Drylab Results
The following pictures are snapshots of sections of our pipeline output. The RNN model is able to run based off of our training dataset “sequences_final.txt”, and is able to put out the specified number of sequences requested in the code. In this instance, we are telling the pipeline that we want three potential PETase mutant candidates, and the pipeline is able to return the three mutants.
To view our full pipeline, training dataset, and sequence outputs, here is our Github.
Figure 1: Running the RNN model and training it with our dataset.
Figure 2: Sequences generated and printed for output.
NanoDrop Kinetic Assay
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).
Figures 3 and 4 show reactions consisting of sample, reaction buffer, and PET plastic film. All measurements were performed on a NanoDrop One. Reaction buffer plus PET plastic film was used as a blank and Dialysis buffer plus PET plastic film acted as a negative control. (Note that the uninduced sample in Figure 3 was noticeably higher in absorbance due to the high concentration of cells.)
Figure 3: Plot of absorbance taken at 260 nm for crude soluble wild-type PETase sample in terms of A260 vs. enzyme sample quantity.
Figure 4: Plot of absorbance taken at 260 nm for purified wild-type PETase sample in terms of A260 vs. enzyme sample quantity.
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.
SDS-PAGE
Figure 5: 7/15/21. First SDS-PAGE ran on E. coli BL21 expression of PETase. We used Coomassie Blue dye to stain and Destain. There was no clear data from the gel.
Figure 6: 7/17/21. For this Gel, we switched the staining method to ThermoFisher SimplyBlueTM SafeStain and destained it with water and 20% NaCl Solution. This provided us with a clearer staging of the gel.
Figure 7: 7/24/21. Staining Method: ThermoFisher SimplyBlueTM SafeStain and destained with water and 20% NaCl Solution. Lanes as follows -- Lane 15: Bio-Rad Kaleidoscope Ladder, Lane 14: Insoluble Fraction, Lane 12: Insoluble Fraction, Lane 11: Insoluble Fraction, Lane 10: Insoluble Fraction, Lane 9: Soluble Fraction, Lane 8: Soluble Fraction, Lane 6: Soluble Fraction, Lane 4: Positive Control Soluble, Lane 2: Positive Control Insoluble, Lane 1: Bio-Rad All Blue Ladder
Figure 8: 7/28/21. Lanes as follows -- C: control, I: Insoluble Fraction, S: Soluble Fraction. Staining Method: ThermoFisher SimplyBlueTM SafeStain and destained with water and 20% NaCl Solution. We used Used 20 ul well 12% Gel.
Figure 9: 9/25/21. Successful Induction and expression of PETase in E. coli C41.
Figure 10: 10/9/21. SDS-PAGE Post FPLC Troubleshooting performed. Lanes as follows -- 1: Kaleidoscope Ladder, 4: Centrifuged Pellet Before FPC, 7 & 8: Supernatant from FPLC, 12-15: FPLC Samples. Issues encountered included high centrifugation speed which resulted in lost soluble supernatant. FPLC Method settings were also off, wells were overflowing and fractions mixed.
Figure 11: 10/18/21. Successful FPLC purification and dialysis of PETase. Lanes as follows -- Lane 1: Kaleidoscope Ladder, Lane 2: Induced Total (Crude soluble and insoluble), Lane 3: Uninduced (Crude Sample to IPTG), Lanes 9-13: FPLC samples
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.
Figure 12: Results of Induction Troubleshooting Assay
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.
Figure 13: Crude Degradation Assay 500 uL
Figure 14: Crude Degredation Assay 750 uL
Figure 15: Crude C Degredation Assay 500 uL
Figure 16: Crude C Degredation Assay 750 uL
Figure 17: Crude C Degredation Assay 100 and 250 uL