High-efficiency transformation of bacterial cells by electroporation

Abstract

We have developed a method for efficiently generating transient pores in the outer membranes ofEscherichia coli K-12 derivatives by using a new type of electroporation apparatus. The pores are large enough and persist long enough to facilitate the equilibration of plasmid molecules between the intracellular and extraceilular spaces. The method has been used to transform bacterial cells with an efficiency greater than 109 transformants per ,ug of plasmid. It has also been used to extract intact plasmid from transformed cells with efficiencies comparable to those of the traditional alkaline lysis or CsCl equilibrium density gradient techniques. The technique is simple and rapid, allowing a transformation or the preparation of microgram quantities of plasmid to be accomplished in minutes.

SpsA, a novel pneumococcal surface protein with specific binding to secretory Immunoglobulin A and secretory component

“…Plasmid DNA was isolated using the Qiagen Plasmid Kit (Qiagen). Transformation of E. coli with pneumococcal spsA gene fragments cloned into pQE30 vector (Qiagen) was achieved by electroporation (Calvin and Hanawalt, 1988). Nucleotide sequence determination was performed by ABI PRISM dye terminator cycle sequencing (Perkin-Elmer) according to the protocol of the manufacturers with pBK-CMV vector primers T3X (5Ј-AATTAACCCTCACTAAAGG-G-3Ј) and T7X (5Ј-TAATACGACTCACTATCGGG-3Ј).…”

Biolistic transformation of prokaryotes: factors that affect biolistic transformation of very small cells

“…Also, transformation has been demonstrated in some Gram-negative bacteria and in protoplasts of Gram-positive bacteria using the electroporation method (Bonnassie et al, 1990;Calvin & Hanawalt, 1988;Fiedler & Wirth, 1988). In this paper we describe a widely applicable transformation method which requires no pretreatment of cells but directly bombards bacterial cells spread on the surface of selective medium with DNA-coated tungsten particles.…”

Direct transfer of genetic material between two Escherichia coli K12 strains by electroporation

“…1,2 Electroporation is also an increasing recognized method for introducing DNA into bacterial cells and for promoting plasmid loss from bacteria. 3,4 In particular this technique was employed in transformation experiments to stimulate the uptake of plasmid DNA, and in direct transfer of genetic material to recipient cells not only among organisms of the same species, but also among bacteria genetically not correlated to each other. 2 Many different experimental conditions were also explored to enhance the efficiency of DNA and proteins exchange.…”

“…The strong reduction of the number of recombinants when DNAse was added suggests that the free genetic material is digested by the enzyme, The low number of recombinants still obtained under these experimental conditions might be due to the presence of residual DNA not completely eliminated by the enzyme. On the other hand, the pores appear stable and can be seen under an electronic microscope, [2][3][4][5][6][7][8][9][10][11][12][13] bacterial cells that are not totally killed by the treatment and required about 1 hour to resume their original growth rate, 11 might have enough time to acquire the residual genetic material or establish a direct contact between each other. It is also possible that when pores are formed might fit together with those of the partner cells establishing a direct cell to cell connection enabling all cytoplasmic material to be exchange.…”