Nanodrop Kinetic Assay Results
Due to our complications in our purification of wild-type PETase, we decided to begin our testing on our crude unpurified soluble sample. From our literature review, we decided to use a NanoDrop method for evaluating the enzymatic degradation kinetics of PET films as described by Zhong-Johnson et al [1]. Specifically, we utilized a “bulk absorbance method” in which all degradation products contribute to the absorbance.
We initially took an assay of our crude soluble sample as we were still in the process of conducting FPLC and purifying the wild type PETase. We used a Thermomixer set to 200 rpm and incubated our samples at a temperature of 40 °C, which we chose based on a publication reviewing the current state of PET degrading enzymes [2]. From this assay, we were able to determine the ideal quantities of protein to use for testing the enzymatic degradation kinetics of wild-type PETase. Additionally, after leaving these samples at room temperature overnight, we found that the samples with higher concentrations of protein (≥750 μl) had noticeably higher degradation within the tube (see Crude PET Degradation Visual Assay).
Despite some inconsistencies in our results due to readings taken by the NanoDrop, we were able to observe an overall upward trend in absorbance at 260 nm for our protein sample using the “bulk absorbance method” as depicted by Figures 1 and 2, which suggests that an increase in PET degradation products is occurring over time. However, as shown in Figure 2, the 900 μl protein sample displayed an inconsistent trend in absorbance at 260 nm, which is likely due to the small quantity of reaction buffer (100 μl) in the tube. Moreover, the purified wild-type PETase produced a more consistent trend with the results found by Zhong-Johnson et al., in which the absorbance at 260 nm fell within the range of 0.0 to 1.0.
While we have only included our wild-type PETase results above, we are currently in the process of testing our mutated PETase sequences and intend to test them via the NanoDrop Assay in the near future.
Reference:
- Zhong-Johnson, En Ze, et al. “An Absorbance Method for Analysis of Enzymatic Degradation Kinetics of Poly(Ethylene Terephthalate) Films.” Scientific Reports, vol. 11, no. 1, 2021, https://doi.org/10.1038/s41598-020-79031-5.
- Kawai, Fusako. “The Current State of Research on Pet Hydrolyzing Enzymes Available for Biorecycling.” Catalysts, vol. 11, no. 2, 2021, p. 206., https://doi.org/10.3390/catal11020206.
SDS-PAGE
Wet Lab Data Induction Troubleshooting Assay
In this assay we had two tubes of BL21 E. coli cells with our plasmid of interest in them. Both tubes were grown in LB overnight; however, the tube to the left was induced with IPTG beforehand and the tube to the right was not. The uninduced tube showed a higher visual concentration of cells and led us to conclude that that induction of the PETase protein hindered the growth of the BL21.
Wet Lab Data Crude PET Degradation Visual Assay
In this assay we set up microcentrifuge tubes with various concentrations of crude C41 cell extract with PETase, ¼ hole punch of pet film, and reaction buffer. We let the tubes sit overnight at room temperature and observed the results the following day.