Construction of Long Chromosomal Deletion Mutants of Escherichia coli and Minimization of the Genome
Genetic information consists of protein- and RNA-coding genes that exist in a range of sizes and noncoding cis- and trans-acting sequence elements. The use of long chromosomal deletion mutations is a powerful method for identifying essential genetic information through experimental reduction of the genome to its minimal gene set. Taking advantage of recent technical advances, we constructed sequence-specific long deletion mutations of the Escherichia coli chromosome. In a recent report (1), we described a set of E. coli medium-scale deletions (MDs) and large-scale deletions (LDs). Several LD mutations were combined to generate an engineered strain lacking ∼30% of the parental chromosome. We then constructed another set of deletion mutations, MDs and small-scale deletions (SDs), and identified additional essential genetic regions using complementation analysis. To delete the remaining essential chromosomal regions, we developed an Flp recombinase target (FRT)-based system of site-specific recombination to move chromosomal regions onto mini-F plasmids in vivo. In this report, we describe the details of the construction of several of these types of large chromosomal deletion mutants.
Key Wordschromosome deletions Escherichia coli essential genes FLP recombinase red
- 7.Ayres, E. K., Thomson, V. J., Merino, G., Balderes, D., and Figurski, D. H. (1993) Precise deletions in large bacterial genomes by vector-mediated excision (VEX). The trfA gene of promiscuous plasmid RK2 is essential for replication in several gram-negative hosts. J. Mol. Biol. 230, 174–185.CrossRefPubMedGoogle Scholar
- 12.Murayama, N., Shimizu, H., Takiguchi, S., Baba, Y., Amino, H., Horiuchi, T., et al. (1996) Evidence for involvement of Escherichia coli genes pmbA, csrA and a previously unrecognized gene tldD, in the control of DNA gyrase by letD (ccdB) of sex factor F. J. Mol. Biol. 256, 483–502.CrossRefPubMedGoogle Scholar