Whole-genome detection of conditionally essential and dispensable genes in Escherichia coli via genetic footprinting.

ABSTRACT

We present a whole-genome approach to genetic footprinting in Escherichia coli using Tn5-based transposons to determine gene essentiality. A population of cells is mutagenized and subjected to outgrowth under selective conditions. Transposon insertions in the surviving mutants are detected using nested polymerase chain reaction (PCR), agarose gel electrophoresis, and software-assisted PCR product size determination. Genomic addresses of these inserts are then mapped onto the E. coli genome sequence based on the PCR product lengths and the addresses of the corresponding genome-specific primers. Gene essentiality conclusions were drawn based on a semiautomatic analysis of the number and relative positions of inserts retained within each gene after selective outgrowth.