CN114480464B

CN114480464B - Double plasmid construction method of vibrio parahaemolyticus CRISPRi

Info

Publication number: CN114480464B
Application number: CN202210235334.1A
Authority: CN
Inventors: 王小元; 孟祥宇; 王建莉; 周晴; 黄丹阳
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-03-11
Filing date: 2022-03-11
Publication date: 2023-10-03
Anticipated expiration: 2042-03-11
Also published as: CN114480464A

Abstract

The application discloses a method for constructing double plasmids of vibrio parahaemolyticus CRISPRi, belonging to the fields of molecular biology and biotechnology. The application discloses a construction method of CRISPRi tool plasmids including pSg and pBAD33T-d9 plasmids. The application provides a construction method of 2 plasmids of vibrio parahaemolyticus CRISPRi, which constructs CRISPRi tool plasmids through an overlap PCR and one-step cloning method. Meanwhile, the application of the double plasmids in the research of the gene functions of the vibrio parahaemolyticus is provided, and the functions of genes, especially key genes, can be more rapidly researched or the influence of a plurality of genes on the vibrio parahaemolyticus can be interfered at the same time. Provides a new method and technology for researching the functions of the vibrio parahaemolyticus genes, and promotes the research and development of the vibrio parahaemolyticus functional genes.

Description

Double plasmid construction method of vibrio parahaemolyticus CRISPRi

Technical Field

The application relates to a method for constructing double plasmids of vibrio parahaemolyticus CRISPRi, belonging to the fields of molecular biology and biotechnology.

Background

CRISPR is known as a clustered regularly interspaced short palindromic repeat. The CRISPR/Cas9 gene editing technique is to use the fused crRNA and tracrRNA as a single guide RNA (sgRNA), with a primordial spacer adjacent motif (PAM) present at the 3' end, a 20bp specific sequence, to direct the nuclease Cas protein 9 (Cas 9) to a target site of any DNA sequence (Jiang Y, chen B, span C, et al, multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System [ J ]. Applied & Environmental Microbiology,2015,81 (7): 2506.). Has been used gradually since 2013, and gene editing systems such as human cells, arabidopsis thaliana, saccharomyces cerevisiae, escherichia coli, corynebacterium glutamicum, and the like have been constructed in animals, plants, and microorganisms. In E.coli, the dCAS9-sgRNA complex inhibits transcription by blocking the binding of RNA polymerase to the promoter DNA or by blocking the extension of transcription. CRISPRi has been used for genetic circuit control in model strains such as E.coli and regulation of gene expression in M.tuberculosis (Mycobacterium tuberculosis) and to alter metabolic pathways by changing metabolic flux through CRISPRi studies have shown that the CRISPRi system has been successfully employed in the gram positive bacteria Bacillus subtilis (Bacillus subtilis), the human microbiota Bacteroides symbiotic bacteria (Bacterioides sp.) and in the order of actinomycetes producing medically relevant secondary metabolites. However, the CRISPR technology has not been applied to Vibrio parahaemolyticus at present because the double plasmid cannot coexist in the Vibrio parahaemolyticus cells (Liu Weiji. CRISPR System application research in Vibrio parahaemolyticus [ D ]. Shanghai ocean university, 2017.).

The CRISPR interference (CRISPRi) technique derived from the CRISPR/Cas9 technique is that two bases in the Cas9 protein sequence are mutated to catalytically inactive Cas9 (dCas 9), i.e. the amino acid sequence is mutated to D10A and H840A, but still recognize and bind to sgrnas. The attachment of dCAS9 protein sterically blocks normal transcription of the gene, thus inhibiting gene transcription (Larson M H, gilbert L A, wang X, et al CRISPR interference (CRISPRi) for sequence-specific control of gene expression [ J ]. Nature Protocols,2013,8 (11): 2180-2196.Doi: 10.1038/nprot.2013.132).

Disclosure of Invention

In order to solve the technical problems, the application can stably transcribe the needed sgRNA under any environment after connecting the sgRNA targeting sequence to the 16srRNA promoter by a homologous recombination method. The dCAS9 gene is put on a pBAD33T vector which can be subjected to joint transduction after transformation, and the induction expression of arabinose is utilized, so that the influence on the growth of thalli is reduced. The system can more rapidly study the functions of genes, especially key genes, or simultaneously interfere with the influence of multiple genes on vibrio parahaemolyticus.

