Development of a broad-host-range sacB-based vector for unmarked allelic exchange

被引：103

作者：

Marx C.J. ^{[1
]}

机构：

[1] Department of Organismic and Evolutionary Biology, Harvard University, 3083 Biological Laboratories, Cambridge, MA 02138

来源：

BMC Research Notes | / 1卷 / 1期

基金：

美国国家科学基金会;

关键词：

Negative Selection; Donor Plasmid; Donor Vector; Allelic Exchange; Antibiotic Marker;

D O I：

10.1186/1756-0500-1-1

中图分类号：

学科分类号：

摘要：

Background: Although genome sequences are available for an ever-increasing number of bacterial species, the availability of facile genetic tools for physiological analysis have generally lagged substantially behind traditional genetic models. Results: Here I describe the development of an improved, broad-host-range "in-out" allelic exchange vector, pCM433, which permits the generation of clean, marker-free genetic manipulations. Wild-type and mutant alleles were reciprocally exchanged at three loci in Methylobacterium extorquens AM1 in order to demonstrate the utility of pCM433. Conclusion: The broad-host-range vector for marker-free allelic exchange described here, pCM433, has the advantages of a high copy, general Escherichia coli replicon for easy cloning, an IncP oriT enabling conjugal transfer, an extensive set of restriction sites in its polylinker, three antibiotic markers, and sacB (encoding levansucrase) for negative selection upon sucrose plates. These traits should permit pCM433 to be broadly applied across many bacterial taxa for marker-free allelic exchange, which is particularly important if multiple manipulations or more subtle genetic manipulations such as point mutations are desired. © 2008 Marx; licensee BioMed Central Ltd.

引用

共 18 条

[1]

Herring C.D., Glasner J.D., Blattner F.R., Gene replacement without selection: Regulated suppression of amber mutations in Escherichia coli, Gene, 311, pp. 153-163, (2003)

[2]

Ayres E.K., Thomson V.J., Merino G., Balderes D., Figurski D.H., Precise deletions in large bacterial genomes by vector-mediated excision (VEX). the trfA gene of promiscuous plasmid RK2 is essential for replication in several gram-negative hosts, J Mol Biol, 230, pp. 174-185, (1993)

[3]

Hoang T.T., Karkhoff-Schweizer R.R., Kutchma A.J., Schweizer H.P., A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: Application for isolation of unmarked Pseudomonas aeruginosa mutants, Gene, 212, pp. 77-86, (1998)

[4]

Marx C.J., Lidstrom M.E., Broad-host-range cre-lox system for antibiotic marker recycling in gram-negative bacteria, BioTechniques, 33, pp. 1062-1067, (2002)

[5]

Hamilton C.M., Aldea M., Washburn B.K., Babitzke P., Kushner S.R., New method for generating deletions and gene replacements in Escherichia coli, J Bacteriol, 171, pp. 4617-4622, (1989)

[6]

Maloy S.R., Nunn W.D., Selection for loss of tetracycline resistance by Escherichia coli, J Bacteriol, 145, pp. 1110-1111, (1981)

[7]

Gay P., Le Coq D., Steinmetz M., Ferrari E., Hoch J.A., Cloning structural gene sacB, which codes for exoenzyme levansucrase of Bacillus subtilis: Expression of the gene in Escherichia coli, J Bacteriol, 153, pp. 1424-1431, (1983)

[8]

Peel D., Quayle J.R., Microbial growth on C1 compounds: 1. Isolation and characterization of Pseudomonas AM1, Biochem J, 81, pp. 465-469, (1961)

[9]

Nunn D.N., Lidstrom M.E., Isolation and complementation analysis of 10 methanol oxidation mutant classes and identification of the methanol dehydrogenase structural gene of Methylobacterium sp. strain AM1, J Bacteriol, 166, pp. 581-590, (1986)

[10]

Philippe N., Alcaraz J.P., Coursange E., Geiselmann J., Schneider D., Improvement of pCVD442, a suicide plasmid for gene allele exchange in bacteria, Plasmid, 51, pp. 246-255, (2004)

← 1 2 →