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Screening method of methylated protective strain for expressing restriction enzyme ApaLI

A technology of restriction endonuclease and screening method, which is applied in the field of screening of methylation protection strains, and can solve the problems of rarity and narrow application range.

Pending Publication Date: 2019-12-17
莫纳(连云港)生物科技有限公司 +1
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Problems solved by technology

The disadvantage is that the methylation protection of the host cell DNA can only be specifically protected from the cleavage of the ApaLI restriction enzyme after tedious DNA library construction and screening, and few of them can be used. For the expression of other restriction enzymes, the scope of application is extremely narrow

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  • Screening method of methylated protective strain for expressing restriction enzyme ApaLI
  • Screening method of methylated protective strain for expressing restriction enzyme ApaLI
  • Screening method of methylated protective strain for expressing restriction enzyme ApaLI

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Embodiment Construction

[0054] The specific technical solutions of the present invention will be further described below with reference to the accompanying drawings, so that those skilled in the art can further understand the present invention, without limiting their rights.

[0055] 1. Materials

[0056] 1.1 Strains and plasmids

[0057] Chlorella virus, Acetobacter pasteurianus mogenes

[0058] The strains were all from the American Type Culture Collection (ATCC);

[0059] DH5α was purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

[0060] ER2566 was purchased from Thermo Fisher Scientific Co., Ltd. (Thermo Fisher Scientific);

[0061] pBAD, pACYC184, and pUC19 were purchased from Miaoling Bioplasmid Platform;

[0062] The enzyme activity detection substrate λDNA (HindIII digest) was purchased from Thermo Fisher Scientific Co., Ltd. (ThermoFisher Scientific);

[0063] 1.2 Instruments and reagents

[0064]JN-02C low-temperature ultra-high pressure continuous flow cell disruptor: purch...

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Abstract

The invention discloses a screening method of a methylated protective strain for expressing a restriction enzyme ApaLI. According to the screening method, a gene of methyltransferase M.CviRI is obtained in a genome of a strain C hlorella viruss by using a specific primer of the gene of the methyltransferase M.CviRI, then construction of a recombinant expression strain of the restriction enzyme ApaLI is realized, finally, a recombinant expression plasmid pBAD-R.ApaLI transforms a competent cell [pACYC184-M.CviRI,ER2566] for a screening culture, and the recombinant expression strain of the restriction enzyme ApaLI is obtained. According to the screening method, the efficient recombinant expression of the restriction enzyme ApaLI is realized; an arabinose operon expression system is used forstrictly controlling the background expression level of the restriction enzyme ApaLI; and the screened M.CviRI methylated protective strain can be applied to the recombinant expression of other restriction enzymes with similar recognition sites as well.

Description

technical field [0001] The invention relates to a screening method for strains, in particular to a screening method for methylation-protected bacterial strains expressing restriction endonuclease ApaLI. Background technique [0002] In organisms, there is a class of enzymes that can cut foreign DNA. It can limit the invasion of heterologous DNA and make it lose its vitality, but it has no damage to its own DNA, so that it can protect the original genetic information of cells. role. Since this cutting effect is carried out inside the DNA molecule, it is named restriction endonuclease (restriction enzyme for short). Its discovery led to the birth of DNA recombination technology, which greatly promoted the development of modern molecular biology and genetic engineering, and is an indispensable basic tool for contemporary genetic engineering research. [0003] Due to the "restriction-modification" phenomenon in bacteria, that is, their own DNA is modified by methylases, so as ...

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12Q1/689C12Q1/10C12R1/19
CPCC12N9/22C12N9/1007C12N15/70C12Q1/689
Inventor 张坤晓许恒皓葛绍勇郭馨龚雪梅
Owner 莫纳(连云港)生物科技有限公司
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