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Development of a system for genetic manipulation of the facultative methanotroph methylocella silvestris BL2
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Crombie, Andrew and Murrell, J. C. (J. Colin) (2011) Development of a system for genetic manipulation of the facultative methanotroph methylocella silvestris BL2. In: Rosenzweig, A. C. and Ragsdale, Stephen W., (eds.) Methods in methane metabolism. Part B , Methanotrophy. Methods in enzymology, Volume 495 . Amsterdam, [Netherlands] ; Boston, [Mass.] : Elsevier/Academic Press, pp. 119-133. ISBN 9780123869050
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Abstract
An understanding of the metabolism and metabolic regulation of the facultative methanotroph Methylocella silvestris BL2 is required to understand its role in methane oxidation in the environment, and methods for genetics manipulation are essential tools in these investigations. In addition, the ability to engineer the metabolic capabilities of M. silvestris may well have useful biotechnological applications. We describe a simple and effective method of genetic manipulation for this organism which relies on the electroporation of a linear DNA fragment to introduce chromosomal gene deletions. In a two-step procedure, the gene of interest is first replaced with an antibiotic-resistance cassette which is subsequently removed, resulting in an unmarked gene deletion. This method is illustrated by the deletion of isocitrate lyase, which abolished growth on one-carbon and severely disabled growth on two-carbon compounds. Subsequent complementation with the wild-type gene and promoter restored growth, demonstrating stable transcription from the broad-host-range plasmid employed.
| Item Type: | Book Item | ||||
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| Alternative Title: | |||||
| Subjects: | Q Science > QH Natural history > QH426 Genetics Q Science > QR Microbiology |
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| Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) | ||||
| Library of Congress Subject Headings (LCSH): | Methanotrophs, Genetic engineering | ||||
| Series Name: | Methods in enzymology | ||||
| Publisher: | Elsevier/Academic Press | ||||
| Place of Publication: | Amsterdam, [Netherlands] ; Boston, [Mass.] | ||||
| ISBN: | 9780123869050 | ||||
| Book Title: | Methods in methane metabolism. Part B , Methanotrophy | ||||
| Editor: | Rosenzweig, A. C. and Ragsdale, Stephen W. | ||||
| Official Date: | 2011 | ||||
| Dates: |
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| Volume: | Volume 495 | ||||
| Number of Pages: | 309 | ||||
| Page Range: | pp. 119-133 | ||||
| Status: | Peer Reviewed | ||||
| Publication Status: | Published |
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