Process of producing highly transformable cells and cells produced thereby

• US 4,981,797 A
• Filed: 11/14/1988
• Issued: 01/01/1991
• Est. Priority Date: 08/08/1985
• Status: Expired due to Term

• Current Assignee: Life Technologies Incorporated (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Jessee, Joel A., Bloom, Fredric R.
• Primary Examiner(s): Schwartz, Richard A.
• Assistant Examiner(s): Peet, Richard C.
• Application Number:
  US07/273,646
• Time in Patent Office: 778 Days
• Field of Search: 435/91, 435/170, 435/172.1, 435/172.3, 435/252.3, 435/849, 435/252.8, 435/320, 935/52, 935/55, 935/56, 935/58, 935/66, 935/72, 935/73
• US Class Current: 435/252.8
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: Y10S 435/849 Escherichia coli

Abstract

Process for producing transformable, competent cells including the steps of growing the cells in a growth conducive medium at a temperature of less than 37° C. and freezing the cells, and cells produced by the process.

Claims

1. A process for producing transformable E. coli cells of improved competence by a process comprising the following steps in order:

(a) growing E. coli cells in a growth-conducive medium at a temperature of 18°

C. to 32°

C.;

(b) rendering said E. coli cells competent; and

(c) freezing the cells.

2. The process according to claim 1 wherein the cells are E. coli cells having competency for plasmid DNA.

3. The process according to claim 1 wherein the cells are E. coli cells of at least one of the following strains:

RR1, HB101, DH1, DH5.

4. The process according to claim 1 wherein the growth conducive medium is a complex medium containing Mg.

5. The process according to claim 1 wherein the cells are E. coli, the step of growing the cells includes growing the cells within the range 18°

C. to 32°

C. for a period within a range of from 1 to 20 hours.

6. The process according to claim 1 wherein the cells are E. coli, the step of growing the cells includes growing the cells in the range of 18°

C. to 32°

C. until the optical density at 550 nm reaches the range of 0.5 to 1.0.

7. The process according to claim 1 and further including the step of washing the cells in a buffer prior to the freezing step.

8. The process according to claim 1 wherein the cells are E. coli, and the step of freezing the cells comprises freezing the cells at approximately -70°

C.

9. The process according to claim 1 wherein the cells are E. coli, and the step of freezing the cells comprises freezing the cells in a dry ice-ethanol bath at approximately -70°

C.

10. The process according to claim 1 and further including the step of storing the cells at -70°

C. after the freezing step.

11. The process according to claim 1 wherein the cells are E. coli, and the step of growing the cells includes growing the cells in the presence of sodium chloride.

12. The process according to claim 1 wherein the cells are E. coli, and the step of growing the cells includes growing the cells in the presence of sodium succinate.

13. The process according to claim 1 and further including the steps of thawing and refreezing the cells after said freezing step.

14. The process according to claim 1, wherein the process further comprises the following step between step (a) and step (b):

(a'"'"') harvesting the cells by centrifugation.

15. The process according to claim 14, wherein the process further comprises the following step between step (a'"'"') and step (b):

(a") rapidly resuspending the cells in an appropriate buffer.

16. The process of claim 1, wherein said E. coli are rendered competent in step (b) by washing the cells with a buffer comprising potassium acetate, KCl, MnCl₂.4H₂ O, CaCl₂.2H₂ O, hexamine cobalt chloride and redistilled glycerol.

17. Transformable E. coli cells of improved competence produced by the following steps in order:

(a) growing E. coli cells in a growth-conducive medium at a temperature of 18°

C. to 32°

C.;

(b) rendering said E. coli cells competent; and

(c) freezing the cells.

18. The competent transformable cells according to claim 17 wherein the cells are E. coli having a competency for plasmid DNA, and a transformation efficiency of at least 1×

10⁹ transformants per microgram of plasmid DNA.

19. The competent transformable cells according to claim 17 wherein the cells are E. coli having high transformation efficiencies, and are made by the further steps of repeatedly thawing and freezing the cells after said freezing step without significantly diminishing the transformation efficiencies.

20. The competent transformable cells according to claim 17 wherein the cells are frozen competent E. coli strain RR1.

21. The competent transformable cells according to claim 17 wherein the cells are frozen competent E. coli strain HB101.

22. The competent transformable cells according to claim 17 wherein the cells are frozen competent E. coli strain DH1.

23. The competent transformable cells according to claim 17 wherein the cells are frozen competent E. coli strain DH5.

24. The competent transformable E. coli cells of claim 17, wherein said E. coli are rendered competent in step (b) by washing the cells in a buffer comprising potassium acetate, KCl, MnCl₂.4H₂ O, CaCl₂.2H₂ O, hexamine cobalt chloride and redistilled glycerol.