22] Bacterial transformation using temperature-sensitive mutants deficient in peptidoglycan synthesis

Abstract

This chapter discusses bacterial transformation using temperature-sensitive mutants deficient in peptidoglycan synthesis. Bacterial transfection and transformation are essential processes in molecular cloning experiments. The frequency of transformation is determined by the uptake of deoxyribonucleic acid (DNA) molecules and by the restriction system of the recipient cell. This chapter describes a novel bacterial spheroplast transformation system that is capable of high transfection frequencies, high frequency of regenerating viable cells, and applicable for all bacterial species, containing peptidoglycan. Using a variety of bacterophage DNAs, high transfection frequencies in spheroplasts of Escherichia coli (tsPG – ) are observed. These transfection frequencies are comparable to those obtained with lysozyme-treated spheroplasts and higher than those obtained with calcinated cells. The tsPG – can be used for the transformation with plasmid and chromosomal DNA. The unique advantage of the tsPG – spheroplast system is its high efficiency (95–99%) in generating viable cells at 30°. In principle, this method can be applied to select competent cells for transformation from any bacterial species that contain peptidoglycan.