Development of a system for genetic manipulation of the facultative methanotroph methylocella silvestris BL2
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, S. W., (eds.) Methods in methane metabolism. Part B , Methanotrophy. Methods in enzymology, Vol.495 . Amsterdam, [Netherlands] ; Boston, [Mass.] : Elsevier/Academic Press, pp. 119-133. ISBN 9780123869050Full text not available from this repository.
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|
|Divisions:||Faculty of Science > Life Sciences (2010- )|
|Series Name:||Methods in enzymology|
|Place of Publication:||Amsterdam, [Netherlands] ; Boston, [Mass.]|
|Book Title:||Methods in methane metabolism. Part B , Methanotrophy|
|Editor:||Rosenzweig, A. C. and Ragsdale, S. W.|
|Number of Pages:||309|
|Page Range:||pp. 119-133|
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