CN-122012564-A - Visual gene knockout system and application thereof

CN122012564ACN 122012564 ACN122012564 ACN 122012564ACN-122012564-A

Abstract

The invention provides a visual gene knockout system and application thereof. The visual gene knockout system comprises a recombinant suicide plasmid vector, wherein the recombinant suicide plasmid vector comprises (1) an upstream homologous arm sequence and a downstream homologous arm sequence of a gene to be knocked out, and (2) an expression cassette, wherein the expression cassette comprises a promoter element and a fluorescent protein gene, and the promoter element is a constitutive promoter. According to the invention, a fluorescent protein expression cassette is introduced into a suicide plasmid-mediated homologous recombination gene knockout system, so that a target gene-expected knockout recombinant strain can generate a definite fluorescent signal, and a screening result can be directly judged by naked eye observation or fluorescent detection equipment, thereby avoiding the defect of judging by only relying on a molecular detection means.

Inventors

LIN LIANGCAI
He Manying
LIU GAOWEN
CHEN HUAZHAN

Assignees

天津科技大学
中国科学院深圳先进技术研究院

Dates

Publication Date: 20260512
Application Date: 20260119

Claims (8)

1. A visual gene knockout system comprising a recombinant suicide plasmid vector, said recombinant suicide plasmid vector comprising: (1) An upstream homology arm sequence and a downstream homology arm sequence of the gene to be knocked out; (2) An expression cassette comprising a promoter element and a fluorescent protein gene, the promoter element being a constitutive promoter.
2. The visual gene knockout system of claim 1, wherein the nucleotide sequence of the promoter element is set forth in SEQ ID No. 3.
3. The visual gene knockout system of claim 1, wherein the fluorescent protein gene is any one of sfGFP gene, mCherry gene and BFP gene.
4. The visual gene knockout system of claim 1, wherein the recombinant suicide plasmid vector has pK18mobsacB as a backbone.
5. The visual gene knockout system of claim 1, wherein the upstream homology arm sequence and the downstream homology arm sequence of the gene to be knocked out are 800-1500 bp in length.
6. A visual gene knockout screening method for copper bacteria of hook worm is characterized by comprising the following steps: (1) Sequentially connecting an upstream homologous arm sequence of the gene to be knocked out, a promoter element, a fluorescent protein gene and a downstream homologous arm sequence of the gene to be knocked out, and introducing the connected sequences into a framework to obtain a recombinant plasmid; (2) Transforming the recombinant plasmid in the step (1) into escherichia coli to obtain a donor strain; (3) And (3) joining the donor strain in the step (2) with the copper greedy fungus, and screening out the transformant with fluorescent target gene knockout.
7. The use of the visual gene knockout system of any one of claims 1-5 in editing a copper species of ancylostoma.
8. The use of claim 7, wherein the gene editing is gene knockout.

Description

Visual gene knockout system and application thereof Technical Field The invention relates to the field of genetic engineering, in particular to a visual gene knockout system and application thereof. Background Polyhydroxyalkanoates (PHA) are natural polyester materials synthesized by microorganisms under specific conditions and stored in cells, have good biocompatibility and complete biodegradability, have mechanical properties and thermal properties similar to those of partial petroleum-based plastics (such as polypropylene), and are considered as one of the most promising degradable plastics. Therefore, efficient, low cost production of PHA is of great importance to drive industrialization of biobased materials. Currently, PHAs are produced mainly by means of microbial fermentation. Among the various PHA-producing bacteria reported, copper (Cupriavidus necator) H16, a model strain that has been most extensively studied and used. The strain not only can efficiently synthesize PHA by utilizing various organic carbon sources such as saccharides, grease, organic acid and the like, but also has unique autotrophic growth capacity, and can fix carbon dioxide and synthesize cell substances and PHA by using hydrogen, carbon dioxide and oxygen as substrates through the Calvin cycle. The characteristics enable the copper greedy fungus H16 to have remarkable advantages in the aspects of reducing the raw material cost, reducing the carbon emission and fitting with the development strategy of carbon neutralization. In order to deeply analyze the synthesis and regulation mechanism of PHA, and to improve the yield of PHA, regulate the monomer composition or reduce the production cost by means of metabolic engineering, accurate and efficient gene editing of the copper greedy fungus H16 is an indispensable research means. Among them, gene knockout is an essential technique for studying gene function, weakening competing metabolic pathways, and reconstructing metabolic networks, and is important in genetic engineering of the strain. At present, the gene knockout method which is most widely applied and relatively mature in technology aiming at the copper greedy fungus H16 is a suicide plasmid-mediated homologous recombination method. The method generally adopts a suicide plasmid incapable of autonomous replication in host bacteria as a vector, constructs a recombinant fragment containing an upstream and downstream homologous sequence of a target gene and a resistance selection marker on the plasmid, and introduces the recombinant fragment into a copper bacteria cell of the ancylostoma. Subsequently, gene knockout is achieved by replacing or deleting the target gene with the resistance gene depending on the homologous recombination event. Finally, it is often necessary to identify the obtained strain by means of PCR amplification in combination with electrophoretic analysis to confirm whether the target gene has been successfully knocked out. The above methods are conventional techniques for performing gene knockout operations by those skilled in the art. However, existing suicide plasmid-mediated homologous recombination gene knockout methods still have the following disadvantages: (1) The screening process is tedious and the period is long. From the beginning of the conjugation transfer to the acquisition of positive clones verified by PCR, usually 10-14 days are required, and in the process, plate streaking, strain culture and molecular biology operations are required to be repeatedly carried out, so that time and labor are consumed; (2) The identification mode has lower efficiency and limited reliability. At present, judgment is mainly carried out by depending on PCR and electrophoresis results, but because the genome structure of the copper bacteria of the hook worm is complex and has higher sequence homology, nonspecific bands or primer dimers are easy to generate in the PCR amplification process, so that the electrophoresis background is complex, the results are difficult to judge, further sequencing and confirmation are often needed, and the experimental cost and the error risk are increased; (3) The screening flux is low. The method is difficult to perform rapid preliminary screening on a large number of candidate colonies, and usually only a small number of colonies can be randomly selected for PCR verification, so that the efficiency and the scale of the gene knockout experiment are limited. Therefore, there is a need to provide a screening means that enables a rapid and intuitive determination of homologous recombination events of copper greedy bacteria H16. Disclosure of Invention Aiming at the defects in the prior art, the invention provides a visual gene knockout system and application thereof. The invention provides a visual gene knockout system, which comprises a recombinant suicide plasmid vector, wherein the recombinant suicide plasmid vector comprises: (1) An upstream homology arm sequence and a downstream