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METHOD FOR PRODUCING TRANSFORMED CELL meetings

Foreign code F170008938
File No. (S2015-0632-N0)
Posted date Jan 20, 2017
Country WIPO
International application number 2016JP061420
International publication number WO 2016163466
Date of international filing Apr 7, 2016
Date of international publication Oct 13, 2016
Priority data
  • P2015-078489 (Apr 7, 2015) JP
Title METHOD FOR PRODUCING TRANSFORMED CELL meetings
Abstract The purpose of the present invention is to provide an easy and high-throughput method whereby a DNA can be transferred between different hosts. To achieve this purpose, provided is a method for producing a transformed cell of a yeast or Escherichia coli , said method being characterized in that the yeast or Escherichia coli is transformed using a vector-containing liquid which contains (A) a lytic solution containing a Bacillus subtilis lysate and a vector released therefrom and (B) additive(s) selected from the group consisting of a nuclease inhibitor, a protein-denaturing agent, a nuclease and a combination of two or more of the same.
Outline of related art and contending technology BACKGROUND ART
Recombinant DNA in an experiment, between different host DNA as a technique of moving, transformed, via phage transduction, and the bonding transfer on the method. For example, in the case of using a transformation method, different host replication using a shuttle plasmid vector capable, in a host that was copied in the first DNA 1 once collected, then purified by use of a general-purpose. However, in this method, to recover DNA in a host such as 1 of the first, essential step for purification, a large number of operation and the period of time. Further, the size of DNA is large, it is very difficult to recover and purify DNA is known to be expressed and, to be recovered in the target DNA may not be completely.
The first DNA 2 moving to the host, the host of the first DNA 1 does not need to be recovered and purified from as a method, the first host 1 is a way to protoplasts. However, in this method, the operation of protoplasts for control is necessary in slightly, but anyone is conducted in a simple method, they are less general (non-patent document 1). Further, simply mixing 2 only one host DNA bond capable of moving the transfer method using a (non-patent document 2) is also known, due to the very limited host range is difficult to handle, a large number of genes involved in the bonding to precisely control is required, generally-used method does not.
Scope of claims (In Japanese)[請求項1]
(A)溶菌した枯草菌及びそれから放出されたベクターを含む溶菌液、並びに
(B)ヌクレアーゼ阻害剤、タンパク質変性剤、ヌクレアーゼ、及びそれらの2つ以上の組み合わせからなる群から選択される添加剤、
を含むベクター含有液を用いて、酵母又は大腸菌を形質転換することを特徴とする、形質転換細胞の製造方法。
[請求項2]
前記ヌクレアーゼ阻害剤が、アウリントリカルボン酸、界面活性剤、キレート剤、還元剤、バニジルヌクレオチド、過酸化水素、又は抗ヌクレアーゼ抗体である請求項1に記載の形質転換細胞の製造方法。
[請求項3]
前記タンパク質変性剤がタンパク質分解酵素又は界面活性剤である、請求項1又は2に記載の形質転換細胞の製造方法。
[請求項4]
前記ヌクレアーゼが、エキソヌクレアーゼI、エキソヌクレアーゼIII、マングビーンヌクレアーゼ、又はS1ヌクレアーゼである、請求項1~3のいずれか一項に記載の形質転換細胞の製造方法。
[請求項5]
前記形質転換が、ベクター含有液及び酵母または大腸菌コンピテントセルとの混合、又はベクター含有液及び酵母または大腸菌の混合液へのエレクトロポレーションによって行われる、請求項1~4のいずれか一項に記載の形質転換細胞の製造方法。
[請求項6]
前記溶菌が、枯草菌培養液の静置、ファージの感染、界面活性剤の添加、又はそれらの2つ以上の組み合わせによって行われる、請求項1~5のいずれか一項に記載の形質転換細胞の製造方法。
[請求項7]
請求項1~6のいずれか一項に記載の製造方法によって得られる形質転換細胞。
[請求項8]
請求項1~6のいずれか一項に記載の製造方法によって得られるライブラリー。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • TOKYO INSTITUTE OF TECHNOLOGY
  • KEIO UNIVERSITY
  • Inventor
  • KANEKO Shinya
  • ITAYA Mitsuhiro
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JP KE KG KN KP KR KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG
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