Rapid growing microorganisms for biotechnology applications

• US 6,709,852 B1
• Filed: 06/22/2000
• Issued: 03/23/2004
• Est. Priority Date: 06/22/1999
• Status: Expired due to Term

• Current Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Lin, Jhy-Jhu, Schmidt, Brian J., Bloom, Fredric R.
• Primary Examiner(s): Guzo, David
• Assistant Examiner(s): LAMBERTSON, DAVID A
• Application Number:
  US09/599,452
• Time in Patent Office: 1,370 Days
• Field of Search: 435/69.1, 435/91.1, 435/243, 435/252.1, 435/252.33
• US Class Current: 435/243
• CPC Class Codes: C12N 1/205 Bacterial isolates
• CPC Class Codes: C12N 1/36 Adaptation or attenuation of cells
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/72 Expression systems using regulatory sequences derived from the lac-operon
• CPC Class Codes: C12R 2001/19 Escherichia coli

Abstract

The present invention provides novel rapidly growing microorganisms and methods for their use in cloning or subcloning nucleic acid molecules. The rapid growing microorganisms of the present invention form colonies more rapidly than microorganisms typically used in molecular biology and thus provide a significant improvement in in vitro cloning methods used extensively in molecular biology. The invention also relates to kits and compositions used in the methods of the invention.

Claims

1. An isolated E. coli lacking endogenous plasmids and having a growth rate that is at least 5% greater than the growth rate of at least one microorganism selected from the group consisting of E. coli MM294, DH5α

and DH10B.

2. The isolated E. coli of claim 1, wherein said isolated E. coli are E. coli strain W or strain C.

3. The isolated E. coli of claim 1, wherein said isolated E. coli have a growth rate that is at least 5% greater than the growth rate of E. coli MM294.

4. The isolated E. coli of claim 1, wherein said isolated E. coli have a growth rate that is at least 5% greater than the growth rate of E. coli DH5α

.

5. The isolated E. coli of claim 1, wherein said isolated E. coli have a growth rate that is 5 to 200% greater than the growth rate of E. coli MM294.

6. The isolated E. coli of claim 1, wherein said isolated E. coli have a growth rate that is at least 25% greater than the growth rate ofE. coli MM294.

7. The isolated E. coli of claim 1, wherein said isolated E. coli have a growth rate that is at least 50% greater than the growth rate of E. coli MM294.

8. The isolated E. coli of claim 1 wherein said isolated E. coli have a growth rate that is at least 100% greater than the growth rate of E. coli MM294.

9. A composition comprising E. coli, wherein said E. coli lack endogenous plasmids and have a growth rate that is at least 5% greater than the growth rate of at least one microorganism selected from the group consisting of E. coli MM294, DH5α

and DH10B.

10. The composition of claim 9, further comprising a component selected from the group consisting of a glycerol solution and a competence buffer.

11. The composition of claim 9, further comprising at least one component selected from the group consisting of one or more DNA fragments, one or more ligase enzymes, one or more vectors, one or more buffering salts, and one or more recombination proteins.

12. The composition of claim 9, wherein said E. coli have a growth rate that is at least 5% greater than the growth rate of E. coli MM294.

13. The composition of claim 9, wherein said E. coli have a growth rate that is at least 5% greater than the growth rate of E. coli DH5α

.

14. The composition of claim 9, wherein said E. coli have a growth rate that is 5 to 200% greater than the growth rate of E. coli MM294.

15. The composition of claim 9, wherein said E. coli have a growth rate that is at least 25% greater than the growth rate of E. coli MM294.

16. The composition of claim 9, wherein said E. coli have a growth rate that is at least 50% greater than the growth rate of E. coli MM294.

17. The composition of claim 9, wherein said E. coli have a growth rate that is at least 100% greater than the growth rate of E. coli MM294.

18. A kit for cloning comprising a container containing E. coli lacking endogenous plasmids and having a growth rate that is at least 5% greater than the growth rate of at least one microorganism selected from the group consisting of E. coli MM294, DH5α

and DH10B.

19. The kit of claim 18, wherein said E. coli have a growth rate that is at least 5% greater than the growth rate of E. coli MM294.

20. The kit of claim 18, wherein said E. coli have a growth rate that is at least 5% greater than the growth rate of E. coli DH5α

.

21. The kit of claim 18, wherein said E. coli have a growth rate that is 5 to 200% greater than the growth rate of E. coli MM294.

22. The kit of claim 18, wherein said E. coli have a growth rate that is at least 25% greater than the growth rate of E. coli MM294.

