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SUMMARY:Systematic Analysis of Genetic Interaction of Escherichia coli - P
 rof Hirotada Mori\, Nara Institute of Science and Technology\, Nara 630-01
 01\, Japan
DTSTART:20080725T100000Z
DTEND:20080725T110000Z
UID:TALK12966@talks.cam.ac.uk
CONTACT:M. Madan Babu
DESCRIPTION:Robustness is an important fundamental property of biological 
 systems. Common mechanisms that give rise to robustness depend on alternat
 ive or bypass pathways of metabolism and other redundant biological proces
 ses. Also\, elucidation of epistatic relationships among genes can give in
 sight into the understanding of physiological networks. Synthetic lethalit
 y or sickness by double gene knockout mutations is one of the most powerfu
 l methods for analyzing robustness. To make this possible in\nEshcrichia c
 oli\, which is one of the best organisms to understand cellular systems co
 mprehensively based on the vast amount of accumulation of biological knowl
 edge\, we setup an easy and reliable system for construction double knocko
 ut strains by conjugation and for analysis of their growth effects.\n\nWe 
 previously established the comprehensive single gene knockout library\, ca
 lled Keio collection (Baba et al\, 2006) To combine the second deletion mu
 tation with a single gene deletion of Keio collection\, we initiated const
 ruction of a second single gene deletion library carrying a different (chl
 oramphenicol) resistance cassette\nwith additional features\, including (1
 ) turbo GFP fusion with the initiation codon of the target gene\, (2) a mo
 dified 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 s
 ingle deletions to make double knockout mutants. To do this\, a fragment c
 arrying tra genes and oriT of wild-type F plasmid\, which are essential fo
 r conjugation and transfer\, were combined with oriRg replicon. This plasm
 id can replicate in cells supplying pir gene product.\n\nThe chromosomal f
 ragment of the target site of integration of modified F\, is cloned by hom
 ologous recombination. The resultant pseudo F plasmid is transferred by co
 njugation to the newly established single gene deletion mutant and selecte
 d Hfr strain integrated F plasmid by antibiotic resistance selection. In t
 hese ways\, we are now developing the high-throughput system of conjugatio
 n.\n\nI would like to summarize our analyses after genome project of E. co
 li and will report the present situation in Systems approaches\, focusing 
 on genetic interaction.\n
LOCATION:Structural Studies Coffee room\, MRC Laboratory of Molecular Biol
 ogy
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