Chapter 19 : Recombineering in Prokaryotes

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Ebook: Choose a downloadable PDF or ePub file. Chapter is a downloadable PDF file. File must be downloaded within 48 hours of purchase

Buy this Chapter
Digital (?) $15.00

Preview this chapter:
Zoom in

Recombineering in Prokaryotes, Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555816506/9781555813079_Chap19-1.gif /docserver/preview/fulltext/10.1128/9781555816506/9781555813079_Chap19-2.gif


This chapter describes the present state of recombineering in and details the essential elements of the system. The success of this technology has encouraged its development for use in other organisms. The chapter discusses that goal and some obstacles that must be overcome in order to fully utilize recombineering in other prokaryotes. It emphasizes on the biochemistry of the proteins used for recombineering, the genetic manipulations possible, and the relative efficiencies of each. Several different types of genetic construction can be engineered by recombineering, and the desired genetic product dictates the type of DNA substrate used in the recombineering reaction. The following four types of substrates have been used successfully in laboratories: PCR products; short, partially dsDNA created by annealing ss-oligos; gapped linear plasmid DNA; and ssDNA oligonucleotides. Exo and Beta are needed to process the PCR product prior to its incorporation into the chromosome, and Gam is needed to prevent the degradation of the linear dsDNA by the RecBCD nuclease and possibly by the SbcCD nuclease. Recombineering in more distantly related bacteria will be facilitated by the identification and characterization of Red-like functions in phages from those organisms. Other requirements for the ready use of recombineering are those in common with other modes of genetic analysis, such as the development of systems permitting regulated gene expression and means of easily introducing DNA into the organism under study.

Citation: Thomason L, Costantino N, Sawitzke J, Datta S, Bubunenko M, Court D, Myers R, Oppenheim A. 2005. Recombineering in Prokaryotes, p 383-399. In Waldor M, Friedman D, Adhya S (ed), Phages. ASM Press, Washington, DC. doi: 10.1128/9781555816506.ch19

Key Concept Ranking

Genetic Elements
Herpes simplex virus 1
Highlighted Text: Show | Hide
Loading full text...

Full text loading...


1. Alonso, J. C.,, G. Luder,, and T. A. Trautner. 1992. Intramolecular homologous recombination in Bacillus subtilis 168. Mol. Gen. Genet. 236:6064.
2. Anderson, D. G.,, and S. C. Kowalczykowski. 1997. The translocating RecBCD enzyme stimulates recombination by directing RecA protein onto ssDNA in a χ-regulated manner. Cell 90:7786.
3. Antoine, R.,, and C. Locht. 1992. Isolation and molecular characterization of a novel broad-host-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from gram-positive organisms. Mol. Microbiol. 6:17851799.
4. Appasani, K.,, D. S. Thaler,, and E. B. Goldberg. 1999. Bacteriophage T4 gp2 interferes with cell viability and with bacteriophage lambda Red recombination. J. Bacteriol. 181:13521355.
5. Armstrong, K. A.,, R. Acosta,, E. Ledner,, Y. Machida,, M. Pancotto,, M. McCormick,, H. Ohtsubo,, and E.A. Ohtsubo. 1984.A 37 × 103 molecular weight plasmid-encoded protein is required for replication and copy number control in the plasmid pSC101 and its temperature-sensitive derivative pHS1. J. Mol. Biol. 175:331348.
6. Ayora, S.,, R. Missich,, P. Mesa,, R. Lurz,, S. Yang,, E. H. Egelman,, and J. C. Alonso. 2002. Homologous-pairing activity of the Bacillus subtilis bacteriophage SPP1 replication protein G35P. J. Biol. Chem. 277:3596935979.
7. Barbour, S. D.,, H. Nagaishi,, A. Templin,, and A. J. Clark. 1970. Biochemical and genetic studies of recombination proficiency in Escherichia coli. II. Rec+ revertants caused by indirect suppression of rec mutations. Proc. Natl. Acad. Sci.USA 67:128135.
8. Biek, D. P.,, and S. N. Cohen. 1986. Identification and characterization of recD, a gene affecting plasmid maintenance and recombination in Escherichia coli. J. Bacteriol. 167:594603.
9. Brooks, K.,, and A. J. Clark. 1967. Behavior of λ bacteriophage in a recombination-deficient strain of Escherichia coli. J. Virol. 1:283293.
10. Bunny, K.,, J. Liu,, and J. Roth. 2002. Phenotypes of lexA mutations in Salmonella enterica: evidence for a lethal lexA null phenotype due to the Fels-2 prophage. J. Bacteriol. 184:62356249.
11. Carter, D. M.,, and C. M. Radding. 1971. The role of exonuclease and β protein of phage λ in genetic recombination. II. Substrate specificity and the mode of action of lambda exonuclease. J. Biol. Chem. 246:25022512.
12. Cassuto, E.,, T. Lash,, K. S. Sriprakash,, and C. M. Radding. 1971. Role of exonuclease and β protein of phage λ in genetic recombination.V. Recombination of λ DNA in vitro. Proc. Natl. Acad. Sci. USA 68:16391643.
13. Cassuto, E.,, and C. M. Radding. 1971. Mechanism for the action of λ exonuclease in genetic recombination. Nat. New Biol. 229:1316.
14. Chalker, A. F.,, D. R. Leach,, and R. G. Lloyd. 1988. Escherichia coli sbcC mutants permit stable propagation of DNA replicons containing a long palindrome. Gene 71:201205.
15. Cohen, A.,, and A. J. Clark. 1986. Synthesis of linear plasmid multimers in Escherichia coli K-12. J. Bacteriol. 167:327335.
16. Copeland, N. G.,, N. A. Jenkins,, and D. L. Court. 2001. Recombineering: a powerful new tool for mouse functional genomics. Nat. Rev. Genet. 2:769779.
17. Costantino, N.,, and D. L. Court. 2003. Enhanced levels of lambda Red-mediated recombinants in mismatch repair mutants. Proc. Natl. Acad. Sci. USA 100:1574815753.
18. Court, D. L.,, and A. B. Oppenheim,. 1983. Phage lambda’s accessory genes, p. 251277. In R. W. Hendrix,, J. W. Roberts,, F. W. Stahl,, and R. A. Weisberg (ed.), Lambda II. Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
19. Court, D. L.,, J. A. Sawitzke,, and L. C. Thomason. 2002. Genetic engineering using homologous recombination. Ann. Rev. Genet. 36:361388.
20. Cromie, G. A.,, C. B. Millar,, K. H. Schmidt,, and D. R. Leach. 2000. Palindromes as substrates for multiple pathways of recombination in Escherichia coli. Genetics 154:513522.
21. Datsenko, K.A.,, and B. L. Wanner. 2000.Onestep inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97:66406645.
22. Derbise, A.,, B. Lesic,, D. Dacheux,, J. M. Ghigo,, and E. Carniel. 2003.A rapid and simple method for inactivating chromosomal genes in Yersinia. FEMS Immunol. Med. 38:113116.
23. Dodd, I. B.,, A. J. Perkins,, D. Tsemitsidis,, and J. B. Egan. 2001. Octamerization of lambda CI repressor is needed for effective repression of PRM and efficient switching from lysogeny. Genes Dev. 15:30133022.
24. El Karoui, M.,, D. Ehrlich,, and A. Gruss. 1998. Identification of the lactococcal exonuclease/ recombinase and its modulation by the putative Chi sequence. Proc. Natl. Acad. Sci.USA 20:626631.
25. Ellis, H. M.,, D. Yu,, T. DiTizio,, and D. L. Court. 2001. High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc. Natl. Acad. Sci.USA 98:67426746.
26. Gay, P.,, D. Le Coq,, M. Steinmetz,, T. Berkelman,, and C. I. Kado. 1985. Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria. J. Bacteriol. 164:918921.
27. Gibson, F. P.,, D. R. F. Leach,, and R. G. Lloyd. 1992. Identification of sbcD mutations as cosuppressors of recBC that allow propagation of DNA palindromes in Escherichia coli K-12. J. Bacteriol. 174:12221228.
28. Gillen, J. R.,, D. K. Willis,, and A. J. Clark. 1981. Genetic analysis of the RecE pathway of genetic recombination in Escherichia coli K-12. J. Bacteriol. 145:521532.
29. Goryshin, I.Y.,, J. Jendrisak,, L. M. Hoffman,, R. Meis,, and W. S. Reznikoff. 2000. Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes. Nat. Biotechnol. 18:97100.
30. Gottesman, M. M.,, M. E. Gottesman,, S. Gottesman,, and M. Gellert. 1974. Characterization of bacteriophage λ reverse as an Escherichia coli phage carrying a unique set of host-derived recombination functions. J. Mol. Biol. 88:471487.
31. Hall, S. D.,, M. F. Kane,, and R. D. Kolodner. 1993. Identification and characterization of the Escherichia coli RecT protein, a protein encoded by the recE region that promotes renaturation of homologous single-stranded DNA. J. Bacteriol. 175:277287.
32. Halpern, D.,, A. Gruss,, J. P. Claverys,, and M. El Karoui. 2004. rexAB mutants in Streptococcus pneumoniae. Microbiology 150:24092414.
33. Hashimoto-Gotoh, T.,, F. C. Franklin,, A. Nordheim,, and K. N. Timmis. 1981. Specificpurpose plasmid cloning vectors. I. Low copy number, temperature-sensitive, mobilization-defective pSC101-derived containment vectors. Gene 16:227235.
34. Helm, R. A.,, A. G. Lee,, H. D. Christman,, and S. Maloy. 2003. Genomic rearrangements at rrn operons in Salmonella. Genetics 165:951959.
35. Hendrix, R. W. 2002. Bacteriophages: evolution of the majority. Theor. Popul. Biol. 61:471480.
36. Hill, S. A.,, M. M. Stahl,, and F. W. Stahl. 1997. Single-strand DNA intermediates in phage λs Red recombination pathway. Proc. Natl. Acad. Sci. USA 94:29512956.
37. Iyer, L. M.,, E. V. Koonin,, and L. Aravind. 2002. Classification and evolutionary history of the single- strand annealing proteins, RecT, Redβ, ERF, and RAD52. BMC Genomics 3:811.
38. Joseph, J. W.,, and R. Kolodner. 1983. Exonuclease VIII of Escherichia coli. I. Purification and physical properties. J. Biol. Chem. 258:1041110417.
39. Joseph, J. W.,, and R. Kolodner. 1983. Exonuclease VIII of Escherichia coli. II. Mechanism of action. J. Biol. Chem. 258:1041810424.
40. Kaiser, K.,, and N. E. Murray. 1979. Physical characterization of the “Rac prophage” in E. coli K12. Mol. Gen. Genet. 175:159174.
41. Karakousis, G.,, N. Ye,, Z. Li,, S. K. Chiu,, G. Reddy,, and C. M. Radding. 1998.The β protein of phage λ binds preferentially to an intermediate in DNA renaturation. J. Mol. Biol. 276:721731.
42. Karu, A. E.,, Y. Sakaki,, H. Echols,, and S. Linn. 1975.The γ protein specified by bacteriophage λ. Structure and inhibitory activity for the RecBC enzyme of Escherichia coli. J. Biol. Chem. 250:73777387.
43. Keim, P.,, and K. G. Lark. 1990.The RecE recombination pathway mediates recombination between partially homologous DNA sequences: structural analysis of recombination products. J. Struct. Biol. 104:97106.
44. Kmiec, E.,, and W. K. Holloman. 1981. β protein of bacteriophage λ promotes renaturation of DNA. J. Biol. Chem. 256:1263612639.
45. Kovall, R.,, and B.W. Matthews. 1997.Toroidal structure of λ-exonuclease. Science 277:18241827.
46. Kulkarni, S. K.,, and F. W. Stahl. 1989. Interaction between the sbcC gene of Escherichia coli and the gam gene of phage λ. Genetics 123:249253.
47. Kusano, K.,, K. Nakayama,, and H. Nakayama. 1989. Plasmid-mediated lethality and plasmid multimer formation in an Escherichia coli recBC sbcBC mutant. Involvement of RecF recombination pathway genes. J. Mol. Biol. 209:623634.
48. Lee, E. C.,, D. Yu,, J. Martinez de Velasco,, L. Tessarollo,, D. A. Swing,, D. L. Court,, N. A. Jenkins,, and N.G. Copeland. 2001.A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 73:5665.
49. Li, X. T.,, N. Costantino,, L.Y. Lu,, D. P. Liu,, R. M. Watt,, K. S. Cheah,, D. L. Court,, and J. D. Huang. 2003. Identification of factors influencing strand bias in oligonucleotide-mediated recombination in Escherichia coli. Nucleic Acids Res. 31:66746687.
50. Lin-Chao, S.,, W. T. Chen,, and T. T. Wong. 1992. High copy number of the pUC plasmid results from a Rom/Rop-suppressible point mutation in RNA II. Mol. Microbiol. 6:33853393.
51. Little, J. W. 1967.An exonuclease induced by bacteriophage λ. II. Nature of the enzymatic reaction. J. Biol. Chem. 242:679686.
52. Lupski, J. R.,, J. R. Roth,, and G. M. Weinstock. 1996. Chromosomal duplications in bacteria, fruit flies, and humans. Am. J. Hum. Genet. 58:2127.
53. Matsuura, S.,, J. Komatsu,, K. Hirano,, H. Yasuda,, K. Takashima,, S. Katsura,, and A. Mizuno. 2001. Real-time observation of a single DNA digestion by λ exonuclease under a fluorescence microscope field. Nucleic Acids Res. 29:E79.
54. Mikhailov, V. S.,, K. Okano,, and G. F. Rohrmann. 2003. Baculovirus alkaline nuclease possesses a 5′→3′ exonuclease activity and associates with DNA-binding protein LEF-3. J. Virol. 77:24362444.
55. Modrich, P.,, and R. Lahue. 1996. Mismatch repair in replication fidelity,genetic recombination,and cancer biology. Annu. Rev. Biochem. 65:101133.
56. Muniyappa, K.,, and C. M. Radding. 1986.The homologous recombination system of phage λ. Pairing activities of β protein. J. Biol. Chem. 261:74727478.
57. Murphy, K. C. 1991. λ Gam protein inhibits the helicase and χ-stimulated recombination activities of Escherichia coli RecBCD enzyme. J. Bacteriol. 173:58085821.
58. Murphy, K. C. 1998. Use of bacteriophage λ recombination functions to promote gene replacement in Escherichia coli. J. Bacteriol. 180:20632071.
59. Murphy, K. C.,, and K. G. Campellone. 2003. Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli. BMC Mol. Biol. 4:1122.
60. Murphy, K. C.,, K. G. Campellone,, and A. R. Poteete. 2000. PCR-mediated gene replacement in Escherichia coli. Gene 246:321330.
61. Muyrers, J. P.,, Y. Zhang,, G. Testa,, and A. F. Stewart. 1999. Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res. 27:15551557.
62. Muyrers, J. P.,, Y. Zhang,, F. Buchholz,, and A. F. Stewart. 2000. RecE/RecT and Redα/Redβ initiate double-stranded break repair by specifically interacting with their respective partners. Genes Dev. 14:19711982.
63. Muyrers, J. P.,, Y. Zhang,, V. Benes,, G. Testa,, W. Ansorge,, and A. F. Stewart. 2000. Point mutation of bacterial artificial chromosomes by ET recombination. EMBO Rep. 1:239243.
64. Myers, R. S.,, and K. E. Rudd. 1998. Mining DNA sequences for molecular enzymology: the Red Alpha superfamily defines a set of recombination nucleases,p. 4950. Proceedings of the 1998 Miami Nature Biotechnology Winter Symposium. University of Miami School of Medicine, Miami, Fla.
65. Mythili, E.,, K. A. Kumar,, and K. Muniyappa. 1996.Characterization of the DNA-binding domain of β protein, a component of phage λ Red pathway, by UV catalyzed cross-linking. Gene 182:8187.
66. Noirot, P.,, and R. D. Kolodner. 1998. DNA strand invasion promoted by Escherichia coli RecT protein. J. Biol. Chem. 273:1227412280.
67. Oppenheim, A. B.,, A. J. Rattray,, M. Bubunenko,, L. C. Thomason,, and D. L. Court. 2004. In vivo recombineering of bacteriophage λ by PCR fragments and single-strand oligonucleotides. Virology 319:185189.
68. Parker, B.O.,, and M. G. Marinus. 1992. Repair of DNA heteroduplexes containing small heterologous sequences in Escherichia coli. Proc. Natl. Acad. Sci. USA 89:17301734.
69. Passy, S. I.,, X. Yu,, Z. Li,, C. M. Radding,, and E. H. Egelman. 1999. Rings and filaments of β protein from bacteriophage lambda suggest a superfamily of recombination proteins. Proc. Natl. Acad. Sci. USA 96:42794284.
70. Perkins, T. T.,, R. V. Dalal,, P. G. Mitsis,, and S. M. Block. 2003. Sequence-dependent pausing of single lambda exonuclease molecules. Science 301:19141918.
71. Poteete, A. R.,, and A. C. Fenton. 1983.DNAbinding properties of the Erf protein of bacteriophage P22. J. Mol. Biol. 163:257275.
72. Radding, C. M.,, J. Rosenzweig,, F. Richards,, and E. Cassuto. 1971. Separation and characterization of exonuclease, β protein, and a complex of both. J. Biol. Chem. 246:25102512.
73.Reuven N. B., A. E. Staire, R. S. Myers, and S. K. Weller. 2003.The herpes simplex virus 1 alkaline nuclease and single-stranded DNA binding protein mediate strand exchange in vitro. J. Virol. 77:74257433.
74. Russell, C. B.,, D. S. Thaler,, and F. W. Dahlquist. 1989. Chromosomal transformation of Escherichia coli recD strains with linearized plasmids. J. Bacteriol. 171:26092613.
75. Sakaki, Y. 1974. Inactivation of the ATP-dependent DNase of Escherichia coli after infection with double- stranded DNA phages. J. Virol. 14:16111612.
76. Sanderson, K. E.,, P. R. MacLachlan,, and A. Hessel. 1995. Electrotransformation in Salmonella. Methods Mol. Biol. 47:115123.
