INFORMATION CHANGE THE WORLD

International Journal of Information Technology and Computer Science(IJITCS)

ISSN: 2074-9007 (Print), ISSN: 2074-9015 (Online)

Published By: MECS Press

IJITCS Vol.3, No.2, Mar. 2011

Applied an Efficient Site-directed Mutagenesis Method into Escherichia coli

Full Text (PDF, 330KB), PP.24-30


Views:75   Downloads:0

Author(s)

Muqing Qiu

Index Terms

Overlapping PCR, gene replacement, ompR gene, site-directed mutagenesis

Abstract

A new technique for conducting site-directed mutagenesis was developed. This method allows the color selection of mutants through the simultaneous activation or deactivation of the α-peptide of ß-galactosidase. The method can efficiently create mutations at multiple sites simultaneously and can be used to perform multiple rounds of mutation on the same construct. In this paper, in order to develop an efficient site-directed mutagenesis method in vivo, the tests were tested by the following methods. The methods that the fragment knock-out ompR gene was constructed through overlapping PCR, digested by Notand SalⅠⅠ, ligated to plasmid pKOV were applied. The recombination plasmid was transformed into Escherichia coli WMC-001 strain, integrated into the genomic DNA through two step homologous recombination. The Escherichia coli WMC-001/ompR- mutant was obtained due to gene replacement. The fragment of the mutant ompR gene was amplified through overlapping PCR, cloned into pKOV vector. The recombinant plasmid was introduced into Escherichia coli WMC-001/ompR- mutant. The Escherichia coli WMC-001/ompR mutant was also obtained due to gene replacement. Results: The site-directed mutagenesis has been successfully constructed in the ompR gene by sequencing. Conclusion: The method is effective for construction of gene site-directed mutagenesis in vivo.

Cite This Paper

Muqing Qiu, "Applied an Efficient Site-directed Mutagenesis Method into Escherichia coli", International Journal of Information Technology and Computer Science(IJITCS), vol.3, no.2, pp.24-30, 2011. DOI: 10.5815/ijitcs.2011.02.04

Reference

[1]Kunkel, T.A., Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. U.S.A. 1985, vol..82, pp. 488–492

[2]Kunkel, T.A., Bebenek, K., McClary, J., Efficient site-directed mutagenesis using uracil-containing DNA. In: Methods in Enzymology. Academic Press, Inc, 1991, vol.22, pp. 125–139.

[3]Sugimoto, M., Esaki, N., Tanaka, H., Soda, K., A simple and efficient method for the oligonucleotide-directed mutagenesis using plasmid DNA template and phosphorothioate-modified nucleotide. Anal. Biochem. 1989, .vol.179, pp. 309–311.

[4]Silaty, S.N., Fung, M., Shen, S.H., Lebel, S., Site-directed mutagenesis by coomplementary-strand synthesis using a closing oligonucleotide and double-stranded DNA templates. Anal. Biochem. 1990, vol.185, pp. 194–200.

[5]Lewis, M.K., Thompson, D.V., Efficient site directed in vitro mutagenesis using ampicillin selection. Nucl. Acids Res. 1990, vol.18, pp. 3439–3443.

[6]Deng, W.P., Nickoloff, J.A., Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal. Biochem. 1992, vol.200, pp. 81–88.

[7]Wong, F., Komaromy, M., Site-directed mutagenesis using thermostable enzymes. BioTechniques, 1995, vol.18, pp. 19-25.

[8]Ohmori, H., A new method for strand discrimination in sequencedirected mutagenesis. Nucl. Acids Res., 1994, vol.22, pp. 884–885.

[9]Li, F., Liu, S.L., Mullins, J.I., Site-directed mutagenesis using uracilcontaining double-stranded DNA templates and DpnI digestion. Biotechniques, 1999, vol.27, pp. 734–738.

[10]Xin, W., Huang, D.W., Zhang, Y.M., Geng, L., DNA mutagenesis using T4 DNA polymerase and DpnI restriction endonuclease. Anal. Biochem. 2004, vol.329, pp. 151–153.

[11]Lesley, S.A., Bohnsack, R.N., Site-directed mutagenesis using the altered sites II systems. Promega Notes, Promega Corporation, Madison, WI. , 1994, vol.46, pp. 6-10.

[12]Weiner, M.P., Costa, G.L., Schoettlin, W., Cline, J., Mathur, E., Bauer, J.C., Site-directed mutagenesis of double-stranded DNA by the polymerase chain reaction. Gene, 1994, vol.151, pp. 119–123.

[13]Lu, L., Patel, H., Bissler, J.J.,. Optimizing DpnI digestion conditions to detect replicated DNA. Biotechniques, 2002, vol.33, pp. 316–318.

[14]Chiu, J., March, P.E., Lee, R., Tillett, D., Site-directed, ligaseindependent mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h. Nucl. Acids Res. 2004, vol.32, pp. 174-178.

[15]Seyfang, A., Jin, J.H., Multiple site-directed mutagenesis of more than 10 sites simultaneously and in a single round. Anal. Biochem. 2004, vol.324, pp. 285–291.

[16]Weiner, M.P., Costa, G.L., Schoettlin, W., Cline, J., Mathur, E., Bauer, J.C., Site-directed mutagenesis of double-stranded DNA by the polymerase chain reaction. Gene , 1994, vol.151, pp. 119–123.

[17]Wu, W., Jia, Z., Liu, P., Xie, Z., Wei, Q., A novel PCR strategy for hight-efficiency, automated site-directed mutagenesis. Nucl. Acids Res. 2005, vol. 33, pp.110-115.

