Team:Austin UTexas/Results
Plastic Results
We implemented several assays to assess our plasmid’s effectiveness in degrading PET. For these assays, we made measurements using several positive and negative controls in order to compare our system's performance . All assays were tested using samples cultured in either media or buffer containing a PET film, over the course of three to seven days at 30°C or 16°C.
Negative Control First, we created two negative controls for comparison of our experimental results. Our first control was PET film immediately after collection, with no exposure to any kind of incubation or media at all. The second control consisted of PET film placed in cell culture media, which was incubated for three days. No cells nor purified enzyme were included, so no degradation was expected from this sample. PET film typically has a smooth surface on a microscopic level, but degradation of PET by PETase can cause the plastic to develop a rough, scarred surface. Scanning Electron Microscopy (SEM) was conducted on these samples to determine the baseline for PET's texture on a microscopic level, while also determining whether incubation led to any change in texture that could be misinterpreted as degradation of the film. Results for SEM for negative controls were as follows:
Figure 1: SEM images of negative controls (A) PET film prior to any exposure. (B) PET film with exposure to cell culture media without PETase.
In these cases, no difference was observed. Shown left is an image of PET film not exposed to any medium nor incubated. Shown right is an image of PET film exposed to LB growth medium for three days. No difference is observed between these samples, showing that our construct is necessary to detect a change in surface topography.
Positive Control As a positive control, we incubated PET film in LB media with purified wildtype PETase. LB media was used to match the conditions used to test our actual phagemid system. The reaction proceeded for three days before measurements were taken. This control was useful for understanding how our experimental samples compare to more concentrated PETase. Similarly, SEM was conducted on this sample and appeared as follows:
Figure 2: SEM image of positive control. Purified WT PETase after incubation for 36h at 30°C at concentration of 200nM in 200uL reaction volume
PET film incubated with purified PETase showed a marked change in surface texture. Holes appear in the plastic’s surface as PETase acts upon it, showing that degradation has occurred. This sample shows that our construct should cause a similar effect if it functions correctly.
High performance liquid chromatography (HPLC) is a common technique used to identify and quantify components in a mixture. In this assay, HPLC was used to identify and quantify the plastic degradation products, TPA and MHET. These monomers are released upon PETase activity. Thus, greater concentrations of PET monomers released would indicate greater degradation. HPLC measurements were taken for both the purified enzyme, WT PETase, and our second positive control, I. sakaiensis at incubation temperatures of 16°C and 30°C. Data was taken at various time points and a linear regression was performed, as indicated by the graph below :
Figure 3: HPLC measurements of positive controls purified WT PETase in LB media and I. Sakaiensis culture at 30°C and 16°C.
Purified WT PETase performed as expected, with the most degradation over time. Similarly, the other positive control, the I. sakaiensis culture also performed as expected. While there were very few PET monomers detected, this is largely due to the fact thatI. Sakaiensis uses these PET monomers as a carbon source and therefore would not be detected in solution. This does not mean degradation has not occurred. Instead, to confirm PET degradation, SEM images and weight changes were assessed.
Experimental Samples Experimental samples included cells containing our construct. Our construct contained BBa_K3601017, a form of PETase and MHETase. This plasmid was expressed by E. coli strain C41(DE3) cells incubated in cell culture media for three days.
Figure 4: SEM image of experimental sample. PET film incubated with c41(DE3) cells expressing our plasmid construct.
Another sample was allowed to incubate for seven days instead. This sample contained our PETase-expressing construct. In this case, degradation is so strong that holes in the plastic’s surface eventually widen to encompass the entire surface.
Results from the SEM data validate the activity of our plastic-degrading plasmids compared to the negative controls. This indicates a strong potential for using our P1 system to deliver these plasmids to a variety of bacteria within the bacteriophage's broad host range in order to degrade plastic in the environment. Next steps for the project include using the P1 phage system to deliver these plasmids and assessing activity through HPLC and weight changes. This degradation activity of our proposed system will be measured against the positive controls of the purified enzyme WT PETase and I. sakaiensis to determine the effectiveness of our system.
