Methods for lyophilizing competent cells

• US 7,648,832 B2
• Filed: 08/31/2005
• Issued: 01/19/2010
• Est. Priority Date: 02/12/1997
• Status: Expired due to Fees

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Jessee, Joel A., Trinh, Thuan, Bloom, Fredric R.
• Primary Examiner(s): Afremova; Vera
• Application Number:
  US11/214,800
• Publication Number:
  US 20060014255A1
• Time in Patent Office: 1,602 Days
• Field of Search: 435/260
• US Class Current: 435/260
• CPC Class Codes: A01N 1/02 Preservation of living parts
• CPC Class Codes: A01N 1/0284 Temperature processes, i.e. using a designated change in temperature over time
• CPC Class Codes: C12N 1/04 Preserving or maintaining viable microorganisms immobilised microorganisms C12N11/00
• CPC Class Codes: Y10S 435/849 Escherichia coli

Abstract

This invention relates to a method for producing cells which are competent for transformation and which may be stably stored for extended periods of time at various temperatures. The method involves growing cells in a growth conducive medium, rendering said cells competent, and lyophilizing said competent cells. The invention further relates to competent cells produced by such a method, to methods of transforming said cells with a DNA molecule, and to a method of producing a desired protein or polypeptide from said transformed cells.

Claims

1. A method of preparing lyophilized gram negative prokaryotic competent cells, comprising first rendering gram negative prokaryotic cells competent, then lyophilizing said competent cells, wherein said lyophilized competent cells exhibit a transformation efficiency of between about 1×

10⁵ and 1×

10⁹ transformants per microgram of DNA upon rehydration.

2. The method of claim 1, wherein said cells are selected from the group consisting of Escherichia, Klebsiella, Salmonella and Pseudomonas.

3. The method of claim 2, wherein said cells are Escherichia cells.

4. The method of claim 3, wherein said Escherichia cells are E. coli cells.

5. The method of claim 4, wherein said cells are selected from the group consisting of DH5, DH5α

, DH10B, HB101, RR1, JV30, DH11S, DM1, DH10B/p3, DH5α

MCR, DH5α

F′

IQ, DH5α

F′

, SCS1, Stb12, DH12S, DH5α

-E, DH10BAC, XL-1Blue MRF, XL2-Blue MRF, XL1-Blue MR, SURE Strain, SURE 2 Strain, XL-1 Blue, XL2-Blue, AG1, JM101, JM109, JM110/SCS110, NM522, TOPP strains, ABLE Strains, SL1-Red, BL21 Strains, TK BI Strain and derivatives thereof.

6. The method of claim 1, wherein said cells have a transformation efficiency of about 1×

10⁵ transformants per microgram of DNA.

7. The method of claim 1, wherein said cells have a transformation efficiency of about 1×

10⁶ transformants per microgram of DNA.

8. The method of claim 1, wherein said cells have a transformation efficiency of about 1×

10⁷ transformants per microgram of DNA.

9. The method of claim 1, wherein said cells have a transformation efficiency of about 1×

10⁸ transformants per microgram of DNA.

10. The method of claim 1, wherein said cells have a transformation efficiency of about 1×

10⁹ transformants per microgram of DNA.

11. A method of preparing lyophilized gram negative prokaryotic competent cells, comprising first rendering gram negative prokaryotic cells competent, then lyophilizing said competent cells, wherein said lyophilized competent cells upon rehydration exhibit a transformation efficiency after storage for 30 days to 120 days at −

20°

C. that is about 40% to 100% of the transformation efficiency that said cells exhibit upon rehydration immediately after lyophilization.

12. The method of claim 11, wherein said cells are selected from the group consisting of Escherichia, Klebsiella, Salmonella and Pseudomonas.

13. The method of claim 12, wherein said cells are Escherichia cells.

14. The method of claim 13, wherein said Escherichia cells are E. coli cells.

15. The method of claim 14, wherein said cells are selected from the group consisting of DH5, DH5α

, DH10B, HB101, RR1, JV30, DH11S, DM1, DH10B/p3, DH5α

MCR, DH5α

F′

IQ, DH5α

F′

, SCS1, Stb12, DH12S, DH5α

-E, DH10BAC, XL-1Blue MRF, XL2-Blue MRF, XL1-Blue MR, SURE Strain, SURE 2 Strain, XL-1 Blue, XL2-Blue, AG1, JM101, JM109, JM110/SCS110, NM522, TOPP strains, ABLE Strains, SL1-Red, BL21 Strains, TK Bi Strain and derivatives thereof.