23. The kit of claim 18, wherein said E. coli have a growth rate that is at least 50% greater than the growth rate of E. coli MM294.

24. The kit of claim 18, wherein said E. coli have a growth rate that is at least 100% greater than the growth rate of E. coli MM294.

25. The kit of claim 18, wherein said E. coli are competent.

26. The kit of claim 25, wherein said E. coli are chemically competent.

27. The kit of claim 25, wherein said E. coli are electrocompetent.

28. The kit of claim 18, further comprising one or more vectors.

29. The kit of claim 28, further comprising at least one component selected from the group consisting of one or more restriction enzymes, one or more ligase enzymes, and one or more polymerases.

30. The kit of claim 29, further comprising a container containing a recombination protein.

31. An E. coli having deposit number NRRL B-30143 and derivatives thereof.

32. An E. coli having deposit number NRRL B-30144 and derivatives thereof.

33. An E. coli having a growth rate that is at least 5% greater than the growth rate of at least one microorganism selected from the group consisting of E. coli MM294, DH5α

and DH10B, wherein said E. coli has been made competent.

34. The E. coli of claim 33, wherein said E. coli are E. coli strain W or strain C.

35. The E. coli of claim 33, wherein said E. coli have a growth rate that is at least 5% greater than the growth rate of E. coli MM294.

36. The E. coli of claim 33, wherein said E. coli have a erowth rate that is at least 5% greater than the growth rate of E. coli DH5α

.

37. The E. coli of claim 33, wherein said E. coli have a growth rate that is 5 to 200% greater than the growth rate of E. coli MM294.

38. The E. coli of claim 33, wherein said E. coli have a growth rate that is at least 25% greater than the growth rate of E. coli MM294.

39. The E. coli of claim 33, wherein said E. coli have a growth rate that is at least 50% greater than the growth rate of E. coli MM294.

40. The E. coli of claim 33, wherein said E. coli have a growth rate that is at least 100% greater than the growth rate of E. coli MM294.

41. The E. coli of claim 33, wherein said E. coli are chemically competent.

42. The E. coli of claim 33, wherein said E. coli are electrocompetent.

43. An E. coli having a growth rate that is at least 5% greater than the growth rate of at least one microorganism selected from the group consisting of NRRL B-30143, and NRRL B-30144, wherein said E. coli has been made competent.

44. A method of producing E. coli for cloning, comprising:

(a) obtaining E. coli having endogenous plasmids and having a growth rate that is at least 50% greater than the growth rate of at least one microorganism selected from the group consisting of E. coli MM294, DH5α and

DH10B; and

(b) curing said E. coli of endogenous plasmids.

45. The method of claim 44, wherein said E. coli are E. coli strain W or strain C.

46. The method according to claim 44, wherein said E. coli have a growth rate that is at least 5% greater than the growth rate of E. coli MM294.

47. The method according to claim 44, wherein said E. coli have a growth rate that is at least 5% greater than the growth rate of E. coli DH5α

.

48. The method of claim 44, wherein said E. coli have a growth rate that is 5 to 200% greater than the growth rate of E. coli MM294.

49. The method of claim 44, wherein said E. coli have a growth rate that is at least 25% greater than the growth rate of E. coli MM294.

50. The method of claim 44, wherein said E. coli have a growth rate that is at least 50% greater than the growth rate of E. coli MM294.

51. The method of claim 44, wherein said E. coli have a growth rate that is at least 100% greater than the growth rate of E. coli MM294.

52. A method of making competent E. coli, comprising:

(a) obtaining E. coli having a growth rate that is at least 5% greater than the growth rate of at least one microorganism selected from the group consisting of E. coli MM294, DH5α and

DH10B; and

(b) treating said E. coli to make it competent.

53. The method of claim 52, further comprising the step of curing said E. coli of endogenous plasmids.

54. The method of claim 52, wherein said E. coli are E. coli strain W or strain C.

55. The method of claim 52, wherein said E. coli have a growth rate that is at least 5% greater than the growth rate of E. coli MM294.

56. The method of claim 52, wherein said E. coli have a growth rate that is at least 5% greater than the growth rate of E. coli DH5α

.

57. The method of claim 52, wherein said E. coli have a growth rate that is 5 to 200% greater than the growth rate of E. coli MM294.

58. The method of claim 52, wherein said E. coli have a growth rate that is at least 25% greater than the growth rate of E. coli MM294.

59. The method of claim 52, wherein said E. coli have a growth rate that is at least 50% greater than the growth rate of E. coli MM294.

