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Bacillus coli-streptomycete-pseudomonas shuttling expressing BAC vector and construction method thereof

A Pseudomonas and vector technology, applied in the field of genetic engineering, can solve the problems of not screening recombinant clone markers, affecting gene expression efficiency, etc.

Inactive Publication Date: 2008-11-12
广州冀源生物科技有限公司
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  • Abstract
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Problems solved by technology

The disadvantage is that the vector is still a single copy; or there is no obvious marker for screening recombinant clones; and the transformed Pseudomonas genome contains many foreign genes, which may affect the expression efficiency of genes cloned on the vector

Method used

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  • Bacillus coli-streptomycete-pseudomonas shuttling expressing BAC vector and construction method thereof
  • Bacillus coli-streptomycete-pseudomonas shuttling expressing BAC vector and construction method thereof
  • Bacillus coli-streptomycete-pseudomonas shuttling expressing BAC vector and construction method thereof

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Embodiment 1

[0063]Referring to Figure 1, genes or fragments such as pUC18, Am, Crp, PP_0423', int, attP, OriT were cloned into pECBAC1 by conventional molecular biology and recombinant engineering methods, and the chloramphenicol resistance gene part on pECBAC1 was removed.

[0064] Linking pECBAC1 and pUC18 to obtain high-copy BAC vector pEPUBAC

[0065] Both pECBAC1 and pUC18 were digested with BamHI, precipitated with ethanol, dissolved in an appropriate amount of double distilled water, and connected overnight at 16°C with T4 DNA ligase. The connection solution was transformed into competent cells of E.coli DH10B, and the transformation solution was spread on On LB solid plates containing chloramphenicol 12.5 μg / ml, 50 μg / ml ampicillin, 20 μg / ml IPTG and 40 μg / ml Xgal, culture overnight at 37°C. Pick the white colony, extract the plasmid, and verify it by digestion. The correct clone is named pEPUBAC.

[0066] Removal of the XbaI site outside the pEPUBAC multiple cloning site

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Abstract

The invention relate to a shuttle expression BAC vector among colon bacillus, streptomyces and pseudomonad, and a construction method thereof. The BAC vector contains a replicon of pUC18, a penbritin resistance gene, a conjugal transfer fragment oriT, an attP locus, an integrase gene int, an Am resistance gene, a 5' end of a PP_0423' gene of the pseudomonad, a crp gene of the pseudomonad, a redF gene of the BAT vector, duplicate field OriS, a repA gene, a sopA gene, a sopB gene, a sopC gene, a cos locus, and a loxP locus. The method starts from pECBAC1, effectively combines and optimizes a necessary original of heterologous expression, applies means such as gene clone and recombination engineering and so on, successfully constructs the shuttle expression BAC vector, and provides the convenience for biosynthetic gene clusters with the heterologous expression and high throughput screening.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a BAC vector shuttled and expressed among three hosts of Escherichia coli-Streptomyces-Pseudomonas and its construction method. Background technique: [0002] Microorganisms are a major source of drugs used clinically. Between 1981 and 2006, 34% of small-molecule drugs approved by the FDA were natural products or their direct semi-synthetic derivatives of microbial origin; of a total of 155 anticancer drugs, 47% were natural products or their direct semi-synthetic derivatives thing. With the rapid development of microbial molecular biology, more and more microbial genomes have been analyzed, and the full utilization of microbial genome resources has become a hot spot in the field of microbial pharmacology. [0003] Among many genetic engineering methods, the heterologous expression of biosynthetic gene clusters has been proven to be the most effective so far. Heterologous ex...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/65
Inventor 尚广东黄慧颖宋杰
Owner 广州冀源生物科技有限公司
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