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http://dx.doi.org/10.4014/jmb.0800.261

Construction of Recombinant Lactobacillus casei Strains Using Splicing by Overlap Extension  

Jeong, Do-Won (Department of Agricultural Biotechnology, Seoul National University)
Lee, Jong-Hoon (Department of Food Science and Biotechnology, Kyonggi University)
Lee, Hyong-Joo (Department of Agricultural Biotechnology, Seoul National University)
Publication Information
Journal of Microbiology and Biotechnology / v.18, no.12, 2008 , pp. 1953-1957 More about this Journal
Abstract
Recombinant Lactobacillus strains have been constructed using gene splicing by overlap extension (SOE). Primers were designed of which one end of an amplified product contained complementary sequences for an end of other amplified fragment. For efficient matching, we used an asymmetric PCR step that was effective at generating an excess of strands that would anneal in the final PCR. CP12, a recombinant fragment consisting of the integrase gene and attachment site of the bacteriophage A2, was constructed and inserted into the genome of Lactobacillus casei ATCC 393, yielding Lb. casei ATCC 393::XCP12. Another recombinant Lb. casei strain was constructed, where the egfp gene was a part of the construction. The EGFP produced from Lb. casei ATCC 393::XCEGFP14 was detected by Western blot hybridization. This simple and widely applicable approach has significant advantages over standard recombinant DNA techniques for Lactobacillus species.
Keywords
Splicing by overlap extension; Lactobacillus; recombination; integration;
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1 De Ruyter, P. G., O. P. Kuipers, and W. M. De Vos. 1996. Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Appl. Environ. Microbiol. 62: 3662-3667
2 Gao, F. S., Q. M. Fang, Y. G. Li, X. S. Li, H. F. Hao, and C. Xia. 2006. Reconstruction of a swine SLA-I protein complex and determination of binding nonameric peptides derived from the foot-and-mouth disease virus. Vet. Immunol. Immunopathol. 113: 328-338   DOI   ScienceOn
3 Jeong, D. W., J. H. Lee, K. H. Kim, and H. J. Lee. 2006. A food-grade expression/secretion vector for Lactococcus lactis that uses an alpha-galactosidase gene as a selection marker. Food Microbiol. 23: 468-475   DOI   ScienceOn
4 Mason, C. K., M. A. Collins, and K. Thompson. 2005. Modified electroporation protocol for lactobacilli isolated from the chicken crop facilitates transformation and the use of a genetic tool. J. Microbiol. Methods 60: 353-363   DOI   ScienceOn
5 Gosalbes, M. J., C. D. Esteban, J. L. Galan, and G. Perez- Martinez. 2000. Integrative food-grade expression system based on the lactose regulon of Lactobacillus casei. Appl. Environ. Microbiol. 66: 4822-4828   DOI   ScienceOn
6 Zhu, H., Y. Wang, M. Jiang, S. Ji, X. Bai, and C. Ruan. 2005. Generation and characterization of a recombinant single chain Fv antibody to von Willebrand factor A1 domain from phage display library. Thromb. Res. 116: 385-391   DOI   ScienceOn
7 Flicek, P. and M. R. Brent. 2006. Using several pair-wise informant sequences for de novo prediction of alternatively spliced transcripts. Genome Biol. 7(Suppl 1): S8   DOI
8 Lin, M. Y., S. Harlander, and D. Savaiano. 1996. Construction of an integrative food-grade cloning vector for Lactobacillus acidophilus. Appl. Microbiol. Biotechnol. 45: 484-489
9 Shareck, J., Y. Choi, B. Lee, and C. B. Miguez. 2004. Cloning vectors based on cryptic plasmids isolated from lactic acid bacteria: Their characteristics and potential applications in biotechnology. Crit. Rev. Biotechnol. 24: 155-208   DOI   ScienceOn
10 Xu, Z., Z. Zhong, L. Huang, L. Peng, F. Wang, and P. Cen. 2006. High-level production of bioactive human beta-defensin-4 in Escherichia coli by soluble fusion expression. Appl. Microbiol. Biotechnol. 72: 471-479   DOI   ScienceOn
11 Lu, Z., T. Masaki, Y. Shoyama, and H. Tanaka. 2006. Construction and expression of a single chain Fv fragment against pharmacologically active paeoniflorin in Escherichia coli, and its potential use in an enzyme-linked immunosorbent assay. Planta Med. 72: 151-155   DOI   ScienceOn
12 Jeong, D. W., Y. C. Choi, J. M. Lee, J. H. Kim, J. H. Lee, K. H. Kim, and H. J. Lee. 2006. Isolation and characterization of promoters from Lactococcus lactis ssp. cremoris LM0230. Food Microbiol. 23: 82-89   DOI   ScienceOn
13 Mercenier, A., H. Muller-Alouf, and C. Grangette. 2000. Lactic acid bacteria as live vaccines. Curr. Issues Mol. Biol. 2: 17-25
14 Horton, R. M., H. D. Hunt, S. N. Ho, J. K. Pullen, and L. R. Pease. 1989. Engineering hybrid genes without the use of restriction enzymes: Gene splicing by overlap extension. Gene 77: 61-68   DOI   ScienceOn
15 Kullen, M. J. and T. R. Klaenhammer. 2000. Genetic modification of intestinal lactobacilli and bifidobacteria. Curr. Issues Mol. Biol. 2: 41-50
16 Takala, T. M., P. E. Saris, and S. S. Tynkkynen. 2003. Foodgrade host/vector expression system for Lactobacillus casei based on complementation of plasmid-associated phospho-betagalactosidase gene lacG. Appl. Microbiol. Biotechnol. 60: 564- 570   DOI
17 Martin, M. C., J. C. Alonso, J. E. Suarez, and M. A. Alvarez. 2000. Generation of food-grade recombinant lactic acid bacterium strains by site-specific recombination. Appl. Environ. Microbiol. 66: 2599-2604   DOI   ScienceOn
18 Pouwels, P. H. and S. Chaillou. 2003. Gene expression in lactobacilli, pp. 143-188. In B. J. B. Wood and P. J. Warner (eds). Genetics of Lactic Acid Bacteria. Kluwer Academic, New York