Nanodrop Kinetic Assay Results
Due to our complications in our purification of 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. 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. From this assay, we were able to determine the ideal quantities of protein to use for testing the enzymatic degradation kinetics of PETase. Additionally, after leaving these samples at room temperature
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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.