Results

PFAS Detection

Generated Part

The prmA promoter-mRFP insert was ordered and synthesized through IDT (order number: 16173335). Part was confirmed by gel electrophoresis (below) and inserted into pSB1C3 and pSRKBB (a modified Rhodococcus jostii plasmid).

Characterization of BBa_E1010: A mRFP

Fig. 1: Gel of ordered part

Positive and Negative Controls

The positive control is the pink streak and the negative control is the opaque streak. The positive control contains a lacI promoter and the red fluorescent protein (mRFP). The promoter is constitutive, and as such, continually transcribes mRFP, exhibited by the red-pink coloring. The negative control contains mRFP but does not contain a promoter. The mRFP gene is never expressed in the negative control. Both of these controls were used for comparison with prmA-mRFP (the promoter-gene used in testing with PFOA and PFOS.)

Fig. 2: Positive and Negative controls for RFP fluorescence

Characterization

BBa_E1010 is a Red Fluorescent Protein biobrick that has been used in other iGEM projects as a reporter. We utilized this part as our quick and rapid way to detect expression. In order to better understand BBa E1010 as a reporter, we further characterized the part by looking at RFP expression over time and at different incubation temperatures. For this inital characterization, E. coli (DH5 alpha) was used as the vector. Detection of RFP was seen at 12 hours of growth with significant increases in RFP color up to 48 hours.

Fig. 3: mRFP expression in E. coli

Additionally, we looked at the growth kinetics in relation to expression of the BBa_E1010 RFP reporter. Overall, expression of the RFP followed the growth curve dynamics very closely. The most growth and RFP expression occurred at 37C incubation, followed by 30C and 23C which both had less growth and reporter expression.

Fig. 4: Quantification of mRFP expression at different incubation temperatures

Part Testing

First, the team put together an E. coli plasmid that contained the prmA-mRFP insert and transformed it into E. coli cells. These cells were grown on LB plates that contained PFOA and PFOS and did not exhibit the red fluorescence pictured in the positive control. The team then exposed these transformed cells to PFOA and PFOS in liquid cultures. Again, the cells did not exhibit any red fluorescence. This could be due to the fact that our insert, which contains a promoter that comes from R. jostii, did not function properly in an E. coli plasmid and cell. It could also be due to the fact that our promoter had mutations (deletions.)

The team then created a Rhodococcus jostii plasmid that contained the same prmA-mRFP insert as the E. coli plasmid and transformed it into R. jostii cells. The team exposed these cells to PFOA and PFOS in the same way as the E. coli (LB plates and liquid cultures). The R. jostii cells, like E. coli, did not exhibit any red fluorescence. This ruled out the possibility that the mRFP gene isn’t being expressed due to being in E. coli, because it was equally non-functional in R. jostii. However, although no visible results were found, the activation of the mRFP by the prmA promoter was measured through qRT-PCR and was found to be up regulated in the presence of PFOA as shown below. This lack of visible fluorescence could be due to a low sensitivity in the promoter region, which could be improved in future products (see future work in project description).

Fig. 5: Quantification of mRFP expression at different incubation temperatures

PFAS Degradation

Biological Degradation Testing

In experiments attempting to grow bacteria in the presence of PFOA and PFOS, agar plates with either of the two PFAS as the carbon source were created supplemented with minimal inorganic additives and a small amount of glucose to induce growth. For 7 similar species to YAB-1, none grew on either PFOA or PFOS plate at any concentration of the PFAS (Data not shown). This suggests that none of these bacteria are PFAS degrading species.

CHP Reaction: Proof of Concept

In optimal settings, CHP reactions with PFOA were reported to degrade 89% of PFOA in 150 minutes. In an attempt to mimic this finding with available resources, the reaction was performed using available 3% hydrogen peroxide, a catalyst solution of FeCl3, and a 11.35 mM stock solution of PFOA in water. Various methods to quantify PFOA in solution were attempted such as fluorine NMR, High Performance Liquid Chromatography tandem Mass Spectrometry (HPLC-MS), and direct MS injection before settling on a modified HPLC-MS system. Unfortunately, due to the acidic nature of PFOA, no silica-based LC columns were usable. A column-less HPLC system acted as an auto sampler which fed into the MS running in Single Ion monitoring (SIM) mode looking at the two common peaks (169.2 & 109 m/z) for PFOA (referred to as SIM2). This allowed for direct injection into the MS with much more precision control over volume and time. As this reaction serves as a proof-of-concept for catalyzed hydrogen peroxide degradation, this level of quantification was deemed appropriate, as shown below in the figure below showing the calibration curve of MS curve integration to concentration of PFOA in the range 5.7uM - 11.35 mM.

Fig. 6: Calibration Curve of PFOA using SIM2 Method (169.2 m/z & 109 m/z) from range 5.7uM - 11.35 mM. MS was in ESI - mode.

In order to determine a maximum ratio of PFOA to peroxide, the variable of RQ (Reactant Quotient) was defined that represented the molar concentration of PFOA divided by the molar concentration of peroxide. Varying the RQ in triplicate across 8 samples (7 standards and 1 blank of RQ = 0) and graphing the values relative to the % PFOA loss after approx. 12 hr generated Fig. 7 below.

Fig. 7: RQ of reaction vs. PFOA loss.

Only 4 standards are shown due to approaching the Limit of Detection of the instrument setup for lower RQ values, causing wild variation between individual samples and generating nonsense data. The maximum RQ was determined to be 0.00451964. This reached a maximum of approximately 75% PFOA degradation. This value of 75% removal was similar to the reported 89% of Shannon M. Mitchell, et. al., and as our experiments used much less concentrated hydrogen peroxide, this lower value was expected. All samples were allowed to react overnight in hopes to increase the degradation amount. This was considerably longer than the reported 150 min reaction time by Shannon M. Mitchell, et. al. likely due to the significant difference in peroxide concentration. This proof of concept was a success, as PFOA was shown to degrade via a CHP reaction, and other peroxide-based reaction can now be considered for future research.

Team:USAFA/Results