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 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 OD600 0.6. These cells are 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 achieves a transformation efficiency of (3.86 ± 0.29) × 105 cfu µg-1 DNA, a 103 -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.
Highlights
Ralstonia eutropha H16 is a Gram‐negative lithoautotrophic β‐proteobacterium found in soil and freshwater habitats
Five common transformation buffers used for cell wash were first investigated, including double‐ distilled water, 10% (v/v) glycerol, 0.3 M sucrose, 0.3 M glucose and 10% (w/v) fructose (0.56 M)
Combinations of sucrose with glycerol or fructose were investigated but both resulted in poorer transformation efficiency
Summary
Ralstonia eutropha H16 ( known as Cupriavidus necator H16) is a Gram‐negative lithoautotrophic β‐proteobacterium found in soil and freshwater habitats. R. eutropha H16 can metabolize a broad range of substrates as well as grow chemolithoautotrophically using CO2 and H2 as its sole carbon and energy source, respectively. These traits have captured the interests of scientists and fueled the research on this non‐pathogenic bacterium for carbon capture and utilization, biopolymer synthesis and biofuel production [3,4,5,6,7]. With its growing industrial application, an efficient transformation method is urgently needed to support easy investigation and fast engineering of R. eutropha H16. Several recent publications included the use of both electroporation and bioconjugation for introducing plasmid DNA into R. eutropha H16, but electroporation was never the sole method used in any of these publications. A likely reason is the low electroporation transformation efficiency of R. eutropha H16, ranging from 3 cfu/μg to 4 x 103 cfu/μg DNA depending on the plasmid used [11,12,13]
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