Cloning and/or sequencing vector

• US 20050130308A1
• Filed: 11/19/2004
• Published: 06/16/2005
• Est. Priority Date: 07/31/1992
• Status: Active Grant

• Current Assignee: Universite libre de Bruxelles
• Original Assignee: Universite libre de Bruxelles
• Inventors: Gabant, Philippe, Bernard, Philippe
• Granted Patent: US 7,176,029 B2
• Time in Patent Office: Days
• 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

1-30. -30. (canceled)

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

introducing said cloning vector into a first prokaryotic cell, said cloning vector comprising a promoter operably linked to a nucleotide sequence encoding a protein which is active as a poison to prokaryotic cells, wherein said poison protein originates from a plasmid which encodes an antidote or antipoison to said poison protein, said cell expressing said antidote or antipoison or a portion thereof that is sufficient to antagonize the killer activity of said poison protein;

propagating said cloning vector in said first prokaryotic cell without a selective pressure that modifies the cytotoxic activity of said vector, thereby expressing said poison protein without killing the cell; and

recovering the cloning vector.

32. The method of claim 31, further comprising introducing the cloning vector lacking a nucleic acid insert into a second prokaryotic cell, said second prokaryotic cell being susceptible to said protein which is active as a poison, thereby preventing growth of said second prokaryotic cell.

33. The method of claim 31, wherein said poison protein is CcdB or an active portion thereof.

34. The method of claim 33, wherein said antidote is CcdA or a portion thereof which is sufficient to antagonize the activity of CcdB or an active portion thereof.

35. The method of claim 34, wherein said first prokyarotic cell contains a mutation in a gene encoding topoisomerase II which renders said cell resistant to CcdB or an active portion thereof.

36. The method of claim 35, wherein said mutation is at position 462 if the gyrA protein.

37. The method of claim 36, wherein the arginine at position 462 is replaced with a cysteine.

38. The method of claim 31, wherein said vector contains the origin of replication from the pUC series of plasmids.

39. The method of claim 31, wherein said origin of replication is the origin of replication from pUC18 or pUC19.

40. The method of claim 31, wherein said poison protein is Kid or an active portion thereof.

41. The method of claim 40, wherein said antidote is Kis or a portion thereof which is sufficient to antagonize the activity of Kid or an active portion thereof.

42. A method of growing cells that comprise a cloning vector expressing the CcdB protein or an active portion thereof without selecting for cells containing mutations in the ccdB gene or an active portion thereof, comprising:

providing the cells with a gene encoding CcdA protein or a portion thereof that is sufficient to antagonize the activity of the CcdB protein or an active portion thereof;

providing said cells with a gene encoding topoisomerase II protein that is resistant to the CcdB protein or an active portion thereof; and

growing said cells while expressing the ccdB gene or an active portion thereof.

43. The method of claim 42, wherein the cells are grown in the absence of a repressor of transcription of the ccdB gene or a gene encoding an active portion of CcdB.

44. The method of claim 42, wherein the topoisomerase II protein has a mutation at position 462 of the gyrA protein.

45. The method of claim 44, wherein the arginine at position 462 is replaced with a cysteine.

46. A method of growing cells that comprise a cloning vector encoding CcdB or an active portion thereof comprising:

providing said cells with a cloning vector with a pUC origin of replication, said cloning vector also encoding the CcdB protein or an active portion thereof; and

providing said cells with a topoisomerase II protein which is resistant to said CcdB protein or an active portion thereof.

47. The method of claim 46, wherein said topoisomerase II protein comprises the gyrA 462 mutation.

48. The method of claim 46, wherein the cells are grown in the absence of a repressor of transcription of the ccdB gene or a gene encoding an active portion of CcdB.