16. The method of claim 11, wherein the transformation efficiency of said cells after storage for 30 days to 120 days at −

20°

C. is about 60% to 100% of the transformation efficiency of said cells immediately after lyophilization.

17. The method of claim 11, wherein the transformation efficiency of said cells after storage for 30 days to 120 days at −

20°

C. is about 70% to 100% of the transformation efficiency of said cells immediately after lyophilization.

18. The method of claim 11, wherein the transformation efficiency of said cells after storage for 30 days to 120 days at −

20°

C. is about 80% to 100% of the transformation efficiency of said cells immediately after lyophilization.

19. A method of preparing lyophilized competent cells, comprising (a) growing said cells in a growth conducive medium, (b) rendering the cells competent, and then (c) lyophilizing said competent cells , wherein said competent cells do not appreciably lose transformation efficiency when stored for extended periods of time.

20. The method of claim 19, wherein said competent cells are lyophilized in the presence of a cryoprotectant.

21. The method of claim 19, wherein said cells are rendered competent by incubating the cells in a competence buffer.

22. The method of claim 21, wherein said cells are incubated in a competence buffer at a temperature between 0°

C. to 4°

C.

23. The method of claim 19, wherein said cells are E. coli.

24. The method of claim 23, wherein said E. coli cells are selected from the group consisting of DH5α

and DH10B.

25. The method of claim 19, wherein said lyophilized competent cells exhibit a transformation efficiency of between about 1×

10⁵ and 1×

10⁹ transformnants per microgram of DNA upon rehydration.

26. The method of claim 19, wherein said lyophilized competent cells upon rehydration and after storage for an extended period of time exhibit a transformation efficiency that is about 40% to 100% of the transformation efficiency of what said cells exhibit when rehydrated immediately after lyophilization without being subjected to storage for an extended period of time.

27. The method of claim 26, wherein said storage is for 30 days to 120 days.

28. The method of claim 26, wherein said storage is at −

20°

C.

29. A composition comprising:

a plurality of competent gram negative bacterial cells; and

at least one cryoprotectant;

wherein said composition is lyophilized, and wherein said competent gram negative bacterial cells, when rehydrated, exhibit a transformation efficiency of between about 10⁵ to about 10⁹ transformants/μ

g DNA.

30. The composition according to claim 29, wherein said competent gram negative bacterial cells are storage stable from room temperature to about −

80°

C.

31. The composition according to claim 29, wherein said competent gram negative bacterial cells are storage stable at −

20°

C.

32. The composition according to claim 29, wherein said competent gram negative bacterial cells are storage stable at −

20°

C. for at least 1 year.

33. The composition according to claim 29, wherein said cryoprotectant is selected from the list trehalose, sucrose, lactose, maltose, mannitol, galactose, ribose, fructose, xylose, mannose, dextrose, glucose, and sorbitol, polyethyleneamine, polyvinylpyrrolidone (PYP), ficoll, acacia gum, albumin, gelatin, and a sugar alcohol.

34. The composition according to claim 29, wherein said gram negative bacterial cells are selected from the group consisting of Escherichia, Klebsiella, Salmonella and Pseudomonas.

35. The composition according to claim 29, wherein said gram negative bacterial cells are Escherichia cells.

36. The method of claim 29, wherein said gram negative bacterial cells are E. coli cells.

37. The method of claim 29, wherein said gram negative bacterial cells are selected from the group consisting of DH5, DH5α

, DH10B, HB101, RR1, JV30, DH11S, DM1, DH10B/p3, DH5α

MCR, DH5α

F′

IQ, DH5α

F′

, SCS1, Stb12, DH12S, DH5α

-E, DH10BAC, XL-1Blue MRF, XL2-Blue MRF, XL1-Blue MR, SURE Strain, SURE 2 Strain, XL-1 Blue, XL2- Blue, AG1, JM101, JM109, JM110/SCS110, NM522, TOPP strains, ABLE Strains, SL1-Red, BL21 Strains, TK Bi Strain and derivatives thereof.