A method for genetic transformation of nonprotoplasted Streptococcus lactis.
Plasmid transformation of whole cells of Streptococcus lactis LM0230 was demonstrated. The procedure required polyethylene glycol and incubation in hypertonic media, but did not require enzymatic cell wall digestion. Conditions were optimized, yielding 5 X 10(5) transformants per micrograms of pSA3 DNA. Variables tested for effect on transformation efficiency included molecular weight, concentration, and pH of polyethylene glycol; cell density; plating media; DNA concentration; heat shock; and incubation of cells in hypertonic buffer. DNAs transformed included pSA3, pVA856, pTV1, and c2 phi. Transformation from DNA-DNA ligation mixes, with DNA not purified through density gradients, and with previously frozen cells was also achieved. The method described here for transformation of nonprotoplasted cells of LM0230 is unique, and to date has not been applied successfully to other lactic acid bacteria.
D003001:Cloning, Molecular; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D006863:Hydrogen-Ion Concentration; D013294:Lactococcus lactis / Q000235:genetics*; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011092:Polyethylene Glycols; D014169:Transformation, Bacterial
D003470:Culture Media; D004269:DNA, Bacterial; D011092:Polyethylene Glycols