Genomic flank-sequencing of plasposon insertion sites for rapid identification of functional genes.
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.
Leveau
Johan H J
JH
Netherlands Institute of Ecology (NIOO-KNAW), Department of Terrestrial Microbial Ecology, Boterhoeksestraat 48, 6666 GA Heteren, The Netherlands. j.leveau@nioo.knaw.nl
Gerards
Saskia
S
Fritsche
Kathrin
K
Zondag
Gerben
G
van Veen
Johannes A
JA
D024509:3' Flanking Region / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D015723:Gene Library; D006090:Gram-Negative Bacteria / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D017422:Sequence Analysis, DNA
D004251:DNA Transposable Elements; D004269:DNA, Bacterial