Team:ASIJ Tokyo/Results

Results

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BIOBRICK CONSTRUCTION

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The goal of our project was to construct a biobrick capable of plastic degradation. We created three biobrick constructs. Each of these constructs contained PETase, ChloroR, osmY, and an Anderson Promoter. The principal difference between these three constructs was the strength of the Anderson promoter incorporated in the construct. Depending on their predicted ability to promote the secretion of the enzyme PETase by the E. coli bacterium, these Anderson promoters were labelled as weak, moderately-strong, or strong. Our team was successful in synthesizing a biobrick, as demonstrated by the expression of the Chloro gene when the construct was transformed into E. Coli cultures. Successful growth signified integration of the PETase gene into the plasmid. However, our experiment was ineffectual in demonstrating a substantial degradation of PET plastic. The differences in mass measurements of PET after allowing the E. coli bacterium to secrete PETase into the system were insignificant. We believe human errors, such as the failure to replace the LB broth suspension at appropriate time intervals to maintain the E. Coli in log phase, were a significant cause of experimental inaccuracies. In addition, time constraints prevented further data collection. We are unsure regarding whether our PETase construct was successfully secreted into the system. One way we could test this would be to tag PETase with c-myc, and perform a Western Blot test.

Table 1: Change in Average Mass of PET Film (g) per Promoter

	Day 0	Day 7	Day 13
Strong	0.05175	0.05175	0.05175
Moderately-Strong	0.071	0.071	0.07125
Weak	0.06675	0.06825	0.06725

Graph 1: Average PET Mass Sample over Time

Future Works

Although we were successful in creating a PETase biobrick, we have yet to collect enough data to find an ideal promoter for the construct. As such, the future proceedings of this project would be in gathering a greater amount of data at more frequent intervals in order to identify an ideal promoter.
In addition, we hope to insert an ampicillin resistance gene in our construct, and test for E. coli growth on an ampicillin plate. Testing for another selection factor would act as a secondary confirmation test for the successful construction of a PETase biobrick.
Determining the Andersen promoter with the highest potential to degrade PET plastic would be the first step regarding real-life applications of our lab. The later steps of the PET degradation process, such as the breakdown of Terephthalic Acid and Ethylene Glycol, may be topics of further investigation.