Comparative genome analysis of three classical E. coli cloning strains designed for blue/white selection: JM83, JM109 and XL1-Blue.

Abstract

The development of the Escherichia coli K-12 laboratory strains JM83, JM109 and XL1-Blue was instrumental in early gene technology. We report the comprehensive genome sequence analysis of JM83 and XL1-Blue using Illumina and Oxford Nanopore technologies and a comparison with both the wild-type sequence (MG1655) and the genome of JM109 deposited at GenBank. Our investigation provides insight into the way how the genomic background that allows blue/white colony selection-by complementing a functionally inactive ω-fragment of β-galactosidase (LacZ) with its α-peptide encoded on the cloning vector-has been implemented independently in these three strains using classical bacterial genetics. In fact, their comparative analysis reveals recurrent motifs: (i) inactivation of the native enzyme via large deletions of chromosomal regions encompassing the lac locus, or a chemically induced frameshift deletion at the beginning of the lacZ cistron, and (ii) utilization of a defective prophage (ϕ80), or an F'-plasmid, to provide the lacZ∆M15 allele encoding its ω-fragment. While the genetic manipulations of the E. coli strains involved repeated use of mobile genetic elements as well as harsh chemical or physical mutagenesis, the individual modified traits appear remarkably stable as they can be found even in distantly related laboratory strains, beyond those investigated here. Our detailed characterization at the genome sequence level not only offers clues about the mechanisms of classical gene transduction and transposition but should also guide the future fine-tuning of E. coli strains for gene cloning and protein expression, including phage display techniques, utilizing advanced tools for site-specific genome engineering.