Abstract
Chemolithoautotrophic bacteria can be of industrial and environmental importance, but they present a challenge for systems biology studies, as their central metabolism deviates from that of model organisms and there is a much less extensive experimental basis for their gene annotation than for typical organoheterotrophs. For microbes with sequenced genomes but unconventional metabolism, the ability to create knockout mutations can be a powerful tool for functional genomics and thereby render an organism more amenable to systems biology approaches. In this chapter, we describe a genetic system for Thiobacillus denitrificans, with which insertion mutations can be introduced by homologous recombination and complemented in trans. Insertion mutations are generated by in vitro transposition, the mutated genes are amplified by the PCR, and the amplicons are introduced into T. denitrificans by electroporation. Use of a complementation vector, pTL2, based on the IncP plasmid pRR10 is also addressed.
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Acknowledgments
For HRB, work conducted by the Joint BioEnergy Institute was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231. Regarding work conducted at Lawrence Livermore National Laboratory, LLNL is operated by Lawrence Livermore National Security, LLC, for the U.S. Department of Energy, National Nuclear Security Administration under Contract DE-AC52-07NA27344.
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Beller, H.R., Legler, T.C., Kane, S.R. (2012). Genetic Manipulation of the Obligate Chemolithoautotrophic Bacterium Thiobacillus denitrificans . In: Navid, A. (eds) Microbial Systems Biology. Methods in Molecular Biology, vol 881. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-827-6_5
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DOI: https://doi.org/10.1007/978-1-61779-827-6_5
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