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.
Our contribution to the original part, part BBa_K2010999, is the addition of the reporter pink chromoprotein and the addition of information on PET biodegradation that we obtained through a bioassay.
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