Surfactants Results
In order to ensure the efficacy of our proteins of interest we purified them in high amounts. We did this by over expressing the genes of interest using IPTG-inducible pET plasmids. We began with BL21 DE3 E. coli attempting to express Ranaspumin-2, iLOV-Ranaspumin-2, Latherin, and the plastic degrading enzyme PETase, but were only able to express iLOV-Rsn2. We purified the iLOV-Rsn2 using 6xHis-tag and verified this process by checking for iLOV fluorescence and running an SDS-PAGE gel (Figure 1). Figure 1 shows the successful purification of iLOV-Rsn2. Significant amounts of proteins were seen in all 3 elutions slightly larger than 25 kDa, consistent with the expected protein size (iLOV-Rsn2 is 26.7 kDa).
Figure 1: SDS-PAGE gel of His-tag purification of iLOV-Rsn2. The lanes from left to right are as follows: low molecular weight protein ladder, protein lysate, initial column flowthrough, wash 1, wash 2, wash 3, elution 1, elution 2, and elution 3.
We tried a variety of other cells that contain T7 machinery (to work with the inducible system and promoter) to express our other proteins of interest (PETase, Rsn2, and Latherin) but were still only able to over express iLOV-Rsn2.
The oil spreading assay directly tests the oil dispersing abilities of a sample. The sample is added to the center of plate containing a thin layer of crude oil covering a much larger volume of water. After a few seconds, an area without oil forms from the point where the sample was added. The area of this "spread" directly correlates to the concentration of surfactant. The Triton X-100 created a large spread while the BSA did not, showing that the assay is a valid method of testing for oil dispersing abilities in a liquid sample. When we tested iLOV-Rsn2, there was a small spread.
Figure 2: Oil spreading assay.Top left: 1 mg/mL BSA (a non-surfactant protein), Top right: 0.1% Triton X-100 (a chemical surfactant), and Bottom: 1 mg/mL iLOV-Rsn2 that we managed to purify.
We hypothesize that this reduced dispersion compared to previous data regarding Rsn2 is due to the iLOV tag. Surfactant proteins' conformations are very important due to the unfolding mechanism they use when reducing the surface tension of an interface, so the addition of the iLOV tag to Rsn2 may interfere with its ability to disperse oil.
A more sensitive test for surfactant abilities is the foam stability or bubbling assay. This assay takes advantage of the surfactant ability to decrease the surface tension at an air-water (or solution) interface. This assay involves bubbling large amounts of air through the sample and then determining the stability of the foam created by checking how long it lasts. Because the air-water interface is more miscible than the oil-water interface, this assay is more sensitive to surfactant abilities than the oil spreading assay. We used the same samples as the oil spreading assay but got much better results. The Triton X-100 created a stable foam with small bubbles that popped at a steady but slow rate until the last few layers, which remained for over an hour. The BSA on the other hand did not create a stable foam, rather forming large bubbles that popped quickly. When we tested iLOV-Rsn2, we observed similar foam creation and stability to Triton X-100.
Figure 3: Timelapse of iLOV-Rsn2 (left) and BSA (right) over 10 minutes
Figure 4: Timelapse of iLOV-Rsn2 (left) and Triton X-100 (right) over 20 minutes
Although we were unable to correctly assemble the phagemid for the surfactant genes Rsn2 or iLOV-Rsn2, we were able to verify that iLOV-Rsn2 maintains some of the surfactant ability of untagged Rsn2. This was shown by both the oil spreading assay and the foam stability/bubbling assay. The oil spreading assay with iLOV-Rsn2 sample showed a nontrivial spread, indicating that iLOV-Rsn2 does display surfactant abilities despite the addition of the iLOV tag potentially affecting its conformation and unfolding mechanism. The foam stability assay further enforced this when the iLOV-Rsn2 sample displayed bubble formation and foam stability similar to the known chemical surfactant Triton X-100. This indicates a strong potential that upon replication with untagged Rsn2, the dispersion may be strong enough to effectively disperse oil slicks.