Methods for lyophilizing competent cells

• US 6,960,464 B2
• Filed: 04/11/2002
• Issued: 11/01/2005
• Est. Priority Date: 02/12/1997
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Jessee, Joel A., Trinh, Thuan, Bloom, Fredric R.
• Primary Examiner(s): Afremova, Vera
• Application Number:
  US10/120,163
• Publication Number:
  US 20020164771A1
• Time in Patent Office: 1,300 Days
• Field of Search: 435/252.1, 435/252.8, 435/260, 435/849, 435/252.33, 435/252.3, 435/243, 424/93.4, 424/93.48
• US Class Current: 435/252.1
• 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. One or more lyophilized competent cells, wherein said cells have been rendered competent prior to being lyophilized, and wherein said cells exhibit a transformation efficiency of at least about 1×

10⁵ transformants per microgram of DNA upon rehydration, wherein said cells are gram negative prokaryotic cells.

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

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

4. The lyophilized competent cells of claim 3, wherein said cells are E. coli cells.

5. The lyophilized competent cells 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, XL1-Blue, XL2-Blue, AG1, JM101, JM109, JM110/SCS110, NM522, TOPP strains, ABLE Strains, XL1-Red, BL21 Strains, TK B1 Strain, and derivatives thereof.

6. The lyophilized competent cells of claim 1, wherein said cells have a transformation efficiency of at least about 1×

10⁶ transformants per microgram of DNA.

7. The lyophilized competent cells of claim 6, wherein said cells have a transformation efficiency of at least about 1×

10⁷ transformants per microgram of DNA.

8. The lyophilized competent cells of claim 7, wherein said cells have a transformation efficiency of at least about 1×

10⁸ transformants per microgram of DNA.

9. The lyophilized competent cells of claim 8, wherein said cells have a transformation efficiency of at least about 1×

10⁹ transformants per microgram of DNA.

10. The lyophilized competent cells of claim 1, wherein the transformation efficiency of said cells after storage for 30 days to 120 days at −

20°

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

11. The lyophilized competent cells of claim 10, 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.

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

200°

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

13. The lyophilized competent cells of claim 12, 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.