Expression and purification of GxcA, a c-type cytochrome involved with metal respiration by the bacterium, Geothrix fermentans
2011-04-13
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Expression and purification of GxcA, a c-type cytochrome involved with metal respiration by the bacterium, Geothrix fermentans
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2011-04-13
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The study of bacteria capable of respiring oxidized metals offers insights into the geochemical cycling of metals, including toxic heavy metal contaminants. These dissimilatory metal-reducing organisms couple oxidation of organic compounds with the reduction of substrates to gain energy. Improving understanding of the various bacterial metal-reducing strategies will increase our ability to produce renewable energy using microbial fuel cell technologies. The bacterium Geothrix fermentanshas long been ignored in the study of metal respiration, but it has recently been shown to employ a unique strategy involving more than one biochemical pathway that appears tuned to use of high potential metals, such as uranium and manganese. Membranes isolated from Fe(III)-respiring G. fermentans contain high levels of a decahemecytochrome, known as GxcA, which is suspected in electron transfer by G. fermentans. As a genetic system for G. fermentans is not yet available, GxcAwas targeted for expression and purification. The DNA sequence for GxcA, containing an in-frame hexahistidinesequence, was first cloned into E. coli, using the inducible expression vector pETlite. Then, the recombinant plasmid was co-transformed into E. coli with pEC86, a plasmid that contains genes for the ccmc-type cytochrome maturation system. Colonies were screened for c-type cytochromes by redox difference absorption analysis and hemestain analysis. Future work will involve GxcApurification for redox and localization experiments to determine GxcA’spotential role in G. fermentans.
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Additional contributors: Misha Mehta-Kolte; Daniel Bond (faculty mentor)
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This project was supported by the Undergraduate Research Opportunities Program (UROP).
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Liu, Joanne. (2011). Expression and purification of GxcA, a c-type cytochrome involved with metal respiration by the bacterium, Geothrix fermentans. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/104494.
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