A simple approach for random genomic insertion–deletions using ambiguous sequences in Escherichia coli

Abstract

Escherichia coli K-12, being one of the best understood and thoroughly analyzed organisms, is the preferred platform for genetic and biochemical research. Among all genetic engineering approaches applied on E. coli, the homologous recombination approach is versatile and precise, which allows engineering genes or large segments of the chromosome directly by using polymerase chain reaction (PCR) products or synthetic oligonucleotides. The previously explained approaches for random insertion and deletions were reported as technically not easy and laborious. This study, first, finds the minimum length of homology extension that is efficient and accurate for homologous recombination, as 30 nt.Second, proposes an approach utilizing PCR products flanking ambiguous NNN-sequence (30-nt) extensions, which facilitate the homologous recombination to recombine them at multiple regions on the genome and generate insertion-deletion mutations. Further analysis found that these mutations were varying in number, that is, multiple genomic regions were deleted. Moreover, evaluation of the phenotype of all the multiple random insertion-deletion mutants demonstrated no significant changes in the normal metabolism of bacteria. This study not only presents the efficiency of ambiguous sequences in making random deletion mutations, but also demonstrates their further applicability in genomics. K E Y W O R D Sambiguous sequences, Escherichia coli, random genomic deletion, red recombination | INTRODUCTIONEscherichia coli, the most completely characterized single-cell life form, is a preferred model organism in bacterial genetic engineering research because of the available whole-genome sequence and described molecular genetics [1,2]. There are several methods available for genome-based engineering and to generate large mutant sets through both random and directed approaches [3][4][5]. Among all the reported approaches, homology-dependent, recombination-mediated, genetic engineering is considered as most efficient that allows engineering genes and segments of the bacterial chromosome directly by using polymerase chain reaction (PCR) products or synthetic oligonucleotides [6,7]. The E. coli KEIO mutant library is the best example of a directed