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Culture medium for inducing plasmid copy number increase and application thereof

A technology of copy number increase and culture medium, which is applied in the field of medium for inducing plasmid copy number increase, and can solve the problems of cumbersome operation of the plasmid copy number induction system

Pending Publication Date: 2020-07-07
JIANGSU GENSCRIPT BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Aiming at the problem of complicated operation of the existing plasmid copy number induction system, the present invention provides a culture medium for inducing plasmid copy number increase and its application, which improves production efficiency while simplifying the operation

Method used

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  • Culture medium for inducing plasmid copy number increase and application thereof
  • Culture medium for inducing plasmid copy number increase and application thereof
  • Culture medium for inducing plasmid copy number increase and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0057] The pCCIBAC plasmid (shown in SEQ ID NO: 1) contains the oriV origin of replication. The pCCIBAC plasmid was transformed into EPI300 Escherichia coli competent by chemical transformation. The transformed EPI300 strain was grown in the initial medium. The initial medium composition was 10g / L tryptone, 5g / L yeast extract and 10g / L sodium chloride, 2g / L glucose and 0.75g / L arabinose. Bacteria were cultured at 220 rpm at 25°C for 5 hours. After inoculation, 6 OD600 cells were taken every 1 h for plasmid extraction. The plasmid was extracted using the standard procedure of the plasmid extraction kit AxyGEN Miniprep Kit (Lot#17718KA1). Plasmid extraction results see figure 1 .

Embodiment 2

[0059] The pCCIBAC plasmid contains the oriV origin of replication. The plasmid was transformed into EPI400 Escherichia coli competent by chemical transformation. The transformed EPI400 strain was grown in the initial medium. The initial medium composition was 5g / L tryptone, 10g / L yeast extract and 5g / L sodium chloride, 1g / L glucose and 0.6g / L arabinose. Bacteria were cultured at 220 rpm and 30°C for 5 hours. After inoculation, 6 OD600 cells were taken every 1 h for plasmid extraction. The plasmid was extracted using the standard procedure of the plasmid extraction kit AxyGEN Miniprep Kit (Lot#17718KA1). Plasmid extraction results see figure 2 .

Embodiment 3

[0061] The pCCIBAC plasmid contains the oriV origin of replication. The pCCIBAC plasmid was transformed into EPI300 Escherichia coli competent by chemical transformation. The transformed EPI300 strain was grown in the initial medium. The initial medium components were 10g / L tryptone, 10g / L yeast extract and 10g / L sodium chloride, 0.5g / L glucose and 0.3g / L arabinose. Bacteria were cultured at 220 rpm at 37°C for 5 hours. After inoculation, 6 OD600 cells were taken every 1 h for plasmid extraction. The plasmid was extracted using the standard procedure of the plasmid extraction kit AxyGEN Miniprep Kit (Lot#17718KA1). Plasmid extraction results see image 3 .

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Abstract

The invention provides a culture medium for inducing plasmid copy number increase and an application thereof. The culture medium for inducing the plasmid copy number increase has the advantages that plasmid extraction concentration is improved by 45-95% in comparison with that of a traditional plasmid copy number induction method, and the plasmid extraction concentration is improved by 110-440% incomparison with that of a culture medium induction method free of glucose and arabinose. The culture medium plays very important roles in induction of the plasmid copy number increase and implementation of high throughput production.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a culture medium for inducing plasmid copy number increase and application thereof. [0002] technical background [0003] In the field of gene synthesis, more than 10% of gene sequences can be toxic to the host Escherichia coli. These toxicities are usually caused by the introduction of heterologous genes into the host and the expression of proteins that affect the physiological metabolism of the host, thereby affecting the growth of the host. In order to resist toxic genes, the host Escherichia coli tends to retain plasmids with gene mutations or gene deletions, resulting in reduced stability of genes and plasmids. The use of low-copy plasmids in production can effectively reduce the expression of toxic genes, while improving the growth vigor and gene stability of the host. However, the yield of low-copy plasmids is low, and extraction is more difficult, which affects ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70
CPCC12N1/20C12N15/70C12R2001/19C12N1/205C12N2500/34C12N2500/74C12N2800/101
Inventor 赵方龙马艳秋王嫚刘凡吴政宪
Owner JIANGSU GENSCRIPT BIOTECH CO LTD
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