Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Construction of a T-Vector Using an Esterase Reporter for Direct Cloning of PCR Products

  • Lim, Ho-Dong (Department of Biological Sciences, College of Natural Sciences, Chonnam National University) ;
  • Cheong, Dae-Eun (Department of Biological Sciences, College of Natural Sciences, Chonnam National University,) ;
  • Shin, Hyun-Jae (Department of Chemical Engineering, Chosun University) ;
  • Kim, Geun-Joong (Department of Biological Sciences, College of Natural Sciences, Chonnam National University)
  • Received : 2010.07.09
  • Accepted : 2010.07.31
  • Published : 2010.11.28
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Abstract

We constructed an efficient T-vector, pTQEST216T that employed an engineered esterase as an indicator for direct cloning of PCR products. After ligation of the XcmI-digested vector with PCR products, this cloning system could easily discriminate positive clones owing to insertional inactivation of the esterase reporter. Additionally, PCR products were efficiently cloned into this vector without the gel purification steps, owing to the well-designed multi-cloning site that was in-frame fused at the circularly permutated gap of the reporter.

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References

  1. Agafonov, D. E., K. S. Rabe, M. Grote, Y. W. Huang, and M. Sprinzl. 2005. The esterase from Alicyclobacillus acidocaldarius as a reporter enzyme and affinity tag for protein biosynthesis. FEBS Lett. 579: 2082-2086. https://doi.org/10.1016/j.febslet.2005.02.059
  2. Cheong, D. E., S. Y. Park, H. J. Shin, and G. J. Kim. 2009. A new cloning system using a mutant esterase containing MCS as an indicator for gene cloning. J. Microbiol. Methods 77: 302- 307. https://doi.org/10.1016/j.mimet.2009.03.010
  3. Cuff, J. A. and G. J. Barton. 2000. Application of multiple sequence alignment profiles to improve protein secondary structure prediction. Protein Struct. Funct. Genet. 40: 502-511. https://doi.org/10.1002/1097-0134(20000815)40:3<502::AID-PROT170>3.0.CO;2-Q
  4. Han, S. S., J. Y. Lee, W. H. Kim, H. J. Shin, and G. J. Kim. 2008. Screening of promoters from metagenomic DNA and their use for the construction of expression vectors. J. Microbiol. Biotechnol. 18: 1634-1640.
  5. Holton, T. A. and M. W. Graham. 1991. A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors. Nucleic Acids Res. 19: 1156. https://doi.org/10.1093/nar/19.5.1156
  6. Inouye, S., H. Ogawa, K. Yasuda, K. Umesono, and J. I. Tsuji. 1997. A bacterial cloning vector using a mutated Aequorea green fluorescent protein as an indicator. Gene 189: 159-162. https://doi.org/10.1016/S0378-1119(96)00753-6
  7. Kovalic, D., J. H. Kwak, and B. Weisblum. 1991. General method for direct cloning of DNA fragments generated by the polymerase chain reaction. Nucleic Acids Res. 19: 4560. https://doi.org/10.1093/nar/19.16.4560
  8. Kwon, J., K. S. Park, S. W. Park, and S. Y. Choi. 1998. T vector for direct selection using green fluorescent proteins. Biotechniques 25: 192-196.
  9. Marchuk, D., M. Drumm, A. Saulino, and F. S. Collins. 1991. Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res. 19: 1154. https://doi.org/10.1093/nar/19.5.1154
  10. Mead, D. A., N. K. Pey, C. Herrnstadt, R. A. Marcil, and L. M. Smith. 1991. A universal method for the direct cloning of PCR amplified nucleic acid. Biotechnology 9: 657-663. https://doi.org/10.1038/nbt0791-657
  11. Park, H. K. and C. Zeng. 2008. A vector for purification-free cloning of polymerase chain reaction products. Anal. Biochem. 377: 108-110. https://doi.org/10.1016/j.ab.2008.02.023