Quick and efficient method for genetic transformation of biopolymer‐producing bacteria |
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| Authors: | Qin Wang Alexander P Mueller Chean Ring Leong Ken'ichiro Matsumoto Seiichi Taguchi Christopher T Nomura |
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| Affiliation: | 1. Department of Chemistry, State University of New York‐College of Environmental Science and Forestry, Syracuse, NY 13210, USA;2. Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, N13W8, Kita‐ku, Sapporo‐shi, Hokkaido 060‐8628, Japan |
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| Abstract: | In order to genetically modify microorganisms capable of producing polyhydroxyalkanoate (PHA) biopolymers, a simple and rapid method to prepare freshly plated Pseudomonas cells for transformation via electroporation was developed. This method can be used to transfer both replicative plasmids and linear DNA to knock out genes into the cells. The transformation efficiencies were in the range of ≥107 transformants µg?1 DNA for replicative plasmids and ≥106 transformants µg?1 DNA for linear DNA, which are comparable with commercially available competent cells. Furthermore, this transformation procedure can be performed in less than 10 min, saving a great deal of time compared with traditional methods. Knockout mutants of several Pseudomonas species were generated by transformation of linear DNA and these mutations were verified by PCR and analysis of PHA content. Copyright © 2009 Society of Chemical Industry |
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| Keywords: | transformation electroporation Pseudomonas putida polyhydroxyalkanoates (PHAs) |
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