Escherichia coli-streptomycete shuttle-type BAC vector and construction method

Abstract

The invention relates to an escherichia coli-streptomycete shuttle-type BAC vector and its construction method. The BAC carrier contains multiple cloning sites (MCS), an Am resistant gene, an intergrase gene int, an integrate site attp, conjugal transfer fragments oriT and oriS, a repA gene, a sopA gene, a sopB gene, a sopC sequence, a cos site and a loxP site of replication region of the BAC vector. Beginning with pBeloBAC11, a Red homologous recombination technology is used to replace the above chloramphenicol resistant gene with DNA fragment of oriT-attp-int-Am for conjugal transfer (oriT), site-specific integration (attp-int) and resistance screening (Am) so as to obtain a vector pBTIBAC1. Thus, a foundation is established for realizing heterogeneous expression of large fragments of gene clusters in streptomycete.

Description

technical field [0001] The invention relates to an Escherichia coli-Streptomyces shuttle type BAC vector and a construction method thereof, in particular to a BAC vector capable of conjugative transfer from Escherichia coli to Streptomyces and integrated into its genome and a construction method thereof, belonging to the field of genetic engineering. Background technique [0002] Streptomyces is the species that produces the most antibiotics in microorganisms. Among the more than 5,000 antibiotics found in the world, more than 60% are synthesized by Streptomyces. As antibiotic production bacteria, Streptomyces has a long history of application in the traditional fermentation industry. The history of Streptomyces, while the development of molecular genetics has shown broad application prospects for the gene recombination of Streptomyces. Since the first report of Streptomyces gene cloning in 1980, Streptomyces genetic engineering has been on the rise. [0003] As an important...

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Problems solved by technology

[0004] At the same time, the Streptomyces gene transfer system has also begun to be established. The earlier documented method is the protoplast transformation method, including PEG-mediated and liposome-assisted methods. However, many obstacles are often encountered in practical applications, mainly due to the special The structural characteristics of the protoplasts are difficult to prepare, or it is difficult to be transformed by foreign plasmids, and the electric shock transformation method is also very inefficient. Since Trien-Cuot et al. first clarified the difference between Escherichia coli and many Gram-positive bacteria Two years later, Mazodier developed a method for conjugative transfer of plasmids between Escherichia coli and Streptomyces, that is, introducing the conjugative transfer cis-acting element oriT on the Escherichia coli-Streptomyces shuttle vector, Transform it into Escherichia coli carrying the RP4 plasmid, and the vector carrying the target gene is conjugatively transferred from Escherichia coli to Streptomyces under the induction of the trans-acting element tra on RP4

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