An Efficient Transformation Method for the Bioplastic-Producing 'Knallgas' Bacterium Ralstonia eutropha H16
| Autor: | Abayomi Oluwanbe Johnson, James Grinham, Kang Lan Tee, Arona Martin Othusitse, Miriam Gonzalez-Villanueva, Tuck Seng Wong |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Microorganism Cupriavidus necator Sodium Chloride Applied Microbiology and Biotechnology Bioplastic Microbiology Calcium Chloride 03 medical and health sciences Plasmid Ralstonia biology Polyhydroxyalkanoates Electroporation General Medicine biology.organism_classification 030104 developmental biology Biochemistry Molecular Medicine Transformation Bacterial Genetic Engineering Bacteria Plasmids Transformation efficiency |
| Zdroj: | Biotechnology Journal. 12:1700081 |
| ISSN: | 1860-6768 |
| DOI: | 10.1002/biot.201700081 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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