[18]R.M. Schaaper, R.L. Dunn, B.W. Glickman, Mechanisms of ultraviolet-induced mutation: mutational spectra in the Escherichia coli lacI gene for a wild-type and an excision-repairdeficient strain, J. Mol. Biol. 1987, vol. 198, pp. 187–202.

[19]R.B. Setlow, W.L. Carrier, F.J. Bollum, Pyrimidine dimers in UV-irradiated poly dI:dC, Proc. Natl. Acad. Sci. USA, 1965, vol. 53, pp. 1111–1118.

[20]A. Burger, D. Fix, H. Liu, J. Hays, R. Bockrath, In vivo deamination of cytosine-containing cyclobutane pyrimidine dimers in E.coli: a feasible part of UV-mutagenesis, Mutat. Res. , 2003, vol.522, pp. 145–156.

[21]D. Fix, R. Bockrath, Thermal resistance to photoreactivation of specific mutations potentiated in E. coli B/r ung by ultraviolet light, Mol. Gen. Genet. , 1981, vol.182, pp. 7–11.

[22]S.K. Banerjee, R.B. Christensen, C.W. Lawrence, J.E. LeClerc, Frequency and spectrum of mutations produced by a single cissyn thymine-thymine cyclobutane dimer in a single-stranded vector, Proc. Natl. Acad. Sci. USA , 1988, vol.85 , pp. 8141–8145.

[23]S.K. Banerjee, A. Borden, R.B. Christensen, J.E. LeClerc, C.W. Lawrence, SOS-dependent replication past a single trans-syn TT cyclobutane dimer gives a different mutation spectrum and increased error rate compared with replication past this lesion in uninduced cells, J. Bacteriol. , 1990, vol.172, pp. 2105–2112.

[24]J.E. LeClerc, A. Borden, C.W. Lawrence, The thymine-thymine pyrimidine-pyrimidone(6-4) ultraviolet light photoproduct is highly mutagenic and specifically induces 3_ thymine-to-cytosine transitions in Escherichia coli, Proc. Natl. Acad. Sci. USA , 1991, vol.88, pp. 9685–9689.

[25]X.D. Zhao, S. Nadji, J.L.F. Kao, J.-S. Taylor, The structure of d(TpA)*, the major photoproduct of thymidylyl-(3-5)-deoxyadenosine, Nucleic Acids Res. , 1996, vol.24, pp. 1554–1560.

[26]S.N. Bose, R.J.H. Davies, S.K. Sethi, J.A. McCloskey, Formation of an adenine-thymine photoadduct in the deoxydinucleoside monophosphate d(TpA) and in DNA, Science , 1983, vol.220, pp. 723–725.

[27]S.N. Bose, R.J. Davies, The photoreactivity of T-A sequences in oligodeoxyribonucleotides and DNA, Nucleic Acids Res. , 1984, vol.12, pp. 7903–7914.

[28]X. Zhao, J.-S. Taylor, Mutation spectra of TA*, the major photoproduct of thymidylyl-(3-5)-deoxyadenosine, in Escherichia coli under SOS conditions, Nucleic Acids Res. , 1996, vol.24, pp. 1561–1565.

[29]D.F. Fix, N-ethyl-N-nitrosourea-induced mutagenesis in Escherichia coli: multiple roles for UmuC protein, Mutat. Res. , 1993, vol.294, pp. 127–138.

[30]Steven Forst, Masayori Inouye. Environmentally regulated gene expression for membrane proteins in Escherichia coli. Ann Rev Cell Biol , 1988,vol. 4, pp. 21-42

[31]Mattison K, Oropeza R, Byers N, et al. A phosphorylation site mutant of OmpR reveals different binding conformations at ompF and ompC.J Mol Biol ,2002, vol.315, pp. 497-511

[32]Jenny G S, Jamie A. A search for amino acid substitutions that universally activate response regulators.Molecular Microbiology, 2004, vol.51, pp. 887-901

[33]Nara F, Matsuyama S, Mizuno T, et al. Molecular analysis of mutant ompR genes exhibiting different phenotypes as to osmoregulation of the ompF and ompC genes of Escherichia coli. Mol Gen Genet, 1986, vol.202, pp.194-199

[34]Takeshi Yoshidal, Ling Qin, Linda A. Transcription Regulation of ompF and ompC by a Single Transcription Factor,OmpR. J Biol Chem, 2006,vol.281, pp.17114-17123

[35]Li J, Li C, Xiao W, et al.Site-directed mutagenesis by combination of homologous recombination and DpnI digestion of the plasmid template in Escherichia coli. Anaal Biochem,2008, vol.373, pp. 89-391

[36]Link AJ, Phillips D, Church GM.Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli:application to open reading frame characterization. J Bacteriol, 1997, vol.179, pp. 6228-6231

[37]Joseph Sambrook, David W.Russell. Molecular Cloning 3,Huang PT translated,Beijing:science press,2002,pp.96-98

[38]Hamiliton C M, Aldea M, Washburnet B K, et al.New method for generating deletions and gene replacements in Escherichia coli. J Bacteriology, 1989, vol.171, pp. 4617-4622

[39]Smith G R.Homologous recombination near and far from DNA breaks: Alternative roles and contrasting views. Annual Review of Genetics, 2001, vol.35, pp. 243-274

[40]Ling M M and Brian H R. Approaches to DNA Mutagenesis:An Overview.Anal Biochem, 1997, vol.254, pp. 157-178