Development of an Efficient Procedure for the Construction of Metagenomic Library from Environment Samples

효율적인 Metagenomic Library의 제작 방법 탐구

  • Lim Dongbin (Department of Bioinformatics and Life Science, Soongsil University)
  • 임동빈 (숭실대학교 자연과학대학 생명정보학과)
  • Published : 2004.12.01

Abstract

I investigated an effective way to generate a metagenomic library from DNA prepared from environental samples. The sizes of DNA extracted from environmental samples were usually in the range of 10 to 100 kbp as estimated from $0.4\%$ agarose gel electrophoresis. Because of this small size, a fosmid, rather than BAC, was chosen as a vector. It was found that, for the successful generation of metagenomic library, the selection of DNA with the sized of about 40 kbp was critical and, therefore, a simple agarose gel electrophoresis system was developed to select this size of DNA. By the procedure described in this report, I obtained metagenomic libraries containing 25,000 fosmid clones, which corresponded to 1,000 Mb of metagenomic DNA.

Keywords

metagenome;metagenomic library;environmental DNA;fosmid

References

  1. Voget S., C. Leggewie, A. Uesbeck, C. Raasch, K.E Jaeger, W.R. Streit. 2003. Prospecting for novel biocatalysts in a soil metagenome. Appl. Environ. Microbiol. 69, 6235-6242
  2. Gabor E.M., W.B. Alkema, and D.B. Janssen. 2004. Quantifying the accessibility of the metagenome by random expression cloning techniques. Environ. Microbiol. 6, 879-886
  3. Daniel R. 2004. The soil metagenome-a rich resource for the discovery of novel natural products. Curr. Opin. Biotechnol. 15, 199- 204
  4. Collins J, and H.J. Bruning. 1978. Plasmids useable as gene-cloning vectors in an in vitro packaging by coliphage $\lambda$: 'cosmids'. Gene 4, 85-107
  5. Shizuya H, B. Birren, U.J. Kim, V. Mancino, T. Slepak, Y. Tachiiri, and M. Simon. 1992. Cloning and stable maintenance of 300 kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc. Natl. Acad. Sci. USA. 89, 8794-8797
  6. Wild J. and W. Szybalski. 2004. Copy-control tightly regulated expression vectors based on pBAC/oriV. Methods Mol. Biol. 267, 155-167
  7. Murotsu T., H. Tsutsui, and K. Matsubara. 1984. Identification of the minimal essential region for the replication origin of miniF plasmid. Mol. Gen. Genet. 196, 373-378
  8. Thomas C.M. and A.A. Hussain. 1984. The korB gene of broad host range plasmid RK2 is a major copy number control element which may act together with trfB by limiting trfA expression. EMBO J. 3, 1513-1519
  9. Streit W.R. and R.A. Schmitz, 2004. Metagenomics-the key to the uncultured microbes. Curr. Opin. Microbiol. 7, 492-498
  10. Rondon M.R., P.R. August, A.D. Bettermann, S.F. Brady, T.H. Grossman, M.R. Liles, K.A. Loiacono, B.A. Lynch, I.A. MacNeil, C. Minor, C.L. Tiong, M. Gilman, M.S. Osburne, J. Clardy, J. Handelsman, and R.M. Goodman. 2000. Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms. Appl. Environ. Microbiol. 66, 2541-2547
  11. Kim U.J., H. Shizuya, P.J. de Jong, B. Birren, and M.I Simon. 1992. Stable propagation of cosmid sized human DNA inserts in an F factor based vector. Nucleic Acids Res. 20, 1083-1085
  12. Streit W.R., R. Daniel, and K.E. Jaeger. 2004. Prospecting for biocatalysts and drugs in the genomes of non-cultured microorganisms. Curr. Opin. Biotechnol. 15, 285-290
  13. Zhou J., M.A. Bruns, and J. M. Tiedje. 1996. DNA recovery from soils of diverse composition. Appl. Environ. Microbiol. 62, 316- 322