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1. WO2020087951 - STRAIN HAVING CCDB NEGATIVE SCREENING EFFECT AND CONSTRUCTION METHOD THEREFOR

Publication Number WO/2020/087951
Publication Date 07.05.2020
International Application No. PCT/CN2019/092640
International Filing Date 25.06.2019
IPC
C12N 1/21 2006.01
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
1Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
20Bacteria; Culture media therefor
21modified by introduction of foreign genetic material
C12N 15/70 2006.01
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
70Vectors or expression systems specially adapted for E. coli
C12R 1/19 2006.01
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
RINDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C-C12Q75
1Microorganisms
01Bacteria or actinomycetales
185Escherichia
19Escherichia coli
Applicants
  • 云舟生物科技(广州)有限公司 YUNZHOU BIOSCIENCES (GUANGZHOU) INC. [CN]/[CN]
Inventors
  • 蓝田 LAN, Tian
  • 施金秀 SHI, Jinxiu
  • 罗燕 LUO, Yan
  • 蒙伟能 MENG, Weineng
Agents
  • 广州三环专利商标代理有限公司 SCIHEAD IP LAW FIRM
Priority Data
201811305441.702.11.2018CN
Publication Language Chinese (ZH)
Filing Language Chinese (ZH)
Designated States
Title
(EN) STRAIN HAVING CCDB NEGATIVE SCREENING EFFECT AND CONSTRUCTION METHOD THEREFOR
(FR) SOUCHE AYANT UN EFFET DE CRIBLAGE NÉGATIF CCDB ET SON PROCÉDÉ DE CONSTRUCTION
(ZH) 一种具有ccdB负筛作用的菌株及其构建方法
Abstract
(EN)
A strain having ccdB negative screening effect and a construction method therefor. The strain is NEB® Stable strain from which ccdA gene and TetR gene in F factor are removed. Said method comprises the following steps: (1) replacing ccdA and TetR genes in the F factor in NEB® Stable strain with FRT-Kan cassette-FRT DNA sequence by using Red/ET homologous recombination technology to obtain a gene edited F factor; and (2) providing a plasmid expressing FLP recombinase, and mediating a recombination reaction by means of the FLP recombinase expressed by the plasmid, and deleting an exogenous sequence between two FRT sites on the gene edited F-factor, so as to obtain the strain having ccdB negative screening effect.
(FR)
L'invention concerne une souche ayant un effet de criblage négatif ccdB et son procédé de construction. La souche en question est la souche NEB® Stable à partir de laquelle le gène ccdA et le gène TetR dans le facteur F sont éliminés. Le procédé comporte les étapes suivantes : (1) le remplacement des gènes ccdA et Tetr dans le facteur F dans la souche NEB® Stable par la séquence d'ADN FRT-Kan cassette FRT en utilisant une technologie de recombinaison homologue rouge/ET pour obtenir un facteur F édité par un gène ; et (2) la fourniture d'un plasmide exprimant la recombinase FLP et la médiation d'une réaction de recombinaison au moyen de la recombinase FLP exprimée par le plasmide et la suppression d'une séquence exogène entre deux sites FRT sur le facteur F édité par le gène, de façon à obtenir la souche ayant un effet de criblage négatif ccdB.
(ZH)
一种具有ccdB负筛作用的菌株及其构建方法,该菌株为去掉了F因子中ccdA基因和TetR基因的NEB® Stable菌株,该方法包括以下步骤:(1)利用Red/ET同源重组技术将NEB® Stable菌株中F因子上的ccdA和TetR基因替换为FRT-Kan cassette-FRT DNA序列,得到基因编辑后的F因子;(2)提供表达FLP重组酶的质粒,通过所述质粒表达的FLP重组酶介导重组反应,删除所述基因编辑后的F因子上两个FRT位点中间的外源序列,即得所述具有ccdB负筛作用的菌株。
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Latest bibliographic data on file with the International Bureau