1. Field of the Invention
The present invention relates generally to the permeabilization of prokaryotic cell walls by electroporation. More particularly, the invention relates to a method and system for the electroporation-enhanced transformation of prokaryotic cells with nucleic acids.
Electroporation has heretofore been employed to assist in the transfection of eukaryotic cells, including intact animal cells and plant protoplasts, both of which are substantially free from rigid cell walls. The cells to be transformed are suspended in a solution between a pair of electrodes, and an electrical pulse is supplied which has the effect of increasing the permeability of the cell membrane to the passage of nucleic acids and other substances. The field strength employed in the electroporation of eukaryotic cells has typically been in the range from about 0.5 to 1 kV/cm.
Electroporation has been used only to a very limited extent in the transformation of prokaryotic cells. The rigid cell wall of prokaryotic cells appears to be generally refractory to the passage of nucleic acids under the conditions of electroporation which have been utilized with eukaryotic cells. Electroporation of prokaryotes has generally been successful only after the rigid cell wall has been at least partially digested.
At present, chemical treatment is the method of choice for promoting transformation of prokaryotic cells by the uptake of exogenous nucleic acids. Such chemical treatment, however, varies widely in effectiveness among different types of prokaryotes. Even with the most amenable bacterial hosts, e.g., E. coli, the efficiency of transformation achieved using chemical treatment methods seldom exceeds about 10.sup.8 transformants/.mu.g of nucleic acids and is frequently much less. Moreover, the chemical treatment methods often require lengthy regeneration periods for the treated hosts and do not always achieve consistent results.
For these reasons, it would be desirable to provide methods and systems for performing electroporation of prokaryotic cells, where said methods and systems are capable of inducing permeability of the rigid cell wall. It would be particularly desirable if such methods were effective with most or all types of prokaryotic cells, were capable of providing a very high efficiency of transformation.
2. Description of the Background Art
A method for the electroporation-assisted transformation of E. coli is described in a brochure entitled "Gene Pulser.TM. Transformation Apparatus Operating Instructions and Applications Guide," distributed by Bio-Rad Laboratories, Inc., Richmond, Calif. 94804. The method employs a cell suspension volume of 0.8 ml and discloses a maximum transformation efficiency of 10.sup.6 cells/.mu.g DNA. Following the instructions in the Bio-Rad Applications Guide, transformation of Lactobacillus casei (Chassy and Flickinger (1987) FEMS Microbiology Lett. 44:173-177) and Streptococcus lactis (Powell et al. (1988) Appl. Environ. Microbiol. 54:655-660) have also been achieved. Harlander (1987) "Transformation of Streptococcus lactis by Electroporation," in Streptococcal Genetics, Freti and Curtis, eds., pp 229-233, describes the electroporation of intact S. lactis, where the cells are subjected to very short (5 .mu.sec) electric pulses of up to 11.6 kV/cm while being centrifuged in a sucrose density gradient. U.S. Pat. No. 4,081,340, describes a method for increasing the permeability of the skin of cells by applying a constant electric field to a flowing suspension of cells. Potter et al. (1984) Proc. Natl. Acad. Sci. USA 81 7161-7165 describes an electroporation cell having opposed electrodes spaced by 0.5 cm and used to transfect mouse lymphocytes and fibroblasts. Although reference is made to the transformation of E. coli cells, no conditions are disclosed for achieving such transformation. Zeitschrift Fur Allgemeine Mikrobiologie (1983) 23:595 describes the electroporation of polyethylene glycol-treated Bacillus protoplasts which resulted in an increased rate of transformation. Miller et al. (1988) Proc. Natl. Acad. Sci. USA 85: 856-860 describes the method of the present invention applied to the transformation of Campylobacter jejuni.
USPTO Backgrounds
USPTO Backgrounds