K-118. Systematic Analysis of Genetic Interaction of Escherichia coli

R. Takeuchi1, F. Yanagihara1, N. Yamamoto1, K. A. Datsenko2, K. Nakahigashi3, A. Typas4, N. J. Krogan4, A. Emilli5, J. Greenblatt5, C. A. Gross4, B. L. Wanner2, H. Mori1;
1Nara Inst. of Sci. and Technology, Nara, JAPAN, 2Purdue Univ., West Lafayette, IN, 3Inst. of Advanced Biosciences, Keio Univ., Tsuruoka, JAPAN, 4Univ. of California, San Francisco, CA, 5Univ. of Toronto, Toronto, ON, CANADA.

Robustness is an important fundamental property of biological systems. Common mechanisms that give rise to robustness depend on alternative or bypass pathways of metabolism and other redundant biological processes. Also, elucidation of epistatic relationships among genes can give insight into the understanding of physiological networks. Synthetic lethality or sickness by double gene knockout mutations is one of the most powerful methods for analyzing robustness. To make this possible in Eshcrichia coli, which is one of the best organisms to understand cellular systems comprehensively based on the vast amount of accumulation of biological knowledge, we setup an easy and reliable system for construction double knockout strains by conjugation and for analysis of their growth effects. We previously established the comprehensive single gene knockout library, called Keio collection (Baba et al, 2006) To combine the second deletion mutation with a single gene deletion of Keio collection, we initiated construction of a second single gene deletion library carrying a different (chloramphenicol) resistance cassette with additional features, including (1) turbo GFP fusion with the initiation codon of the target gene, (2) a modified FLP recombination site (FRT1) site, does not recombine with FRT, and (3) a 20-nt molecular bar code downstream of the targeted gene. We are also developing tools for efficient conjugation for combining different single deletions to make double knockout mutants. To do this, a fragment carrying tra genes and oriT of wild-type F plasmid, which are essential for conjugation and transfer, were combined with oriRg replicon. This plasmid can replicate in cells supplying pir gene product. The chromosomal fragment of the target site of integration of modified F, is cloned by homologous recombination. The resultant pseudo F plasmid is transferred by conjugation to the newly established single gene deletion mutant and selected Hfr strain integrated F plasmid by antibiotic resistance selection. In these ways, we are now developing the high-throughput system of conjugation. We will report the present situation and show preliminary results of analysis of genetic interaction.