Screening method for methylated protective strain expressing restriction endonuclease SacI

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.

Inactive Publication Date: 2018-01-09
江苏愚公生命科技有限公司
View PDF4 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage is that the methylation protection of the host cell DNA can only be specifically protected from the cutting of the SacI 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

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Screening method for methylated protective strain expressing restriction endonuclease SacI
  • Screening method for methylated protective strain expressing restriction endonuclease SacI
  • Screening method for methylated protective strain expressing restriction endonuclease SacI

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0055] 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.

[0056] 1. Materials

[0057] 1.1 Strains and plasmids Arthrobacter luteus, Streptomyces achromogenes

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

[0059] DH5α was purchased from Beijing Quanshijin Biotechnology Co., Ltd., a commercially available product;

[0060] ER2566 was purchased from Thermo Fisher Scientific Co., Ltd. (Thermo Fisher Scientific), a commercially available product; pBAD, pACYC184, and pUC19 were purchased from Miaoling Bioplasmid Platform;

[0061] The enzyme activity detection substrate λDNA (HindIII digest) was purchased from Thermo Fisher Scientific Co., Ltd., a commercially available product (Thermo Fisher Scientific);

[0062] 1.2 Instruments and reagents JN-02C lo...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention provides a screening method for methylated protective strain expressing restriction endonuclease SacI. The method comprises steps: screening and culturing recombinant expression plasmidpBAD-R. SacI transformed competence cells with correct sequencing to obtain a recombinant expression strain of restriction endonuclease SacI by establishment of recombinant expression strain of methyltransferase M. AluI, recombinant expression of restriction endonuclease SacI, and other steps; and performing enlarging cultivation on the strain, and inducing with arabinose to obtain the recombinantprotein of restriction endonuclease SacI. For the method disclosed by the invention, the efficient recombinant expression of the restriction endonuclease SacI can be achieved; the recombinant expression of the restriction endonuclease SacI is carried out by strict control of an arabinose operon expression system on background expression; the method is stable and efficient against digestion of therestriction endonuclease SacI on a host genome DNA; and an M. AluI methylated protection strain is also applicable to recombinant expression of other restriction endonucleases with similar recognition sites.

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 SacI. Background technique [0002] Restriction endonucleases are nucleases that can recognize specific sequences in double-stranded DNA sequences and cut them (Ch.8, eds. De Bruijin, et al., Chapman & Hall, New York, 78-92.1998), and their discovery prompted DNA recombination technology The birth of it has greatly promoted the development of modern molecular biology and genetic engineering, and it is an indispensable basic tool for contemporary genetic engineering research. In bacteria, restriction endonucleases and their corresponding methyltransferases constitute a restriction-modification system to protect microorganisms from foreign phages or plasmids. Restriction enzymes and methyltransferases of the same system have the same recognition sequence on DNA, but their functions a...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/74C12N15/66
Inventor 张坤晓龚雪梅韩挺翰许恒皓
Owner 江苏愚公生命科技有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap