Journal: Microbial Biotechnology

Article Title: Developing High‐Efficiency Electroporation Protocols for Hard‐To‐Transform Halomonas spp.

doi: 10.1111/1751-7915.70285

Figure Lengend Snippet: Preliminary electroporation protocol of Halomonas elongata DSM 2581. Cells were cultured in LB medium supplemented with NaCl (LB60) at 30°C and 225 rpm for 14 h. Electrocompetent cells were prepared by washing the harvested cells with CaCl 2 (50 mM) + NaCl (6%) for 5 min, followed by two sucrose (300 mM) washes. The cell pellet was then resuspended in sucrose and transferred to a 0.2‐cm gap electroporation cuvette, to which the plasmid DNA was subsequently added. Electroporation was performed using a Bio‐Rad MicroPulser, after which cells were recovered in LB60 supplemented with glucose (20 mM) at 30°C and 225 rpm for 90 min. Following recovery, electroporated cells were plated onto LB60 agar plates and incubated at 30°C for 36 h to allow for colony formation. (Created in BioRender. Rios Solis, L. (2025) https://BioRender.com/osb8xfd ).

Article Snippet: Halomonas elongata DSM 2581, Halomonas boliviensis LC1 and Halomonas campaniensis LS21 were obtained from DSMZ.

Techniques: Electroporation, Cell Culture, Plasmid Preparation, Incubation

Journal: Microbial Biotechnology

Article Title: Developing High‐Efficiency Electroporation Protocols for Hard‐To‐Transform Halomonas spp.

doi: 10.1111/1751-7915.70285

Figure Lengend Snippet: Preliminary electroporation tests of Halomonas elongata DSM 2581. (a) Electroporation efficiencies at different voltages (1.5, 1.7, 2.1 and 2.5 kV) and pulse numbers (2× 2.1, 2× 2.5 kV). (b) Electroporation efficiencies from electrocompetent cells prepared with sucrose (300 mM) vs. glycerol (10%). (c) Electroporation efficiencies from electrocompetent cells prepared from cells harvested at 6, 14 and 24 h. Data shown represent mean ± standard deviation from three biological replicates for panels (a) and (b), and four biological replicates for panel (c). Negative control experiments were performed by electroporating electrocompetent cells without the addition of plasmid pSEVA241. (ns, not significant; *** p < 0.001)

Article Snippet: Halomonas elongata DSM 2581, Halomonas boliviensis LC1 and Halomonas campaniensis LS21 were obtained from DSMZ.

Techniques: Electroporation, Standard Deviation, Negative Control, Plasmid Preparation

Journal: Microbial Biotechnology

Article Title: Developing High‐Efficiency Electroporation Protocols for Hard‐To‐Transform Halomonas spp.

doi: 10.1111/1751-7915.70285

Figure Lengend Snippet: Effect of salinity on electroporation efficiency and cell morphology of Halomonas elongata DSM 2581. (a) Electroporation efficiencies from electrocompetent cells prepared from cultures grown in LB medium containing different concentrations of NaCl: 0.5%, 1%, 2% and 6%. Data shown represent geometric mean */ geometric SD factor from three biological replicates. Negative control experiments were performed by electroporating electrocompetent cells without the addition of plasmid pSEVA241. (** p < 0.01; **** p < 0.0001). (b) Transmission electron microscopy imaging of cells cultured for 24 h in LB medium containing 1% NaCl. (c) Transmission electron microscopy imaging of cells cultured for 24 h in LB medium containing 6% NaCl. (d) Transmission electron microscopy imaging of cells cultured for 14 h in LB medium containing 6% NaCl.

Article Snippet: Halomonas elongata DSM 2581, Halomonas boliviensis LC1 and Halomonas campaniensis LS21 were obtained from DSMZ.

