Part:BBa_K4191003

J23119 + B0034 + HAD + B0010

This part includes our new HAD basic part (BBa_K4191004), along with an existing promoter (BBa_J23119) , RBS (BBa_B0034), and terminator (BBa_B0010). Haloacid dehalogenase (HAD) is an enzyme catalyzing the cleavage of carbon-halogen bonds, in this case, the cleavage of the carbon-fluorine bond (Chan et al). This was one of the enzymes we used to degrade the toxicant perfluorooctanoic acid (PFOA).

Using Autodock 4, the interactions between our enzymes and ligands were visualized. The above figure shows the interaction between PFOA and HAD and the conformation at the maximum binding energy (binding energy: -5.8, ligand efficiency: 0.23).

With the computational modeling supporting the defluorination pathway for the enzyme in the literature, we proceeded to test the enzyme in the lab. First, we modified the HAD sequence in the SnapGene Software and codon optimized it for E. coli. To prepare it for loop assembly, we removed restriction sites BsaI and SapI , and added 20 bases of homology to both ends of the gene sequences. Lastly, to ensure that the sequence would be compatible with our u loop system, the overhangs were modified to match with the C-D overhangs specifically for loop assembly.

After the sequence was modified in SnapGene, we ordered it from IDT, and inserted the part into the L0 plasmid vector via the gibson assembly cloning method. Gibson assembly put the sequence in the C-D “slot”, forming an L0 vector plasmid. Next, this component was assembled into a complete plasmid (including promoter, RBS, and terminator components) via the golden-gate assembly method. The golden gate method simultaneously joins multiple DNA fragments into a single plasmid, in our case, the PCA odd-1 backbone (Pollak et al). The promoter (BBa_J23119) was inserted in the A-B, the RBS (BBa_B0034) was inserted in the B-C, and the terminator (BBa_B0010) was inserted in the D-F “slot.”

The plasmid was then electroporated into several cultures of P. putida and E. coli (successful transformation was confirmed through kanamycin resistance, as the plasmid BB has a kanamycin resistant gene). The six cultures were FAcD + PFOA in E coli, HAD + PFOA in E. coli, WT E. coli with PFOA, WT P. putida + PFOA, E. coli engineered with FAcD, and E coli engineered with HAD. The last two cultures were used as controls for our experiment. pH level and optical density were measured at several time points over 3 days (9:00 AM, 12:00 PM, 3:00 PM, 6:00 PM).

The graph below depicts the trends for pH vs Time (hours) for cultures:

In previous literature, lower pH levels have been used to indicate that bacteria are able to successfully degrade PFOA. pH levels decrease because as the enzyme cleaves C-F bonds, HF is the natural byproduct of the defluorination reaction. As the reaction continues and the concentration of HF increases, the concentration of H+ ions also increases (because HF is a weak acid which partially dissociates in solution). An increase in the concentration of H+ causes the pH of the solution to drop (Wackett et al).

With the data we have collected, however, we cannot confidently say that our enzyme insert had an effect on PFOA degradation. A potential source of error may have been the evaporation of some HF, as we worked with the bacteria in an open system. In the future, we hope not only to use a closed system to ensure the dissociation of HF is enabled, but also to find a more accurate method than pH level for detecting the degradation of PFOA (such as DCMS, LCMS, NMR, etc).

References

[1] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3101105/ (Chan et al)

[2] https://www.biorxiv.org/content/10.1101/247593v2 (Pollak et al)

[3] https://sfamjournals.onlinelibrary.wiley.com/doi/full/10.1111/1751-7915.13928 (Wackett et al)