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Gene targeting in cyanobacteria utilizing I-TevI endonuclease

Research Project

Project/Area Number 11660330
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Applied molecular and cellular biology
Research InstitutionSojo University

Principal Investigator

MATSUOKA Masayoshi  Sojo University, Department of Applied Microbial Technology, Associate Professor, 工学部, 助教授 (10121667)

Co-Investigator(Kenkyū-buntansha) OGAWA Takahira  Sojo University, Department of Applied Microbial Technology, Professor, 工学部, 教授 (40029244)
Project Period (FY) 1999 – 2000
Project Status Completed (Fiscal Year 2000)
Budget Amount *help
¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 2000: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1999: ¥1,700,000 (Direct Cost: ¥1,700,000)
Keywordsgene replacement / cyanobacteria / streptomycin / rps12 gene / psbAI / gene targeting / リポソームタンパク質 / ストレプトマイシン耐性 / 遺伝子ターゲッティング / 遺伝子変換 / 蛍光タンパク質
Research Abstract

Chromosomal gene replacement in cyanobacteria often relies upon the availability of drug-resistant markers, and thus multiple replacements have been restricted. We report here a versatile gene replacement system without this restriction in a unicellular cyanobacterium Synechococcus sp. PCC 7942. The strategy is based upon the dominance of streptomycin-sensitive rps12 gene encoding a ribosomal S12 protein over a streptomycin-resistant rps12-R43 allele with Lys43Arg substitution. To demonstrate the utility of this method, a cassette consisting of the wild-type rps 12 gene and a kan gene conferring kanamycin resistance was integrated in the rps12-R43 mutant at the psbAI locus encoding photosystem II D1 protein, resulting in streptomycin-sensitive merodiploids. Despite a spontaneous gene conversion in these merodiploids to form streptomycin-resistant progeny at frequencies ranging from 1 x 10^<-5> to 5 x 10^<-5>, we could induce homologous recombination by transforming the merodiploids with template plasmids carrying psbAI 5' and 3' non-coding sequences flanking the D1-coding sequence which was replaced by either gfp ORF for a green fluorescent protein or a precise deletion. Depending on the replication ability of the template plasmids, at most 3 to 16 % of streptomycin-resistant progeny from the merodiploids after transformation turned out homogenote recombinants with concomitant loss of the kan gene even for polyploid cyanobacteria. The rps 72-mediated gene replacement thus makes it possible to construct mutants free from drug-resistant markers, and opens a way to create cyanobacterial strains bearing an unlimited number of gene replacements.

Report

(3 results)
  • 2000 Annual Research Report   Final Research Report Summary
  • 1999 Annual Research Report
  • Research Products

    (3 results)

All Other

All Publications (3 results)

  • [Publications] Masayoshi Matsuoka,Kazutaka Takahama,and Takahira Ogawa: "Gene replacement in cyanobacteria mediated by a dominant streptomycin-sensitive rps12 gene that allows selection of mutants free from drug-resistant markers"Microbiology. (in press). (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Masayoshi Matsuoka, Kazutaka Takahama, and Takahira Ogawa: "Gene replacement in cyanobacteria mediated by a dominant streptomycin-sensitive rps12 gene that allows selection of mutants free from drug-resistant markers"Microbiology. (in press). (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Masayoshi Matsuoka,Kazutaka Takahama,and Takahira Ogawa: "Gene replacement in cyanobacteria mediated by a dominant streptomycin-sensitive rps12 gene that allows selection of mutants free from drug-resistant markers"Microbiology. (in press). (2001)

    • Related Report
      2000 Annual Research Report

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Published: 1999-04-01   Modified: 2016-04-21  

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