Team:ASIJ Tokyo/Results

Results

Data Table

Mass of PET Plastic(g)	9/10/16	9/17/16	9/23/16
Weakest Promoter, Living E. Coli 1	0.078	0.076	0.074
Weakest Promoter, Living E. Coli 2	0.060	0.059	0.060
Weakest Promoter, Living E. Coli 3	0.060	0.059	0.058
Weakest Promoter, Living E. Coli 4	0.075	0.075	0.075
Moderately Strong Promoter, Living E. Coli 1	0.076	0.076	0.075
Moderately Strong Promoter, Living E. Coli 2	0.065	0.066	0.066
Moderately Strong Promoter, Living E. Coli 3	0.068	0.069	0.069
Moderately Strong Promoter, Living E. Coli 4	0.075	0.074	0.074
Strongest Promoter, Living E. Coli 1	0.067	0.067	0.067
Strongest Promoter, Living E. Coli 2	0.049	0.049	0.048
Strongest Promoter, Living E. Coli 3	0.05	0.051	0.051
Strongest Promoter, Living E. Coli 4	0.041	0.04	0.041
Control, Living E. Coli 1	0.054	0.089	0.088
Control, Living E. Coli 2	0.093	0.071	0.071
Control, Living E. Coli 3	0.082	0.081	0.082
Control, Living E. Coli 4	0.079	0.081	0.080
Weakest Promoter, Enzyme Solution (Dead E.Coli) 1	0.099	0.1	—
Weakest Promoter, Enzyme Solution (Dead E.Coli) 2	0.076	0.075	—
Moderately Strong Promoter, Enzyme Solution (Dead E.Coli) 1	0.057	0.057	—
Moderately Strong Promoter, Enzyme Solution (Dead E.Coli) 2	0.067	0.067	—
Strongest Promoter, Enzyme Solution (Dead E.Coli) 1	0.048	0.049	—
Strongest Promoter, Enzyme Solution (Dead E.Coli) 2	0.058	0.058	—

Graphs

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Analysis/Conclusion and Future Works

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

If the team was to carry out this experiment again, perhaps we would gain more significant evidence than we did. We would check for successful expression of our PETase construct, better tend our E. coli, and gather more data over a longer period of time. This data would then help us decide which promoter is best used to optimize the degradation of PET plastic and know that a PETase construct successfully being secreted by the bacteria. Such a conclusion and results could help us determine the next few steps to take in helping our Earth. Of course, we would still have to determine the implications and consequences of using our bioengineered e. Coli and PETase (such as whether or not there would be any acidic waste products, etc.), but knowing which promoter to use to degrade PET plastic more efficiently would be the first step towards applying our lab to the real world.