Screening method for methylated protective strain expressing restriction endonuclease SacI

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...

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

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