2018 Fiscal Year Final Research Report

Survival strategy of a cell; intra- and inter-species interaction analysis by barcode tchnology in Escherichia coli

Research Project

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Project/Area Number	16H02485
Research Category	Grant-in-Aid for Scientific Research (A)
Allocation Type	Single-year Grants
Section	一般
Research Field	System genome science
Research Institution	Nara Institute of Science and Technology
Principal Investigator	Mori Hirotada 奈良先端科学技術大学院大学, データ駆動型サイエンス創造センター, 教授 (90182203)
Co-Investigator(Kenkyū-buntansha)	山田守山口大学, 大学院創成科学研究科, 教授 (30174741) 川野光興川崎医科大学, 医学部, 講師 (00455338) 松野浩嗣山口大学, 大学院創成科学研究科, 教授 (10181744) 片岡正和信州大学, 学術研究院工学系, 准教授 (90332676) 牧泰史大阪医科大学, 医学部, 講師 (60401733) 田村武幸京都大学, 化学研究所, 准教授 (00437261) 大橋菜摘 (斎藤菜摘) 鶴岡工業高等専門学校, その他部局等, 准教授 (50287546)
Research Collaborator	KOSAKA tomoyuki
Project Period (FY)	2016-04-01 – 2019-03-31
Keywords	大腸菌 / 分子バーコード / population dynamics / 混合培養液 / 遺伝的相互作用 / CRISPRi / 必須遺伝子 / 網羅解析
Outline of Final Research Achievements	We have constructed the single gene knockout library of E. coli with 20nt random sequence as molecular bar-code. And we have established the method to monitor the population changes of each of single gene deletion strains in variety of growth condition using molecular bar-code and NGS technologies. Out of 4000 genes, two independent knockout isolates of 3600 genes using lambda RED method had been constructed. 7000 deletion strains carrying different bar-codes were mixed and grown in variety of different condition, such as long time incubation in LB up to three weeks, sub-lethal level of antibiotics or toxic compounds in LB to monitor their population changes. We successfully showed the valuableness of this new bar-code deletion library to monitor their population changes. And the quantitative trace method of each of genes’ knockout strains in the mixed culture opened the door to the novel analysis way to the resistance, persister or toxicity mechanisms of cells and chemical compounds.
Free Research Field	システム生物学
Academic Significance and Societal Importance of the Research Achievements	全ての欠失株の混合培養による生育モニター手法の確立を行なった。この基盤技術は、これまでの大腸菌の研究の蓄積を活用し、さらに抗生物質や毒性を示す化合物と遺伝子欠失との相互作用解析が可能になったことで、薬剤や毒性物質の細胞内分子機構解明への新たな研究開発の展開を可能にした。