Team:ASIJ Tokyo/Results

Results

Overview

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 Andersen Promoter. The principal difference between three these constructs was the strength of the Andersen promoter incorporated in the construct. Depending on their predicted ability to promote the secretion of the enzyme PETase by the E. coli bacterium, these Andersen promoters were labelled as weak, moderately-strong, or strong. 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 our failure to feed the E. colicells at appropriate intervals, were a main 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.

Analysis/Conclusion

After analyzing our data, we have concluded that our experiment was ineffectual in demonstrating a substantial degradation of PET plastic with the Andersen promoters we chose. However, because we tested the strength of our promoters prior to the degradation phase of our experiment, we have concluded that our promoters were not the cause of the lack in measurable PET plastic degradation. We believe that our lack of conclusive data stems from several human errors. During our experiment, we failed to provide the e. Coli cells with sufficient nutrition at the appropriate times. Moreover, due to time constraints, we did not assay for a secreted PETase in the growth medium. Therefore, we are not sure if our PETase construct was successfully secreted. We would have assayed this by using a c-Myc tag on our PETase construct and by performing a Western Blot. Finally, the large differences in PET film mass for our control group suggest two possible errors: either that our PET film was inexplicably polymerized in one of the groups and degraded in the other, or that there was an error in measurement. Overall, our data gives us little meaningful information about PET degradation by our constructs due to a lack of significant change and lots of haphazard experimental practice. If our team had more time to complete our experiment, we would consider our previous errors more carefully. In particular, we would establish set time periods during which we need to change the growth media—LB broth, in our case—for each e.Coli colony. We would also make sure to replenish the nutrient stock of the e.Coli within a three day time period, rather than our previous five to six day period. Finally, we would allow the PET plastic degradation phase to run for longer than 13 days (our current established time allotted for degradation) in order to produce more accurate results.

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.