In vitro reconstitution of genome self-replication system and its evolution

2019 Fiscal Year Final Research Report

• Project/Area Number: 16KT0076
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Multi-year Fund
• Section: 特設分野
• Research Field: Constructive Systems Biology
• Research Institution: Rikkyo University
• Principal Investigator: SU'ETSUGU Masayuki 立教大学, 理学部, 教授 (00363341)
• Co-Investigator(Kenkyū-buntansha): 高田 啓 立教大学, 理学部, ポストドクトラルフェロー (70747899)
• Project Period (FY): 2016-07-19 – 2020-03-31
• Keywords: 染色体複製 / 自己複製 / 人工細胞

Outline of Final Research Achievements

Self-replication is a fundamental feature of living cells. Cells produce their own copy based on their genetic information. We recently developed an in vitro reconstitution system termed RCR (replication cycle reaction), in which replication cycle of the Escherichia coli chromosome repeats continuously to propagate a mini-chromosome that is a circular DNA having replication origin, oriC. RCR consists of 26 proteins .
In this study, we combined an in vitro transcription-translation reconstitution system, PURE system with RCR (PURE-coupled RCR). All 26 RCR proteins were able to express functionally from DNA in PURE system. We constructed a mini-chromosome encoding the RCR proteins. This mini-chromosome allow us to propagate the circular DNA depending on the transcription-translation from its own DNA. We further developed a system in which the PURE-coupled RCR progress from a single mini-chromosome molecule in water-in-oil emulsion for purpose of in vitro evolution of the mini-chromosome.

Free Research Field

合成生物学

Academic Significance and Societal Importance of the Research Achievements

生命の自己複製能の本質は、自身が持つ遺伝情報に基づいて、多様な機能タンパク質がつくられ、それによって再び自身の遺伝情報が複製されることにある。その繰り返しで、生命は増殖し、また遺伝情報に変異が入れば進化する。本研究では大腸菌から精製された因子を用いて、この自己複製系を再現した。自己複製系では、自己複製DNAからの転写翻訳に依存して複製タンパク質がつくられ、複製タンパク質によって自己複製DNA自身の増殖が促される。今後、DNA複製エラーなどにより遺伝情報の変異を導けば、自己複製DNAを試験管内で進化させ、より自己複製に優位なDNA分子が勝ち残っていく、といった進化実験も可能となると期待される。