Cells resistant to toxic genes and uses thereof

• US 20040053412A1
• Filed: 03/26/2003
• Published: 03/18/2004
• Est. Priority Date: 03/02/1999
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Temple, Gary F., Hartley, James L., Brasch, Michael A.
• Application Number:
  US10/396,696
• Publication Number:
  US 20040053412A1
• Time in Patent Office: Days
• US Class Current: 435/473
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: C12N 15/01 Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
• CPC Class Codes: C12N 9/90 Isomerases (5.)

Abstract

The present invention relates to cells and cell strains that are resistant to the killing effects of one or more toxic genes, particularly those that kill hosts in the absence of a suppressing function, e.g., kicB or ccdB. The host cells may comprise one or more suppression mutations, such as deletional or insertional mutations in gyrA, endA, or recA, or combinations thereof (particularly gyrA/endA or gyrA/recA), which allow cell strains carrying the one or more suppression mutations to survive the presence and/or expression of one or more toxic genes within their genome or in extrachromosomal genetic elements within the host cell. Preferred host cell strains include prokaryotic host cells, particularly specified strains of E. coli containing the gyrA462 mutation and/or one or more additional mutations, such as DB3, DB3.1, DB4 and DB5. The host cells of the invention are useful in producing recombinant genetic constructs, particularly cDNAs and cDNA libraries, via traditional genetic engineering techniques or via recombinational cloning using engineered recombination sites. The host cells are also useful in cloning and propagation of toxic genes that act upon DNA gyrase, such as ccdB.

Claims

1. A mutant host cell containing a gyrA gene, an endA gene, and a recA gene, wherein said gyrA and endA genes contain one or more mutations that render said host cell resistant to the expression of one or more toxic genes.

2. The mutant host cell of claim 1, further comprising one or more mutations in said recA gene.

3. The mutant host cell of claim 1, further comprising one or more mutations in one or more additional genes which render said host cell resistant to the expression of two or more toxic genes.

4. The mutant host cell of claim 1 or claim 2, further comprising one or more genetic elements that enable said mutant host cell to grow on tetracycline-containing culture media.

5. The mutant host cell strain of claim 1 or claim 2, wherein said mutation in said gyrA gene is gyrA462.

6. The mutant host cell strain of claim 1, wherein said mutation in said recA gene is Δ

(srl-recA)1398.

7. The mutant host cell of claim 4, wherein said genetic element is a tetracycline resistance gene or transposon Tn10.

8. The mutant host cell of claim 1, wherein said host cell is an Escherichia coli cell.

9. The mutant host cell of claim 1, wherein said toxic gene is selected from the group consisting of ccdB, kicB, DpnI, an apoptosis-related gene, a retroviral gene, a defensin, a bacteriophage lytic gene, Φ

X E, an antibiotic sensitivity gene, an antimicrobial sensitivity gene, a plasmid killer gene, and a eukaryotic transcriptional vector gene that produces a gene product toxic to bacteria.

10. The mutant host cell of claim 1, wherein said toxic gene is ccdB.

11. The mutant host cell of claim 1, wherein said host cell is selected from the group consisting of a DB3 cell (deposit number NRRL B-30097), a DB3.1 cell (deposit number NRRL B-30098), a DB4 cell (deposit number NRRL B-30106), and a DB5 cell (deposit number NRRL B-30107), or a mutant or derivative thereof.

16. A method of cloning a genetic construct comprising one or more toxic genes, said method comprising introducing said genetic construct into the host cell of claim 1 or claim 2 and cultivating said host cell under conditions favoring the clonal expansion of said host cell.

17. The method of claim 16, wherein said toxic gene is selected from the group consisting of ccdB, kicB, DpnI, an apoptosis-related gene, a retroviral gene, a defensin, a bacteriophage lytic gene, Φ

X E, an antibiotic sensitivity gene, an antimicrobial sensitivity gene, a plasmid killer gene, and a eukaryotic transcriptional vector gene that produces a gene product toxic to bacteria.

18. The method of claim 16, wherein said toxic gene is ccdB.

20. A kit comprising one or more of the mutant host cells of claim 1 or claim 2.

21. The kit of claim 20, further comprising one or more additional components selected from the group consisting of one or more culture media suitable for cultivation of said host cell, one or more selection agents, one or more genetic constructs comprising one or more toxic genes, one or more enzymes, one or more nucleotides, one or more buffers, and the like.

12. Mutant host cell strain DB3 (deposit number NRRL B-30097).

13. Mutant host cell strain DB3.1 (deposit number NRRL B-30098).

14. Mutant host cell strain DB4 (deposit number NRRL B-30106).

15. Mutant host cell strain DB5 (deposit number NRRL B-30107).

19. The method of claim 15, wherein said host cell is selected from the group consisting of a DB3 cell (deposit number NRRL B-30097), a DB3.1 cell (deposit number NRRL B-30098), a DB4 cell (deposit number NRRL B-30106), and a DB5 cell (deposit number NRRL B-30107), or a mutant or derivative thereof.

22. The kit of claim 19, wherein said host cell is selected from the group consisting of a DB3 cell (deposit number NRRL B-30097), a DB3.1 cell (deposit number NRRL B-30098), a DB4 cell (deposit number NRRL B-30106), and a DB5 cell (deposit number NRRL B-30107), or a mutant or derivative thereof.