Cloning and/or sequencing vector

• US 7,176,029 B2
• Filed: 11/19/2004
• Issued: 02/13/2007
• Est. Priority Date: 07/31/1992
• Status: Expired due to Term

• Current Assignee: Universite libre de Bruxelles
• Original Assignee: Universite libre de Bruxelles
• Inventors: Gabant, Philippe, Bernard, Philippe
• Primary Examiner(s): Priebe; Scott D.
• Assistant Examiner(s): BURKHART, MICHAEL D
• Application Number:
  US10/994,208
• Publication Number:
  US 20050130308A1
• Time in Patent Office: 816 Days
• Field of Search: None
• US Class Current: 435/471
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli

Abstract

A cloning and/or sequencing vector enables recombinant clones to be selected directly. The vector encodes a fusion protein which includes a protein poison.

Claims

1. A method for propagating a cloning or sequencing vector that lacks an insert, said method comprising:

introducing said vector into a first prokaryotic cell, said cloning vector comprising a promoter operably linked to a nucleotide sequence encoding a CcdB poison protein, said cell expressing a CcdA antidote protein;

propagating said vector in said first prokaryotic cell, thereby expressing said CcdB poison protein without killing the cell; and

recovering the vector.

2. The method of claim 1, further comprising introducing the vector lacking a nucleic acid insert into a second prokaryotic cell, said second prokaryotic cell being susceptible to said CcdB poison protein, thereby preventing growth of said second prokaryotic cell.

3. The method of claim 1, wherein said first prokaryotic cell comprises a mutation at amino acid position 462 of Escherichia coli GyrA.

4. The method of claim 3, wherein the mutation at amino acid position 462 of E. coli GyrA comprises replacement of arginine with a cysteine.

5. The method of claim 1, wherein said vector comprises an origin of replication from the pUC series of plasmids.

6. The method of claim 5, wherein said origin of replication is the origin of replication from pUC18 or pUC19.

7. A method of growing prokaryotic cells that comprise a cloning vector expressing a CcdB poison protein without selecting for prokaryotic cells containing mutations in the ccdB gene, comprising:

providing the prokaryotic cells with a gene encoding a CcdA antidote protein so as to antagonize the activity of the CcdB poison protein

providing said cells with a gene encoding E. coli GyrA having a mutation at amino acid position 462; and

growing said cells while expressing the CcdB poison protein.

8. The method of claim 7, wherein the mutation at amino acid position 462 of GyrA comprises replacement of arginine with cysteine.

9. A method of growing prokaryotic cells that comprise a cloning vector encoding a CcdB poison protein comprising:

providing said prokaryotic cells with a cloning vector with a pUC origin of replication, said cloning vector also encoding the CcdB poison protein; and

providing said prokaryotic cells with E. coli GyrA having a mutation at amino acid position 462.

10. The method of claim 9, wherein the mutation at amino acid position 462 of GyrA comprises replacement of arginine with cysteine.

11. The method of claim 9, wherein said pUC origin of replication is the origin of replication from pUC 18 or pUC 19.

12. A method for propagating a cloning or sequencing vector that lacks an insert, said method comprising:

introducing said vector into a first prokaryotic cell, said cloning vector comprising a promoter operably linked to a nucleotide sequence encoding a CcdB poison protein, said cell expressing a polypeptide consisting of the 41 -carboxy terminal amino acids of a CcdA antidote protein;

propagating said vector in said first prokaryotic cell, thereby expressing the CcdB poison protein without killing the cell; and

recovering the vector.

13. The method of claim 12 further comprising introducing the vector lacking a nucleic acid insert into a second prokaryotic cell, said second prokaryotic cell being susceptible to the CcdB poison protein, thereby preventing growth of said second prokaryotic cell.

14. The method of claim 12, wherein said first prokaryotic cell comprises a mutation at amino acid position 462 of E. coli GyrA.

15. The method of claim 14, wherein the mutation at amino acid position 462 of GyrA comprises replacement of arginine with a cysteine.

16. The method of claim 12, wherein said vector comprises an origin of replication from the pUC series of plasmids.

17. The method of claim 16, wherein said origin of replication is the origin of replication from pUC 18 or pUC 19.

18. The method of claim 12, wherein said vector comprises said nucleic acid encoding the CcdB poison protein and a nucleic acid encoding the 41 -carboxy terminal amino acids of the CcdA antidote protein.