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METHODS

Chromosomal Deletion Formation System Based on Tn5 Double Transposition: Use For Making Minimal Genomes and Essential Gene Analysis

Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA

In this communication, we describe the use of specialized transposons (Tn5 derivatives) to create deletions in the Escherichia coli K-12 chromosome. These transposons are essentially rearranged composite transposons that have been assembled to promote the use of the internal transposon ends, resulting in intramolecular transposition events. Two similar transposons were developed. The first deletion transposon was utilized to create a consecutive set of deletions in the E. coli chromosome. The deletion procedure has been repeated 20 serial times to reduce the genome an average of 200 kb (averaging 10 kb per deletion). The second deletion transposon contains a conditional origin of replication that allows deleted chromosomal DNA to be captured as a complementary plasmid. By plating cells on media that do not support plasmid replication, the deleted chromosomal material is lost and if it is essential, the cells do not survive. This methodology was used to analyze 15 chromosomal regions and more than 100 open reading frames (ORFs). This provides a robust technology for identifying essential and dispensable genes.

[Supplemental material is available online at www.genome.org and is supplied as an extended table enumerating genes lost in two multiple round deletion strains (20-1 and 20-4). These data are summarized in Table 1.]

View this table: [in this window] [in a new window]   Table 1. Genes Deleted in Strains 20-1 and 20-4

² Corresponding author.

E-MAIL Reznikoff{at}biochem.wisc.edu; FAX (608) 265-2603.

Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.611403. Article published online before print in March 2003.

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