Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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A Modified PCR-Directed Gene Replacements Method Using $lambda$-Red Recombination Functions in Escherichia coli

  • KIM SANG-YOON (Department of Bioindustry and Technology, Sangji University) ;
  • CHO JAE-YONG (Department of Bioindustry and Technology, Sangji University)
  • Published : 2005.12.01
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Abstract

We have developed a modified gene replacement method using PCR products containing short homologous sequences of 40- to 50-nt. The method required $\lambda$-Red recombination functions provided under the control of a temperature-sensitive CI857 repressor expressed from the $P_{lac}$ promoter in the presence of IPTG on an easily curable helper plasmid. The method promoted the targeted gene replacements in the Escherichia coli chromosome after shifting cultures of the recombinogenic host, which carries the helper plasmid, to $42^{\circ}C$ for 15 min. Since this method employs $\lambda$-Red recombination functions expressed from the easily curable helper plasmid, multiple rounds of gene replacements in the E. coli chromosome would be possible. The procedures described herein are expected to be widely used for metabolic engineering of E. coli and other bacteria.

Keywords

References

  1. Bailey, J. E. 1991. Towards a science of metabolic engineering. Science 252: 1668-1674 https://doi.org/10.1126/science.2047876
  2. Datsenko, K. A. and B. L. Wanner. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97: 6640-6645
  3. Fukiya, S., H. Mizoguchi, and H. Mori. 2004. An improved method for deleting large regions of Escherichia coli K-12 chromosome using a combination of Cre/loxP and $\gamma$ Red system. FEMS Microbiol. Lett. 234: 325-331
  4. Guldener, U., S. Heck, T. Fiedler, J. Beinhauer, and J. H. Hegemann. 1996. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res. 24: 2519-2524 https://doi.org/10.1093/nar/24.13.2519
  5. Haldimann, A., L. L. Daniels, and B. L. Wanner. 1998. Use of new methods for construction of tightly regulated arabinose and rhamnose promoter fusions in studies of the Escherichia coli phosphate regulon. J. Bacteriol. 180: 1277- 1286
  6. Hong, S. H., S. Y. Moon, and S. Y. Lee. 2003. Prediction of maximum yields of metabolites and optimal pathways for their production by metabolic flux analysis. J. Microbiol. Biotechnol. 13: 571-577
  7. Lee, J.-W., I.-J. Jun, H. J. Kwun, K. L. Jang, and J. Cha. 2004. Direct identification of Vibrio vulnificus by PCR targeting elastase gene. J. Microbiol. Biotechnol. 14: 284-289
  8. Lieb, M. 1979. Heat-sensitive lambda repressors retain partial activity during bacteriophage induction. J. Virol. 32: 162-166
  9. Mergulhao, F. J. M., G. A. Monteiro, J. M. S. Cabral, and M. Angela Taipa. 2004. Design of bacterial vector systems for the production of recombinant proteins in Escherichia coli. J. Microbiol. Biotechnol. 14: 1-14
  10. Morgan-Kiss R. M., C. Walder, and J. E. Cronan, Jr. 2002. Long-term and homogeneous regulation of the Escherichia coli araBAD promoter by use of a lactose transporter of relaxed specificity. Proc. Natl. Acad. Sci. USA 99: 7373- 7377
  11. Murphy, K. C. 1998. Use of bacteriophage $\lambda$ recombination functions to promote gene replacement in Escherichia coli. J. Bacteriol. 180: 2063-2071
  12. Murphy, K. C. and K. G. Campellone. 2003. Lambda Redmediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli. BMC Mol. Biol. 4: 11 https://doi.org/10.1186/1471-2199-4-11
  13. Murphy, K. C., K. G. Campellone, and A. R. Poteete. 2000. PCR-mediated gene replacement in Escherichia coli. Gene 246: 321-330 https://doi.org/10.1016/S0378-1119(00)00071-8
  14. Muyrers, J. P. P., Y. Zhang, F. Buchholz, and A. F. Stewart. 2000. RecE/RecT and $Rec{\alpha}$/$Red{\beta}$ initiate double stranded break repair by specifically interacting with their respective partners. Genes Dev. 14: 1971-1982
  15. Muyrers, J. P. P., Y. Zhang, G. Testa, and A. F. Stewart. 1999. Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res. 27: 1555-1557 https://doi.org/10.1093/nar/27.6.1555
  16. Palmeros, B., J. Wild, W. Szybalski, S. Le Borgne, G. Hernandez-Chavez, G. Gosset, F. Valle, and F. Bolivar. 2000. A family of removable cassettes designed to obtain antibioticresistant- free genomic modifications of Escherichia coli and other bacteria. Gene 247: 255-264 https://doi.org/10.1016/S0378-1119(00)00075-5
  17. Park, S. J., I.-S. Lee, Y. K. Chang, and S. Y. Lee. 2003. Desulfurization of dibenzothiophene and diesel oil by metabolically engineered Escherichia coli. J. Microbiol. Biotechnol. 13: 578-583
  18. Poteete, A. R., A. C. Fenton, and K. C. Murphy. 1988. Modulation of E. coli RecBCD activity by the bacteriophage $\lambda$ Gam and P22 Abc function. J. Bacteriol. 170: 2012-2021 https://doi.org/10.1128/jb.170.5.2012-2021.1988
  19. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, U.S.A
  20. Sergueev, K., D. Yu, S. Austin, and D. Court. 2001. Cell toxicity caused by products of the $P_L$ operon of bacteriophage lambda. Gene 272: 227-235 https://doi.org/10.1016/S0378-1119(01)00535-2
  21. Shashikant, C. S., J. L. Carr, J. Bhargava, K. L. Bentley, and F. H. Ruddle. 1998. Recombinogenic targeting: A new approach to genomic analysis - a review. Gene 223: 9-20 https://doi.org/10.1016/S0378-1119(98)00369-2
  22. Siegele, D. A. and J. C. Hu. 1997. Gene expression from plasmids containing the araBAD promoter at subsaturating inducer concentrations represents mixed populations. Proc.Natl. Acad. Sci. Proc.Natl. Acad. Sci.94: 8168-8172
  23. Steiner, S. and P. Philippsen. 1994. Sequence and promoter analysis of the highly expressed TEF gene of the filamentous fungus Ashbya gossypii. Mol. Genet. Genomics 242: 263- 271 https://doi.org/10.1007/BF00280415
  24. Yi, J.-H., K.-E. Lee, and S.-G. Choi. 2004. Cloning and expression of a novel chitosanase gene (choK) from $\beta$- Proteobacterium KNU3 by double inverse PCR. J. Microbiol. Biotechnol. 14: 563-569
  25. 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: 5978-5983
  26. Zhang, Y., F. Buchholz, J. P. P. Muyrers, and A. F. Stewart. 1998. A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet. 20: 123-128 https://doi.org/10.1038/2417
  27. Zhang, Y., J. P. P. Muyrers, G. Testa, and A. F. Stewart. 2000. DNA cloning by homologous recombination in Escherichia coli. Nat. Biotechnol. 18: 1314-1317 https://doi.org/10.1038/82449