Enhancing DNA electro-transformation efficiency on a clinical Staphylococcus capitis isolate

• Published in: Journal of microbiological methods 2015-02, Vol.109, p.25-30
• Main Authors: Cui, Bintao, Smooker, Peter M., Rouch, Duncan A., Deighton, Margaret A.
• Format: Article
• Language: English
• Subjects: Bacteremia - microbiology; Biofilm; Cell competency; DNA Restriction-Modification Enzymes - radiation effects; DNA, Bacterial - genetics; Electroporation; Electroporation - methods; Genetic Vectors; Hot Temperature; Humans; Infant, Newborn; Restriction–modification barrier; Staphylococcus - genetics; Staphylococcus - isolation & purification; Staphylococcus - radiation effects; Staphylococcus aureus; Staphylococcus capitis; Transformation efficiency; Transformation, Bacterial
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2014.11.012

Clinical staphylococcus isolates possess a stronger restriction–modification (RM) barrier than laboratory strains. Clinical isolates are therefore more resistant to acceptance of foreign genetic material than laboratory strains, as their restriction systems more readily recognize and destroy foreign DNA. This stronger barrier consequently restricts genetic studies to a small number of domestic strains that are capable of accepting foreign DNA. In this study, an isolate of Staphylococcus capitis, obtained from the blood of a very low birth-weight baby, was transformed with a shuttle vector, pBT2. Optimal conditions for electro-transformation were as follows: cells were harvested at mid-log phase, electro-competent cells were prepared; cells were pre-treated at 55°C for 1min; 3μg of plasmid DNA was mixed with 70–80μL of competent cells (3–4×1010cells/mL) at 20°C in 0.5M sucrose, 10% glycerol; and electroporation was conducted using 2.1kV/cm field strength with a 0.1cm gap. Compared to the conventional method, which involves DNA electroporation of Staphylococcus aureus RN4220 as an intermediate strain to overcome the restriction barrier, our proposed approach exhibits a higher level (3 log10 units) of transformation efficiency. Heat treatment was used to temporarily inactivate the recipient RM barrier. Other important parameters contributing to improved electro-transformation efficiency were growth stage for cell harvesting, the quantity of DNA, the transformation temperature and field strength. The approach described here may facilitate genetic manipulations of this opportunistic pathogen. •Improved electro-transformation efficiency was achieved for a S. capitis clinical biofilm-positive isolate.•The recipient host restriction-modification (RM) barrier was temporarily inactivated by heat treatment.•Parameters for electroporation (cell growth phase, DNA quantity, buffer, temperature, and field strength) were optimized.