Minimization of the Escherichia coli genome using a Tn5-targeted Cre/loxP excision system.
An increasing number of microbial genomes have been completely sequenced, and functional analyses of these genomic sequences are under way. To facilitate these analyses, we have developed a genome-engineering tool for determining essential genes and minimizing bacterial genomes. We made two large pools of independent transposon mutants in Escherichia coli using modified Tn5 transposons with two different selection markers and precisely mapped the chromosomal location of 800 of these transposons. By combining a mapped transposon mutation from each of the mutant pools into the same chromosome using phage P1 transduction and then excising the flanked genomic segment by Cre-mediated loxP recombination, we obtained E. coli strains in which large genomic fragments (59-117 kilobases) were deleted. Some of these individual deletions were then combined into a single "cumulative deletion strain" that lacked 287 open reading frames (313.1 kilobases) but that nevertheless exhibited normal growth under standard laboratory conditions.
Yu
Byung Jo
BJ
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, 305-701, Korea.
Sung
Bong Hyun
BH
Koob
Michael D
MD
Lee
Choong Hoon
CH
Lee
Jun Hyoung
JH
Lee
Won Sik
WS
Kim
Mi Sun
MS
Kim
Sun Chang
SC
D017101:Bacteriophage P1 / Q000201:enzymology; D002460:Cell Line; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular / Q000379:methods*; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000145:classification / Q000235:genetics*; D017353:Gene Deletion; D005818:Genetic Engineering / Q000379:methods*; D016680:Genome, Bacterial; D019426:Integrases / Q000235:genetics; D016296:Mutagenesis; D011995:Recombination, Genetic; D018741:Replication Origin; D013045:Species Specificity; D014161:Transduction, Genetic / Q000379:methods*; D014764:Viral Proteins / Q000235:genetics
D004251:DNA Transposable Elements; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases