. T., & McGeehan, J. (2020). Characterization and engineering of a two-enzyme system for plastics depolymerization. Proceedings of the National Academy of Sciences of the United States of America, 117(41), 25476–25485. https://doi.org/10.1073/pnas.2006753117

BBa K4881028 - Construct 2

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 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

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.

References:

Panda, S., Fung, V. Y. K., Zhou, J., Hong, L., & Zhou, K. (2021). Improving ethylene glycol utilization in Escherichia coli fermentation. Biochemical Engineering Journal, 168, 107957. https://doi.org/10.1016/j.bej.2021107957

BBa K4881029 - Construct 3

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 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. 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.

Usage and Biology

Transformation

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.

References:

Förster, A. H., & Gescher, J. (2014). Metabolic Engineering of Escherichia coli for Production of Mixed-Acid Fermentation End Products. Frontiers in Bioengineering and Biotechnology, 2. https://doi.org/10.3389/fbioe.2014.00016