Phenotypic screening of Eschenchia coli K-12 Tn5 insertion libraries, using whole-genome oligonucleotide microarrays

Kelly M. Winterberg, John Luecke, Amanda Bruegl, William S. Reznikoff

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

Complete genome sequences in combination with global screening methods allow parallel analysis of multiple mutant loci to determine the requirement for specific genes in different environments. In this paper we describe a high-definition microarray approach for investigating the growth effects of Tn5 insertions in Eschenchia coli K-12. Libraries of insertion mutants generated by a unique Tn5 mutagenesis system were grown competitively in defined media. Biotin-labeled runoff RNA transcripts were generated in vitro from transposon insertions in each population of mutants. These transcripts were then hybridized to custom-designed oligonucleotide microarrays to detect the presence of each mutant in the population. By using this approach, the signal associated with 25 auxotrophic insertions in a 50-mutant pool was not detectable following nine generations of growth in glucose M9 minimal medium. It was found that individual insertion sites could be mapped to within 50 bp of their genomic locations, and 340 dispensable regions in the E. coli chromosome were identified. Tn5 insertions were detected in 15 genes for which no previous insertions have been reported. Other applications of this method are discussed.

Original languageEnglish (US)
Pages (from-to)451-459
Number of pages9
JournalApplied and Environmental Microbiology
Volume71
Issue number1
DOIs
StatePublished - Jan 2005
Externally publishedYes

Fingerprint

oligonucleotides
Oligonucleotide Array Sequence Analysis
Libraries
genome
Genome
screening
mutants
Growth
Biotin
Mutagenesis
Population
Genes
Chromosomes
RNA
Escherichia coli
Glucose
gene
biotin
messenger RNA
application methods

ASJC Scopus subject areas

  • Environmental Science(all)
  • Biotechnology
  • Microbiology

Cite this

Phenotypic screening of Eschenchia coli K-12 Tn5 insertion libraries, using whole-genome oligonucleotide microarrays. / Winterberg, Kelly M.; Luecke, John; Bruegl, Amanda; Reznikoff, William S.

In: Applied and Environmental Microbiology, Vol. 71, No. 1, 01.2005, p. 451-459.

Research output: Contribution to journalArticle

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