Transformation of Methanococcus maripaludis and identification of a Pst I-like restriction system |
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| Authors: | Debra L. Tumbula Ronald A. Makula William B. Whitman |
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| Affiliation: | Department of Microbiology, University of Georgia, Athens, GA 30602, USA; Department of Biochemistry, University of Georgia, Athens, GA 30602, USA |
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| Abstract: | Abstract An optimized polyethylene glycol (PEG) method of transformation was developed for Methanococcus maripaludis using the pKAS102 integration vector. The frequency of transformation with 0.8 μg of plasmid and 3×109 cells was 4.8×10−5 transformants cfu−1, or 1.8×105 transformants μg−1, which was four orders of magnitude greater than with the natural transformation method. A Pst I restriction activity in M. maripaludis was also identified. Methylation of the plasmid with Pst I methylase increased the methanococcal transformation frequency at least four-fold. Also, chromosomal DNA from M. maripaludis was resistant to digestion by the Pst I endonuclease. |
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| Keywords: | Methanococcus maripaludis Transformation Restriction endonuclease PstI Mrr Polyethylene glycol |
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