60. The method of claim 52, wherein said E. coli have a growth rate that is at least 100% greater than the growth rate of E. coli MM294.

61. A method of transforming E. coli, comprising:

(a) obtaining competent E. coli;

(b) incubating said E. coli in the presence of one or more vectors under conditions which cause said one or more vectors to be taken up by said E. coli; and

(c) culturing said E. coli;

wherein said E. coli are E. coli having a growth rate that is at least 5% greater than the growth rate of at least one microorganism selected from the group consisting of E. coli MM294, DH5α and

DH10B.

62. The method according to claim 61, wherein said E. coli have a growth rate that is at least 5% greater than the growth rate of E. coli MM294.

63. The method according to claim 61, wherein said E coli have a growth rate that is at least 5% greater than the growth rate of E. coli DH5α

.

64. The method of claim 61, wherein said E. coli have a growth rate that is 5 to 200% greater than the growth rate of MM294.

65. The method of claim 61, herein said E. coli have a growth rate that is at least 25% greater than the growth rate of MM294.

66. The method of claim 61, wherein said E. coli have a growth rate that is at least 50% greater than the growth rate of MM294.

67. The method of claim 61, wherein said E. coli have a growth rate that is at least 100% greater than the growth rate of MM294.

68. The method of claim 61, wherein said E. coli are E. coli strain W or strain C.

69. The method of claim 68, wherein said E. coli do not contain endogenous plasmids.

70. A method of cloning, comprising:

(a) obtaining competent E. coli;

(b) transforming said competent E. coli with at least one vector;

(c) selecting transformed E. coli containing said at least one vector; and

(d) culturing said transformed E. coli;

wherein said E. coli are E. coli having a growth rate that is at least 5% greater than the growth rate of at least one microorganism selected from the group consisting of E. coli MM294, DH5α and

DH10B.

71. The method of claim 70, further comprising the step of isolating said vector from said transformed E. coli.

72. The method of claim 70, wherein the temperature at which said transformed E. coli are cultured is about 42°

C.

73. The method of claim 70, wherein said E. coli have a growth rate that is at least 5% greater than the growth rate of E. coli MM294.

74. The method of claim 70, wherein said E. coli have a growth rate that is at least 5% greater than the growth rate of E. coli DH5α

.

75. The method of claim 70, wherein said E. coli have a growth rate that is 5 to 200% greater than the growth rate of E. coli MM294.

76. The method of claim 70, wherein said E. coli have a growth rate that is at least 25% greater than the growth rate of E. coli MM294.

77. The method of claim 70, wherein said E. coli have a growth rate that is at least 50% greater than the growth rate of E. coli MM294.

78. The method of claim 70, wherein said E. coli have a growth rate that is at least 100% greater than the growth rate of E. coli MM294.

79. The method of claim 70, wherein said E. coli are E. coli strain W or strain C.

80. The method of claim 79, wherein said E. coli do not contain endogenous plasmids.

81. The method of claim 70, wherein the temperature at which said transformed E. coli are cultured is greater than 37°

C.

82. The method of claim 81, wherein the temperature at which said transformed E. coli are cultured is about 42°

C.

83. A method of producing a protein or peptide, comprising:

(a) obtaining competent E. coli;

(b) transforming into said competent E. coli a vector containing a gene encoding a protein or peptide; and

(c) culturing said transformed E. coli under conditions that cause said transformed E. coli to produce said protein or peptide;

wherein said E. coli arc E. coli having a growth rate that is at least 5% greater than the growth rate of at least one microorganism selected from the group consisting of E. coli MM294. DH5α and

DH10B.

84. The method of claim 83, wherein said E. coli have a growth rate that is at least 5% greater than the growth rate of E. coli MM294.

85. The method of claim 83, wherein said E. coli have a growth rate that is at least 5% greater than the growth rate of E. coli DH5α

.

86. The method of claim 83, wherein said E. coli have a growth rate that is 5 to 200% greater than the growth rate of E. coli MM294.

87. The method of claim 83, wherein said E. coli have a growth rate that is at least 25% greater than the growth rate of E. coli MM294.

88. The method of claim 83, wherein said E. coli have a growth rate that is at least 50% greater than the growth rate of E. coli MM294.

89. The method of claim 83, wherein said E. coli have a growth rate that is at least 100% greater than the growth rate of E. coli MM294.

90. The method of claim 83, wherein said E. coli are E. coli strain W or strain C.

91. The method of claim 90, wherein said E. coli do not contain endogenous plasmids.