Techniques: Electroporation, Negative Control, Plasmid Preparation, Transmission Assay, Electron Microscopy, Imaging, Cell Culture

Journal: Microbial Biotechnology

Article Title: Developing High‐Efficiency Electroporation Protocols for Hard‐To‐Transform Halomonas spp.

doi: 10.1111/1751-7915.70285

Figure Lengend Snippet: Further optimisation of electroporation parameters for Halomonas elongata DSM 2581. (a) Comparison of electroporation efficiencies using two different volumes of electrocompetent cell suspension: 40 μL vs. 200 μL. (b) Effect of cell concentration on electroporation efficiency. Electrocompetent cells were resuspended in glycerol (10%) at different optical densities: OD 600 = 10, 20, 40, 60. (c) Comparison of electroporation efficiencies using two different electroporator systems—Bio‐Rad MicroPulser vs. Bio‐Rad Gene Pulser—under different electroporation conditions: Voltage, pulse number and resistance. (d) Table summarising the eight electroporation conditions tested in panel (c), resulting from the constrained mixed‐level orthogonal array design. Data shown represent mean ± standard deviation from three biological replicates. Negative control experiments were performed by electroporating electrocompetent cells without the addition of plasmid pSEVA241. (ns, not significant; * p < 0.05; *** p < 0.001; **** p < 0.0001).

Article Snippet: Halomonas elongata DSM 2581, Halomonas boliviensis LC1 and Halomonas campaniensis LS21 were obtained from DSMZ.

Techniques: Electroporation, Comparison, Suspension, Concentration Assay, Standard Deviation, Negative Control, Plasmid Preparation

Journal: Microbial Biotechnology

Article Title: Developing High‐Efficiency Electroporation Protocols for Hard‐To‐Transform Halomonas spp.

doi: 10.1111/1751-7915.70285

Figure Lengend Snippet: Effect of plasmid source on the electroporation efficiency of Halomonas elongata DSM 2581. (a) Electroporation efficiencies of pSEVA241 purified from either E. coli 10‐beta (NEB) or H. elongata DSM 2581. (b) Electroporation efficiencies of pSEVA231 purified from either E. coli 10‐beta (NEB), E. coli C2925 (NEB) or H. elongata DSM 2581. Data shown represent mean ± standard deviation from three biological replicates. Negative control experiments were performed by electroporating electrocompetent cells without the addition of plasmid pSEVA241 (a) or pSEVA231 (b) (* p < 0.05; *** p < 0.001; **** p < 0.0001).

Article Snippet: Halomonas elongata DSM 2581, Halomonas boliviensis LC1 and Halomonas campaniensis LS21 were obtained from DSMZ.

Techniques: Plasmid Preparation, Electroporation, Purification, Standard Deviation, Negative Control

Journal: Microbial Biotechnology

Article Title: Developing High‐Efficiency Electroporation Protocols for Hard‐To‐Transform Halomonas spp.

doi: 10.1111/1751-7915.70285

Figure Lengend Snippet: Electroporation of Halomonas boliviensis LC1 and Halomonas campaniensis LS21. Electroporation efficiencies of H. boliviensis LC1 and H. campaniensis LS21 transformed with pSEVA231 purified from either E. coli C2925 (NEB) (a) Comparison of electroporation efficiencies from electrocompetent cells prepared from cultures grown in LB medium containing different concentrations of NaCl: 6% vs. 1%. (b) Comparison of electroporation efficiencies using two different electroporator systems—Bio‐Rad MicroPulser vs. Bio‐Rad Gene Pulser—under varying electroporation conditions: Voltage, pulse number and resistance. Parameters for conditions C1, C4, C6 and C7 are detailed in Figure . Data shown represent mean ± standard deviation from three biological replicates. Negative control experiments were performed by electroporating electrocompetent cells without the addition of plasmid pSEVA231. (*** p < 0.001; **** p < 0.0001)

Article Snippet: Halomonas elongata DSM 2581, Halomonas boliviensis LC1 and Halomonas campaniensis LS21 were obtained from DSMZ.

Techniques: Electroporation, Transformation Assay, Purification, Comparison, Standard Deviation, Negative Control, Plasmid Preparation