Team:iTesla-SoundBio/Design

Design

The pcbA genes were discovered in the anaerobic bacterium called Dehalococcoides mccartyi (Yan, 2013). Three strains of this bacterium possess dehalogenase enzymes (pcbA5, pcbA4, and pcbA1) necessary for organohalide respiration, a type of cellular respiration that uses chlorines as electron acceptors. Each of the three enzymes breaks down PCBs as well as PCE (Wang, 2014). Structural homology to pceA suggests that norpseudo B12 is an essential cofactor for this process. Our goal was to express these genes heterologously in E. coli because D. mccartyi has a very long doubling time. We chose the pET29b backbone for expressing our genes because it uses an inducible T7 promoter, a common system for manufacturing recombinant proteins. Once we have the functioning expression system, we plan to test the effectiveness of breaking down PCBs in an aerobic environment.

For future testing of the effectiveness of these enzymes, we decided to use TCE (trichloroethylene) instead of PCB or PCE for several reasons. One reason is that TCE is a molecule produced in the process of breaking down PCBs (generally it goes PCB>PCE>TCE>DCE). PCBs and TCE both have the unsaturated carbons + 2 chlorine structure required for the enzyme to bind to the substrate. If our enzyme isn’t effective in breaking down TCE in an aerobic environment, it’s safe to assume it won’t be able to handle PCBs, and if it is effective, it would merit further testing. Another reason we chose TCE over PCB is the inherent difficulty of working with PCBs. PCBs are very insoluble in water and they tend to stick to surfaces. TCE is more soluble than PCB, but it still has multiple chlorines for the dehalogenase to act on.

There are still challenges in setting up a system for testing our enzymes. Like previously mentioned, the pcbA enzymes all require two iron-sulfur clusters and a cofactor (norpseudo B12) to function. E. coli does not naturally produce this form of B12. One possible solution is to introduce the cofactor into our system exogenously. Another possible complication is that TCE molecules are volatile and easily lost to the air.

References:

Wang S, Chng KR, Wilm A, et al. Genomic characterization of three unique Dehalococcoides that respire on persistent polychlorinated biphenyls. Proceedings of the National Academy of Sciences of the United States of America. 2014;111(33):12103-12108. doi:10.1073/pnas.1404845111.

Yan J, Im J, Yang Y, Loffler FE. Guided cobalamin biosynthesis supports Dehalococcoides mccartyi reductive dechlorination activity. Philosophical Transactions of the Royal Society B: Biological Sciences. 2013;368(1616):20120320-20120320. doi:10.1098/rstb.2012.0320.