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Genetically engineered bacterium of coleophoma sp. with efficient homologous recombination as well as construction method and application of genetically engineered bacterium

A technology of genetic engineering bacteria and homologous recombination, which is applied in the field of genetic engineering bacteria of the genus Coleoptera and its construction, can solve the problems of restricting the application of gene targeting technology, low efficiency of homologous recombination and the like

Active Publication Date: 2021-06-04
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Application Information

AI Technical Summary

Problems solved by technology

In filamentous fungi, the repair of DNA double-strand breaks mainly relies on non-homologous end joining (NHEJ), which leads to the integration of foreign DNA into the genome chromosome mainly by random insertion, and the low efficiency of homologous recombination, which seriously Restricting the Application of Gene Targeting Technology in Filamentous Fungi

Method used

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  • Genetically engineered bacterium of coleophoma sp. with efficient homologous recombination as well as construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium of coleophoma sp. with efficient homologous recombination as well as construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium of coleophoma sp. with efficient homologous recombination as well as construction method and application of genetically engineered bacterium

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Example 1. Construction of the genetically engineered strain Coleophoma sp.-△ku80 knocking out the ku80 gene

[0045] 1. Construct the targeting element of ku80 gene

[0046] according to figure 1 Targeting element strategy map, construction of ku80 gene targeting element, and description of the method for constructing genetic engineering strain Coleophoma sp.-△ku80 according to the present invention.

[0047] According to the sequencing results of Coleophoma sp. genome, primers Uku80-F (sequence shown in SEQ ID NO.1, i.e. 5'-tgcgcgtctgaaatggacac-3'), Uku80-R1 (sequence shown in SEQ ID NO.2, i.e. 5') were designed. '-ctttacgcttgcgatcccgaaGGCCATCTGTAACAAGAATAA-3'), Dku80-F1 (sequence shown in SEQ ID NO.3, ie 5'-ccctgggttcgcaaagataattgCGAGATACAATTACGCCACT-3'), Dku80-R (sequence shown in SEQ ID NO.4, ie 5'- cctcggaatgtctccagaag-3'), using the genome of Coleophoma sp. as a template, using pfu DNA polymerase (Fermentas, Catalog No.: EP0501) for PCR amplification, using pri...

Embodiment 2

[0055] Example 2. Validation of Homologous Recombination Efficiency in Genetic Engineering Bacteria Coleophoma sp.-△ku80 with ku80 Gene Deletion

[0056] 1. Using the geneticin resistance gene neo as a screening tag to construct the ku80 site resistance tag replacement element

[0057] according to image 3 Targeting element strategy map, construction of ku80 site resistance tag replacement element, and description of the method for constructing foreign genes described in the present invention.

[0058] Using the genome of Coleophoma sp. as a template, primers Uku80-F (5'-tgcgcgtctgaaatggacac-3'), Uku80-R2 (sequence shown in SEQ ID NO.9, namely 5'-ggggcttttccttctctagaGGCCATCTGTAACAAGAAT-3'), Dku80-F2 (The sequence is shown in SEQ ID NO.10, that is, 5'-actgacttgtgcgctgcaatCGAGATACAATTACGCCACT-3'), Dku80-R (5'-cctcggaatgtctccagaag-3'), the upstream sequence Uku80-2 with a size of about 1.8kb was obtained by PCR amplification The downstream sequence Dku80-2 is about 1.5 kb in s...

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Abstract

The invention discloses a genetically engineered bacterium of coleophoma sp. with efficient homologous recombination as well as a construction method and application of the genetically engineered bacterium, and belongs to the technical field of gene engineering. The invention aims to solve the problem of how to improve the homologous recombination efficiency of Coleophoma sp.. According to the Coleophoma sp. genetically engineered bacterium for improving the homologous recombination efficiency through ku80 gene deletion, the Coleophoma sp. genetically engineered bacterium serves as a starting bacterium, an exogenous gene in a targeting element is integrated to a genome in a homologous recombination mode in a fixed point mode, the homologous recombination efficiency reaches 90% or above, and a good theoretical basis is provided for an application of a gene targeting technology in filamentous fungi.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a highly efficient homologous recombination genetic engineering bacterium of the genus Phomophora and its construction method and application. Background technique [0002] Cyclic lipopeptides are an important natural product of microorganisms, which have been reported to have a variety of biological activities and have been developed into antifungal drugs, immunosuppressants and other drugs. Coleophoma sp. has been reported to be able to synthesize cyclolipopeptides, but due to the low efficiency of homologous recombination, genetic modification such as gene targeting is difficult, which limits the analysis of the synthesis mechanism and metabolism of cyclolipopeptides. Engineered. The development of a highly efficient genetically modified Coleophoma sp. chassis cell and its method have important application value. [0003] Gene targeting technology is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/80C12N15/90C12N15/65C12N15/64C12R1/645
CPCC12N1/14C12N15/80C12N15/902C12N15/65
Inventor 吕雪峰门萍黄雪年王敏
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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