Construction of E. coli Transposon Library

E. coli K-12 strain BW25113, the parent strain of the Keio library, was used for construction of a transposon library. The strain has the following genotype: rrnB3 ΔlacZ4787 hsdR514 Δ(araBAD)567 Δ(rhaBAD)568 rph-1 (65 (link)). The transposon mutant library was constructed by collaborators from Discuva Ltd., Cambridge, United Kingdom, following a method described for Salmonella Typhi (4 (link)). The main differences were that a mini-Tn5 transposon coding for a chloramphenicol resistance cassette was used. This was amplified by PCR from the cat gene of the plasmid vector pACYC184 (66 (link)) using oligonucleotide primers incorporating the Tn5 transposon mosaic ends. Transposomes were prepared using Tn5 transposase (Epicentre, Madison, WI, USA), and these were introduced into E. coli K-12 strain BW25113 by electrotransformation. Transposon mutants were selected by growth on LB agar supplemented with chloramphenicol. Approximately 5.6 million colonies representing an estimated 3.7 million mutants were pooled and stored in 15% glycerol at −80°C.