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Cloning method of antibiotics biosynthetic gene cluster based on site-specific recombination

A technology for synthesizing genes and cloning methods, applied in the field of genetic engineering, can solve the problems of difficulty in cloning and biosynthesis of gene clusters, and achieve the effect of simple methods

Inactive Publication Date: 2013-04-24
FUDAN UNIV
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Problems solved by technology

[0005] The purpose of the present invention is to overcome the problem that the biosynthetic gene clusters of large fragments are difficult to be cloned by traditional methods, and provide a method for cloning antibiotic synthetic gene clusters based on the Streptomyces phage φBT1 integrase system

Method used

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  • Cloning method of antibiotics biosynthetic gene cluster based on site-specific recombination
  • Cloning method of antibiotics biosynthetic gene cluster based on site-specific recombination
  • Cloning method of antibiotics biosynthetic gene cluster based on site-specific recombination

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Embodiment Construction

[0027] The present invention takes the cloning of the erythromycin biosynthetic gene cluster in Saccharopolyspora rubrum as an example, specifically introduces the method for cloning the antibiotic synthetic gene cluster, and the detailed operation process is as follows:

[0028] 1. Construction of the upstream homologous recombination vector pEry-up of the erythromycin gene cluster

[0029] Saccharopolyspora rubrum Saccharopolyspora erythraeThe genome of a is used as a template, and primers SEQ ID No. 2 and primers SEQ ID No. 3 are used to amplify the upstream homology arm fragment of the erythromycin gene cluster. The fragment size is 1993bp, and SEQ ID No. 2 is designed with MunI restriction endonuclease recognition site, make T-A clone of PCR product fragment and pMD19-T vector (TAKARA company) and transform Escherichia coli, pick positive transformant and extract plasmid DNA, select MunI site and XbaI site on T vector in the adjacent direction Plasmid, named T-ery-up. ...

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Abstract

The invention belongs to the field of biotechnology and genetic engineering, and specifically discloses a cloning method of antibiotics biosynthetic gene cluster based on site-specific recombination. The method comprises the following steps of: based on the site-specific recombination reaction mediated by the integrase encoded by the streptomycete bacteriophage Phi BT1, firstly, executing genetic modification on the strains, which hare generated by the antibiotics, by means of homologous recombination mode; respectively inserting the BAC (Bacterial Artificial Chromosome) vector, the resistant gene and the site-specific recombination sites attB and attP identified by the Phi T1 integrase at two ends of the targeted gene cluster, and extracting chromosome genome segments; under the action of the Phi T1 integrase, causing site-specific recombination reaction between the sites attB and attP in vitro; enabling the target gene cluster to cyclize itself, thereby realizing clone of dozens or even hundreds of kb of large-segment antibiotics biosynthetic gene clusters. The cloning method of antibiotics biosynthetic gene cluster based on site-specific recombination lays good foundation for further genetic modification of the biosynthetic gene clusters, and has better application foreground.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for realizing large fragment biosynthetic gene cluster cloning based on φBT1 integrase system and homologous recombination technology. Background technique [0002] Natural products have always played an important role in the discovery and development of drugs. In the past few decades, it has been the main drug for human treatment of diseases. However, with the widespread use of antibiotics, bacteria have gradually developed drug resistance. At the same time, it has become more and more difficult to screen new natural products from nature with traditional methods, so it is difficult to meet the needs of disease treatment. Therefore, the discovery of new biologically active natural products and their structural analogues has become a research hotspot in the fields of biology, chemistry and medicine. As a traditional method to increase the structu...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/66C12R1/01
Inventor 张渤丁晓明赵国屏
Owner FUDAN UNIV
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