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 approximately 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.
Kato
Jun-ichi
J
Department of Biological Sciences, Graduate School of Science and Engineering, Tokyo Metropolitan University, Minaminohsawa, Hachioji, Tokyo, Japan.
Hashimoto
Masayuki
M
D002872:Chromosome Deletion; D002876:Chromosomes, Bacterial / Q000235:genetics*; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D005798:Genes, Bacterial; D020043:Genes, Essential
D004269:DNA, Bacterial; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase