Tee, K.L., Grinham, J., Othusitse, A.M. et al. (3 more authors) (2017) An efficient transformation method for the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16. Biotechnology Journal, 12 (11). ISSN 1860-6768
Abstract
Ralstonia eutropha H16 (also known as Cupriavidus necator H16) is a Gram-negative lithoautotrophic β-proteobacterium with increasing biotechnological applications, including carbon capture and utilization, biopolymer synthesis and biofuel production. Engineering of this organism is supported by the availability of its genome sequence and suitable plasmid systems. However, the lack of a simple and robust transformation method remains a challenge as it limits both the pace and ease of engineering this organism. To overcome this limitation, a systematic study was performed to evaluate the effects of different parameters on the transformation efficiency of R. eutropha H16. The optimized electroporation protocol uses R. eutropha H16 cells grown to OD600 0.6. These cells were made competent by a 15-min incubation in 50 mM CaCl2 , followed by two cell washes and final resuspension in 0.2 M sucrose prior to electroporation using 2.3 kV. This protocol achieved a transformation efficiency of (3.86 ± 0.29) x 10(5) cfu/μg DNA, a 10(3) -fold improvement compared to a previously published value for the same plasmid. This transformation method is a valuable tool for R. eutropha H16 research and will further enable the development of other advanced molecular biology methods for this industrially relevant microorganism.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2017 Wiley. This is an author produced version of a paper subsequently published in Biotechnology Journal. Uploaded in accordance with the publisher's self-archiving policy. |
Keywords: | Ralstonia eutropha H16; Cupriavidus necator H16; Transformation; Recombinant DNA; Biological carbon capture and utilization |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Engineering (Sheffield) > Department of Chemical and Biological Engineering (Sheffield) |
Funding Information: | Funder Grant number INVISTA UNSPECIFIED |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 09 Aug 2017 09:40 |
Last Modified: | 14 Nov 2023 16:10 |
Published Version: | https://doi.org/10.1002/biot.201700081 |
Status: | Published |
Publisher: | Wiley |
Refereed: | Yes |
Identification Number: | 10.1002/biot.201700081 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:119942 |