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Multi-gene combined editing and screening method for microorganisms

A screening method and multi-gene technology, applied in the biological field, can solve the problems of lack of multi-gene combination editing methods, achieve the effect of wide application, fast and efficient multi-gene editing, and reduce workload

Pending Publication Date: 2021-07-16
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the lack of multi-gene combination editing means in microorganisms (especially Clostridium), the purpose of the present invention is to solve the existing deficiencies, provide a multi-gene combination editing and screening method, and edit and combine multiple genes simultaneously in one round of operation , efficient and rapid screening to obtain a combinatorial library of strains

Method used

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  • Multi-gene combined editing and screening method for microorganisms
  • Multi-gene combined editing and screening method for microorganisms
  • Multi-gene combined editing and screening method for microorganisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Combinatorial editing of two genes for one-time transformation with equivalent equivalents matching multiple recombinant vectors

[0056] Combination editing of two histidine kinase genes cac0323 and cac2730 in Clostridium acetobutylicum, theoretically there are three kinds of combination editing of the two genes. According to the sequence information, with the help of software, design appropriate insertion gene sites, respectively select insertion sites at 420 / 421s and 172 / 173a, and synthesize IBS, EBS1d, EBS2, and EBS primers corresponding to the respective genes to construct binary genes targeting different genes. Type intron fragments to obtain methylated recombinant vectors pSY6-cac0323 and pSY6-cac2730. Using the electric pulse conversion method, using the initial concentration (OD 600 =0.8) cells to prepare Clostridium acetobutylicum competent, the two recombinant vectors constructed above were equivalently compatible, wherein the recombinant vectors pSY6-cac032...

Embodiment 2

[0058] Combined editing of two genes by one-time transformation of multiple recombinant vectors with editing sites and equivalence matching

[0059] Combination editing of two histidine kinase genes cac0323 and cac2730 in Clostridium acetobutylicum, theoretically there are three kinds of combination editing of the two genes. According to the sequence information, use the software to design the appropriate insertion gene site, select the forward insertion at the 420 / 421s and 192 / 193s sites respectively, and synthesize the IBS, EBS1d, EBS2 and EBS primers corresponding to the respective genes to construct different gene targeting The two-type intron fragments obtained methylated recombinant vectors pSY6-cac0323 and pSY6-cac2730. Using the electric pulse conversion method, using the initial concentration (OD 600=0.8) cells to prepare Clostridium acetobutylicum competent, the two recombinant vectors constructed above are equivalently compatible, the volume of the recombinant vect...

Embodiment 3

[0061] Combinatorial editing of three genes in one-time transformation with equivalent equivalents matching multiple recombinant vectors

[0062] Combinatorial editing of three genes in the central carbon metabolism pathway in Clostridium acetobutylicum: lactate dehydrogenase gene ldh (cac0267), phosphate transacetylase gene pta (cac1742) and butyrate kinase gene bukII (cac1660), theoretically three There are 7 types of combinatorial editing for each gene. According to the sequence information, with the help of software, design appropriate insertion gene sites, select the forward insertion sites as 171 / 172, 81 / 82, and 421 / 422, and synthesize IBS, EBS1d, EBS2, and EBS primers corresponding to the respective genes to construct Targeting the type II intron fragments of different genes, the methylated recombinant vectors pSY6-cac0267, pSY6-cac1742 and pSY6-cac1660 were obtained. Using the electric pulse conversion method, using the initial concentration (OD 600 =0.8) cells to pr...

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Abstract

The invention belongs to the technical field of biology and particularly relates to a multi-gene combined editing and screening method for microorganisms. A plurality of recombinant vectors are transformed at one time, each recombinant vector carries one or more target sites, a plurality of target genes and a plurality of target sites can be edited at the same time through one round of operation, according to a random combination principle, strains containing a single gene, different combinations of two genes, different combinations of three genes and different combinations of multiple genes can be rapidly constructed, and a strain library of multi-gene combined editing is obtained. Compared with a conventional method for sequentially editing a plurality of genes round by round, the method is simple to operate, has high efficiency, is time-saving and labor-saving, and has a wide use prospect. For example, on the basis of the combined library, screening of microorganisms with excellent shapes is promoted, functions of homologous or same family genes are favorably researched, and the microorganisms are conveniently modified to obtain new genetic functions and characteristics.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a multi-gene combination editing and screening method for microorganisms. Background technique [0002] Microorganisms capable of synthesizing biofuels have been extensively studied, such as Solventogenic Clostridium and Ethanologenic yeast. Use established genetic tools to engineer these strains to enhance solvent synthesis or to study gene function. For Clostridium acetobutylicum (Clostridium acetobutylicum), which has been extensively studied, genes such as buk, pta, aad, solR and spo0A were edited by homologous recombination to obtain corresponding single-gene edited strains (Dong Hongjun, Clostridium acetobutylicum Transformation of genetic operating system, 2011), the widely used prokaryotic type II intron Lactococcus lactis L1.LtrB has also been modified to knock out a single gene of a variety of Clostridium (Shao et al., Cell Research, 2007(17) , 963; Heap et al...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12Q1/686C12Q1/04C40B50/06C12R1/145
CPCC12N15/74C12Q1/686C40B50/06
Inventor 薛闯朱超程驰杜广庆吴又多康巍
Owner DALIAN UNIV OF TECH
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