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An Efficient Transformation Method for the Bioplastic‐Producing “Knallgas” Bacterium Ralstonia eutropha H16
Author(s) -
Tee Kang Lan,
Grinham James,
Othusitse Arona M.,
GonzálezVillanueva Miriam,
Johnson Abayomi O.,
Wong Tuck Seng
Publication year - 2017
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201700081
Subject(s) - ralstonia , cupriavidus necator , plasmid , transformation (genetics) , bioplastic , electroporation , polyhydroxyalkanoates , bacteria , biology , polyhydroxybutyrate , computational biology , chemistry , biochemistry , dna , gene , genetics , ecology
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 is 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 OD 600 0.6. These cells are made competent by a 15‐min incubation in 50 mM CaCl 2 , followed by two cell washes and final resuspension in 0.2 M sucrose prior to electroporation using 2.3 kV. This protocol achieves a transformation efficiency of (3.86 ± 0.29) × 10 5 cfu µg −1 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.