Genetic modification of unculturable bacteria by whole genome manipulation

2018 Fiscal Year Final Research Report

• Project/Area Number: 26710015
• Research Category: Grant-in-Aid for Young Scientists (A)
• Allocation Type: Partial Multi-year Fund
• Research Field: System genome science
• Research Institution: National Institute of Advanced Industrial Science and Technology
• Principal Investigator: Kakizawa Shigeyuki 国立研究開発法人産業技術総合研究所, 生命工学領域, 主任研究員 (10588669)
• Project Period (FY): 2014-04-01 – 2019-03-31
• Keywords: 細菌 / ゲノム

Outline of Final Research Achievements

Toward cloning of whole genome of a bacterium, Spiroplasma, whole genome of this bacterium was extracted and cut by the rare-cutter restriction enzymes. Then patterns of genome fragments were analyzed by pulse-field gel electrophoresis. These genomic fragments were used for the TAR (Transformation-Associated Recombination)-cloning in yeast cells. Lots of positive clones were obtained, and these clones were checked by several methods. Whole sequences of the insert of several positive clones were analyzed by the Illumina sequencing method. As a result, there was almost no mutation in these inserts, thus this method would be useful for cloning of bacterial genomes.

Free Research Field

細菌ゲノム学

Academic Significance and Societal Importance of the Research Achievements

難培養細菌（難培養性細菌・未培養細菌）と呼ばれる細菌は、培養が不可能もしくは非常に困難な細菌である。難培養細菌は決して珍しいものではなく、環境中の微生物の99%以上は培養できないことが明らかとなっている。細菌のゲノムを自在に改変する技術はいくつか報告されているが、難培養細菌に応用できるものは限られている。本研究では細菌ゲノムを丸ごとクローニングして配列を改変することで、難培養細菌の遺伝子を改変することを目指し、そのための全ゲノムクローニングを行った。得られた結果は、今後の細菌ゲノム研究の発展に寄与すると考えられる。