A rapid and efficient electroporation method for transformation of Halomonas sp. O-1

Abstract

Halomonas sp. O-1 is a halophilic bacterium with a high potential for industrial application due to its natural ability to produce polyhydroxyalkanoates (PHAs) using seawater-based media. However, a major barrier preventing industrial scale implementation of this organism is a lack of molecular methodologies capable of readily transforming members of the Halomonas genus. Currently, the only reliable method used for introducing DNA into Halomonas spp. is bacterial conjugation, a somewhat tedious and time-consuming technique compared to electroporation-based methodologies. Here we describe a rapid and reproducible method for the electroporation of Halomonas sp. O-1 with plasmid DNA. Electrocompetent cells were generated by growing Halomonas sp. O-1 in a yeast extract-tryptone medium with a final salinity of 3.5%, pH of 7.5, followed by several washes using 300mM sucrose. Results show that plasmids containing chloramphenicol (Cm(R)) and gentamicin (Gm(R)) resistance cassettes are suitable antibiotic selection markers for transformation and yields of 10(4) transformants per μg of DNA were obtained. This method is simple to perform and the materials used are readily available in most research labs. Additionally, this plasmid-based transformation procedure has the potential to be adapted for a number of applications including the creation of recombinant stains and the generation of deletion mutants of Halomonas spp.

Establishment of genetic tools for genomic DNA engineering of Halomonas sp. KM-1, a bacterium with potential for biochemical production

“…The high-efficiency transformation of Halomonas sp. O-1 using the electroporation method was previously reported using a broad-host-range vector, pBBR1MCS [ 10 ], but the transformation efficiency of Halomonas sp. KM-1 by the vector was 100 times lower than that reported in a previous report (Additional file 1 : Table S2).…”

“…LB medium (1% tryptone, 0.5% yeast extract, and 1% NaCl) was used to culture E. coli . Halomonas growth medium (HGM) (pH 8.0, 1% tryptone, 0.5% yeast extract, and 3.5% NaCl) was used for the electroporation method of Halomonas transformation [ 10 ]. SOT (pH 9.5, 3% (w/v) sucrose, 2.5% NaCl) and SOT plate (2% agar) media were used for the general culture of Halomonas species [ 7 ].…”

“…Electrocompetent KM-1 cells were prepared using a previously reported method [ 10 ]. Briefly, KM-1 was cultured in HGM at 30 °C under agitation at 250 rpm and collected at an OD 600 of 0.5, by centrifugation for 10 min at 5000× g at 25 °C.…”

“…Intended for the industrial development of Halomonas , several genetic tools and methods have been developed, such as gene transfer by conjugation using a broad-host-range vector in H. elongata and Halomonas subglaciescola [ 9 ], an effective electroporation method for transformation using Halomonas sp. O-1 [ 10 ], and gene disruption and homologous recombination in H. bluephagenesis TD01 using CRISPR-Cas9 systems [ 11 , 12 ]. To select mutants after gene knockin and disruption in yeast, URA5 / pyrE and URA3 / pyrF genes involved in pyrimidine synthesis have been utilized for positive selection together with 5-fluoroorotic acid (5-FOA) [ 13 ].…”

Method Development Progress in Genetic Engineering of Thraustochytrids

“…Similarly, sorbitol is the most used electroporation solution for thraustochytrids, followed by sucrose ( Figure 2 ), indicating that sorbitol can be a prioritized option in establishing the protocols. Although the absence of ions during electric pulse increases the cell viability, washing cells without ions could decrease the cell viability [ 88 ], which further complicates the selection of solutions. Even if the solutions used are non-ionic, the cells’ environment will not be entirely non-ionic due to incomplete washing.…”