Our project design constitutes a synthetic design, which involves the insertion of modified PETase and MHETase into E.Coli, and an engineering design, which plans to place our bacteria into a Ram pump that is to be put into the secondary or aeration stage in the wastewater treatment plant.
I. Synthetic Design: E.coli containing modified Petase
We took the sequence of the original PETase found in Yoshida, et al., 2016 and changed two amino acid sequences at two plastic-binding sites S238F/W159H discovered in an article by Austin, et al., 2018.
We used Gibson assembly to build 3 DNA circuits that contain: (1) Wild-type (Contol) PETase, (2) Modified PETase, (3) Modified PETase and MHETase, which are then transformed into our E. coli cells. We utilized AmilCP reporter gene in our DNA to stain cells that are successfully transformed, "blue".
To test the enzyme performance of the successfully transformed E. coli, we inoculated these microbes with media in the presence of small plastic PET bottle shreds.
To observe the efficiency of the these enzyme performance, we weighed the small plastic particles before and after inoculation with the E. coli that are expected to express PETase that would degrade the PET plastic particles. We also planned to utilize the Scanning electron microscopy for a more accurate observation of the plastic particles’ surfaces that might have been degraded or grazed by our PETase enzymes.
The plastic particles exposed to the (1) Wild-type PETase and those exposed to the (2) Modified PETase are analyzed in terms of their reduction in mass to compare the rate of degradation between our wild-type PETase and our modified PETase that is expected to have a higher PET structure binding and degrading rate. Meanwhile, the plastic particles in the (3) Modified PETase and MHETase allow us to observe the efficiency of the MHETase.
II. Engineering Design: Implementation to Wastewater Treatment Plants
The wastewater treatment plant filtration system could be either a two or three step process. The first stage removes large waste particles for the second stage that biodegrades organic materials and remove toxic microorganism, and the third stage filters out fine particulates. We chose the second step or the aeration tank to be the optimal stage where our microbes can function, as it is the stage that deals with filtration on a molecular or microbial level.
(Center for Sustainable Systems, 2016)
After having discussed with professors and different experts at the water treatment facilities that we visited, we decided not to place our engineered microbes directly into the wastewater due to safety concerns. Instead, it would be better to use an external extension, containing our active microbes, that wastewater can pass through for microplastic particles to be degraded by the E. coli. Then the microplastic-free wastewater can flow back out into the wastewater tanks.
We chose the “Ram pump” as the most fitting extension for this design because it:
Austin, H., Allen, M., Donohoe, B., Rorrer, N., Kearns, F., Silveira, R., . . . Beckham, G. (2018). Characterization and engineering of a plastic-degrading aromatic polyesterase. Proceedings of the National Academy of Sciences of the United States of America, 115(19), 4357. doi:10.1073/pnas.1718804115
Center for Sustainable Systems, University of Michigan. 2016. “U.S. Wastewater Treatment Factsheet.” Pub. No. CSS04-14.
Bornscheuer, U. (2016). Feeding on plastic. Science, 351(6278), 1154-1155. doi:10.1126/science.aaf2853
Yoshida, S., Hiraga, K., Takehana, T., Taniguchi, I., Yamaji, H., Maeda, Y., . . . Oda, K. (2016). A bacterium that degrades and assimilates poly(ethylene terephthalate). Science, 351(6278), 1196-1199. doi:10.1126/science.aad6359