Through wet lab experimentation, we hoped to test the degradation efficiency of the mutated PETase sequences generated by the dry lab team. We began by transforming wild-type PETase into BL21 competent cells and determined if it was successful by looking at colony growth and color changes in overnight cultures. During this process, we researched several past iGEM team’s protocols and noticed that BL21 cells were the most popular choice for protein expression. After a few weeks of experimentation, we noticed that while we were able to successfully transform our PETase sequence into this cell line, when we ran SDS-Page to verify we had our protein of interest, we did not see any bands. After several weeks of troubleshooting data and literature review, we discovered that the BL21 cell line is actually not ideal for expressing toxic proteins such as PETase. We instead began using C41 cells which were very promising for expressing PETase as seen in our SDS-Page images. For future iGEM teams who will work with PETase, this data can help them in the protein purification purification process by potentially saving them from troubleshooting.
Unlike prior iGEM teams, we chose to test measure the enzymatic activity of PETase using a “bulk absorbance” method via a NanoDrop. The “bulk absorbance” method outlined by Zhong-Johnson et al. suggests that all PET degradation products contribute to the absorbanceZhong-Johnson et al., 2021. The only equipment required for this assay is a NanoDrop which could be helpful for future iGEM teams that may not have access to other PET degradation testing equipment such as high performance liquid chromatography (HPLC). Overall, the primary benefits of this absorbance method is that it requires minimal time and resources and is cost-effective.