77. Sergueev, K.,, D. Court,, L. Reaves,, and S. Austin. 2002. E. coli cell-cycle regulation by bacteriophage lambda. J. Mol. Biol. 324:297307.
78. Shulman, M. J.,, L. M. Hallick,, H. Echols,, and E. R. Signer. 1970. Properties of recombination-deficient mutants of bacteriophage λ. J. Mol. Biol. 52:501520.
79. Signer, E. R.,, and J. Weil. 1968. Recombination in bacteriophage λ. I. Mutants deficient in general recombination. J. Mol. Biol. 34:261271.
80. Silberstein, Z.,, and A. Cohen. 1987. Synthesis of linear multimers of OriC and pBR322 derivatives in Escherichia coli K-12: role of recombination and replication functions. J. Bacteriol. 169:31313137.
81. Silberstein, Z.,, S. Maor,, I. Berger,, and A. Cohen. 1990. Lambda Red-mediated synthesis of plasmid linear multimers in Escherichia coli K12. Mol. Gen. Genet. 223:496507.
82. Smith, G. R., 1983. General recombination, p. 175209. In R. W. Hendrix,, J. W. Roberts,, F. W. Stahl,, and R. A. Weisberg (ed.), Lambda II. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
83. Stahl, M. M.,, L. Thomason,, A. R. Poteete,, T. Tarkowski,, A. Kuzminov,, and F. W. Stahl. 1997. Annealing vs. invasion in phage lambda recombination. Genetics 147:961977.
84. Stemmer, W. P.,, A. Crameri,, K. D. Ha,, T. M. Brennan,, and H. L. Heyneker. 1995. Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164:4953.
85. Subramanian, K.,, W. Rutvisuttinunt,, W. Scott,, and R. S. Myers. 2003. The enzymatic basis of processivity in lambda exonuclease. Nucleic Acids Res. 31:15851596.
86. Taylor, A.,, and G. R. Smith. 1980. Unwinding and rewinding of DNA by the RecBC enzyme. Cell 22:447457.
87. Thaler, D. S.,, M. M. Stahl,, and F. W. Stahl. 1987. Evidence that the normal route of replication-allowed Red-mediated recombination involves double-chain ends. EMBO J. 6:31713176.
88. Thomason, L. C.,, M. Bubunenko,, N. Costantino,, H. Wilson,, A. Oppenheim,, and D. L. Court. 2003. Recombineering: genetic engineering in bacteria using homologous recombination, p. In Current Protocols in Molecular Biology. Greene Publishing Associates, Brooklyn, N.Y.
89. Thomason, L. C.,, D. L. Court,, A. R. Datta,, R. Khanna,, and J. L. Rosner. 2004. Identification of the Escherichia coli K-12 ybhE gene as pgl, encoding 6-phosphogluconolactonase. J. Bacteriol. 186:82488253.
90. Toussaint, A.,, J. M. Ghigo,, and G. P. Salmond. 2003.A new evaluation of our life-support system. EMBO Rep. 4:820824.
91. Unger, R. C.,, and A. J. Clark. 1972. Interaction of the recombination pathways of bacteriophage λ and its host Escherichia coli K12: effects on exonuclease V activity. J. Mol. Biol. 70:539548.
92. Uzzau, S.,, N. Figueroa-Bossi,, S. Rubino,, and L. Bossi. 2001. Epitope tagging of chromosomal genes in Salmonella. Proc. Natl. Acad. Sci. USA 98:1526415269.
93. Valla, S.,, K. Haugan,, R. Durland,, and D. R. Helinski. 1991. Isolation and properties of temperature-sensitive mutants of the trfA gene of the broad host range plasmid RK2. Plasmid 25:131136.
94. van Oijen, A. M.,, P.C. Blainey,, D. J. Crampton,, C. C. Richardson,, T. Ellenberger,, and X. S. Xie. 2003. Single-molecule kinetics of λ exonuclease reveal base dependence and dynamic disorder. Science 301:12351238.
95. van Oostrum, J.,, J. L. White,, and R. M. Burnett. 1985. Isolation and crystallization of λ exonuclease. Arch. Biochem. Biophys. 243:332337.
96. Vellani, T. S.,, and R. S. Myers. 2003. Bacteriophage SPP1 Chu is an alkaline exonuclease in the SynExo family of viral two-component recombinases. J. Bacteriol. 185:24652474.
97. Wagner, M.,, Z. Ruzsics,, and U. H. Koszinowski. 2002. Herpesvirus genetics has come of age. Trends Microbiol. 10:318324.
98. Yu, D.,, H. M. Ellis,, E. C. Lee,, N.A. Jenkins,, N. G. Copeland,, and D. L. Court. 2000.An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. USA 97:59785983.
99. Yu, D.,, J. A. Sawitzke,, H. Ellis,, and D. L. Court. 2003. Recombineering with overlapping single-stranded DNA oligonucleotides: testing a recombination intermediate. Proc. Natl. Acad. Sci. USA 100:72077212.
100. Zhang, Y.,, F. Buchholz,, J. P. Muyrers,, and A. F. Stewart. 1998.A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet. 20:123128.
101. Zhang, Y.,, J. P. Muyrers,, G. Testa,, and A. F. Stewart. 2000. DNA cloning by homologous recombination in Escherichia coli. Nat. Biotechnol. 18:13141317.
102. Zhang, Y.,, J. P. Muyrers,, J. Rientjes,, and A. F. Stewart. 2003. Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells. BMC Mol. Biol. 4:114.


