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Genomic flank-sequencing of plasposon insertion sites for rapid identification of functional genes

Leveau, J.H.J. and Gerards, S. and Fritsche, K. and Zondag, G. and Van Veen, J.A. (2006) Genomic flank-sequencing of plasposon insertion sites for rapid identification of functional genes. Journal of Microbiological Methods, 66, 276-285. ISSN 0167-7012.

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Official URL: http://dx.doi.org/10.1016/j.mimet.2005.12.010

Abstract

Plasposons are modified mini-Tn5 transposons for random mutagenesis of Gram-negative bacteria. Their unique design allows for the rescue cloning and sequencing of DNA that flanks insertion sites in plasposon mutants. However, this process can be laborious and time-consuming, as it involves genomic DNA isolation, restriction endonuclease treatment, subsequent religation, transformation of religated DNA into an Escherichia coli host, and re-isolation as a plasmid, which is then used as a template in sequencing reactions with primers that read from the plasposon ends into the flanking DNA regions. We describe here a method that produces flanking DNA sequences directly from genomic DNA that is isolated from plasposon mutants. By eliminating the need for rescue cloning, our protocol dramatically reduces time and effort, typically by 2 to 3 working days, as well as costs associated with digestion, ligation, transformation, and plasmid isolation. Furthermore, it allows for a high-throughput automated approach to analysis of the plasposome, i.e. the collective set of plasposon insertion sites in a plasposon mutant library. We have tested the utility of genomic flank-sequencing on three plasposon mutants of the soil bacterium Collimonas fungivorans with abolished ability to degrade chitin. [KEYWORDS: Chitinase ; Collimonas fungivorans ; Genomic sequencing ; Mini-Tn5 transposon ; Plasposome ; Plasposon]

Item Type:Article
Institutes:Nederlands Instituut voor Ecologie (NIOO)
ID Code:11984
Deposited On:23 Nov 2011 01:00
Last Modified:31 Mar 2014 10:40

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