Traceless modification method of chromosome

A modification method and chromosome technology, applied in the field of chromosome genome modification, can solve the problems of no positive screening, time-consuming labor intensity, etc.

Inactive Publication Date: 2010-11-24
苏州神洲基因有限公司
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Problems solved by technology

[0006] The above methods have obvious drawbacks, they involve two crossover processes, there is no positive selection in the final recombination step, and they are limited to work in glkA or pyrF null mutant strains
In addition, each knockout step requires repeated plasmid transformation and knockout, which is time-consuming and labor-intensive

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  • Traceless modification method of chromosome
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Embodiment Construction

[0024] The present invention seamlessly modifies host chromosomes by using positive selection and reverse selection markers. First, the target gene is replaced with a DNA fragment containing a positive resistance marker, recombinants are detected in selective medium, and then selected by I-SceI-induced double-strand breaks and counter-selectable marker gene sacB without any selectable marker or foreign DNA The final recombinant of the sequence.

[0025] According to the DNA recombination and selection mechanism in the target cell body, the present invention reduces some unnecessary steps such as plasmid transformation and repeated screening.

[0026] The present invention uses a counter-selectable marker gene, such as the sacB gene of Bacillus subtilis, to make a large number of bacteria sensitive to sucrose. The SacB gene encodes fructan sucrase, which catalyzes the hydrolysis or polymerization of sucrose to form the lethal product fructan. E. coli and many Gram-negative ba...

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Abstract

The invention provides a traceless modification method of a chromosome, comprising the following steps: firstly building a recombinant box composed of a positive selection marker, a counterselection marker, an endonuclease I-SceI, an induced promoter, an I-SceI recognition site and a target nucleic acid displacement sequence which is homologous with a target chromosome nucleic acid sequence; inputting DNA fragments into a host containing recombinant plasmids by electrotransformation; knocking out the following gene sequences from an expected recombinant through a double-strand break repair phenomenon mediated by I-SceI: the positive selection marker, the induced promoter, an I-SceI gene and the counterselection marker; and finally screening the final recombinant from a sucrose medium. The method can help insert, knock out or replace the target gene of the chromosome without any selection markers or exogenous DNA sequences remained, thus being an effective method for traceless modification of the DNA sequence of a host cell.

Description

technical field [0001] The present invention relates to a method for inserting, knocking out or replacing one or more target genes on a chromosome by means of gene recombination, especially a method for modifying without leaving any screening marks or exogenous DNA sequences on the original chromosome The invention belongs to the technical field of chromosome genome transformation. Background technique [0002] Chromosomal genome modification technologies, such as gene interference, foreign gene insertion, gene mutation and other technologies, are very important for the fields of bioenergy, metabolic engineering and agricultural engineering. Red / ET recombination engineering (Recombineering) is a new type of genetic engineering technology established in recent years. It is a high-efficiency in vivo homologous recombination reaction mediated by recombinant proteins (Exo, Beta, Gam) produced by phage. Sequences for gene insertion, knockout, replacement, etc. do not need to sel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N1/21
Inventor 王德明
Owner 苏州神洲基因有限公司
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