Generic image for table

Typical recombination efficiencies for standard recombineering reactions

DY330, Δ() gal490 (λI857-bioA) ( ).

DY411, W3110 (λI) < > with 34 bp of deleted ( ).

DY378, W3110 (λI) ( ).

HME6, W3110 Δ()I).The oligo creates a T-C mismatch that is well repaired by the MMR system ( ).

Citation: Thomason L, Costantino N, Sawitzke J, Datta S, Bubunenko M, Court D, Myers R, Oppenheim A. 2005. Recombineering in Prokaryotes, p 383-399. In Waldor M, Friedman D, Adhya S (ed), Phages. ASM Press, Washington, DC. doi: 10.1128/9781555816506.ch19
Generic image for table

Recombination efficiencies of various recombineering systems with ss-oligos and dsDNA

W3110, K-12 IN().

TS616, serovar Typhimurium LT2 ::Tn from Genetic Stock Center.

NA, not applicable.

From reference .

Minimal prophage on plasmid (see text and Color Plate 19).The data in the table were generated with the Cm plasmid.

From reference .

ND, not determined.

From reference .

From reference .

10 mM arabinose.

1 mM arabinose.

Citation: Thomason L, Costantino N, Sawitzke J, Datta S, Bubunenko M, Court D, Myers R, Oppenheim A. 2005. Recombineering in Prokaryotes, p 383-399. In Waldor M, Friedman D, Adhya S (ed), Phages. ASM Press, Washington, DC. doi: 10.1128/9781555816506.ch19

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error