アブストラクト
| Date | May 12, 2008 |
| Speaker | Dr. Hirotada Mori, Nara Institute of Science and Technology |
| Title | Systematic Analysis of Genetic Interaction of Escherichia coli |
| Abstract | 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. I would like to summarize our analyses after genome project of E. coli and will report the present situation in Systems approaches, focusing on genetic interaction. |