RRC ID 1641
Author Goryshin IY, Naumann TA, Apodaca J, Reznikoff WS.
Title Chromosomal deletion formation system based on Tn5 double transposition: use for making minimal genomes and essential gene analysis.
Journal Genome Res.
Abstract In this communication, we describe the use of specialized transposons (Tn5 derivatives) to create deletions in the Escherichia coli K-12 chromosome. These transposons are essentially rearranged composite transposons that have been assembled to promote the use of the internal transposon ends, resulting in intramolecular transposition events. Two similar transposons were developed. The first deletion transposon was utilized to create a consecutive set of deletions in the E. coli chromosome. The deletion procedure has been repeated 20 serial times to reduce the genome an average of 200 kb (averaging 10 kb per deletion). The second deletion transposon contains a conditional origin of replication that allows deleted chromosomal DNA to be captured as a complementary plasmid. By plating cells on media that do not support plasmid replication, the deleted chromosomal material is lost and if it is essential, the cells do not survive. This methodology was used to analyze 15 chromosomal regions and more than 100 open reading frames (ORFs). This provides a robust technology for identifying essential and dispensable genes.
Volume 13(4)
Pages 644-53
Published 2003-4
DOI 10.1101/gr.611403
PII GR-6114R
PMID 12654720
PMC PMC430159
MeSH Chromosome Deletion* Chromosome Mapping / methods Chromosomes, Bacterial / genetics DNA Transposable Elements / genetics* Escherichia coli / genetics* Escherichia coli / growth & development Genes, Bacterial / genetics Genes, Essential / genetics* Genome, Bacterial* Lac Operon / genetics Oligonucleotide Array Sequence Analysis / methods Plasmids / genetics Recombination, Genetic / genetics*
IF 9.944
Times Cited 31