The present application provides a CRISPRi system comprising a plasmid carrying an sgRNA expression cassette and a dCas9 expression vector; the sgRNA expression cassette comprises a vibrio parahaemolyticus 16s rRNA upstream homology arm, a sgRNA and a vibrio parahaemolyticus 16s rRNA downstream homology arm; the dCas9 expression vector comprises a dCas9 gene and an origin of replication element.

In one embodiment of the application, the plasmid carrying the sgRNA expression cassette is a pDS132 plasmid.

In one embodiment of the present application, the dCas9 expression vector uses pBAD33 plasmid as a vector.

In one embodiment of the application, the nucleotide sequence of the upstream homology arm of the vibrio parahaemolyticus 16s rRNA is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream homology arm of the vibrio parahaemolyticus 16s rRNA is shown as SEQ ID NO. 2.

In one embodiment of the present application, the nucleotide sequence of dCAS9 gene is shown in SEQ ID NO.3

In one embodiment of the application, the origin of replication element comprises oriT.

In one embodiment of the present application, the nucleotide sequence of the oriT gene fragment is shown in SEQ ID NO. 4.

In one embodiment of the application, the sgRNA expression cassette and dCas9 expression vector carry a resistance gene thereon.

The application also provides a method for constructing the CRISPRi system, which comprises the following steps:

(1) Designing the sgRNA of a target gene, connecting an upstream homology arm of the 16s rRNA of the vibrio parahaemolyticus at the upstream of the sgRNA, and connecting a downstream homology arm of the 16s rRNA of the vibrio parahaemolyticus at the downstream of the sgRNA to construct an sgRNA expression cassette;

(2) Constructing the sgRNA expression cassette in the step (1) to a pDS132 plasmid to obtain a plasmid pSg-EN carrying the sgRNA expression cassette;

(3) Transforming the recombinant plasmid pSg-EN in (2) into an escherichia coli competent cell to obtain escherichia coli/pSg 132-EN;

(4) Constructing an origin of replication element to plasmid pBAD33 to obtain recombinant plasmid pBAD33T;

(5) The dCS 9 gene was ligated to the recombinant plasmid pBAD33T of (4) to give a dCS 9 expression vector pBAD33T-d9.

In one embodiment of the present application, in step (1), the nucleotide sequence of the upstream homology arm of the 16s rRNA of Vibrio parahaemolyticus is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream homology arm of the 16s rRNA of Vibrio parahaemolyticus is shown as SEQ ID NO. 2.

In one embodiment of the present application, in step (5), the nucleotide sequence of the dCas9 gene is shown in SEQ ID No. 3.

In one embodiment of the present application, in step (4), the origin of replication original comprises oriT.

In one embodiment of the application, in steps (3) and (6), the E.coli includes, but is not limited to, S17 (λpir) or CC118 (λpir).

The application also provides an application of the CRISPRi system in interfering with target genes in vibrio parahaemolyticus, which comprises the following specific steps:

(3) Transforming the plasmid pSg-EN in (2) into an E.coli competent cell to obtain E.coli/pSg 132-EN;

(5) Ligating the dCas9 gene to the recombinant plasmid pBAD33T of (4) to obtain a dCas9 expression vector pBAD33T-d9;

(6) Transforming the dCAS9 expression vector pBAD33T-d9 in the step (5) into competent cells of the escherichia coli to obtain escherichia coli/pBAD 33T-d9;

(7) E.coli/pSg-EN in (3) is joined with Vibrio parahaemolyticus, and coated on a culture medium containing polymyxin B and gentamicin to obtain Vibrio parahaemolyticus ATCC33846: pSg132-EN;

(8) Coupling the escherichia coli/pBAD 33T-d9 of (6) with the vibrio parahaemolyticus ATCC 33846::: pSg-EN of (7), and coating the coupled product on a medium containing gentamicin and chloramphenicol to obtain vibrio parahaemolyticus ATCC 33846:::: pSg-en+pbad 33T-d9;

(9) The Vibrio parahaemolyticus ATCC33846: pSg132-EN+pBAD33T-d9 of (8) is inoculated into a culture medium containing arabinose for culture, and the Vibrio parahaemolyticus with reduced target gene expression level is obtained.

In one embodiment of the present application, in steps (3) and (6), the E.coli includes, but is not limited to, S17 (λpir), CC118 (λpir).

In one embodiment of the present application, in the step (7), the medium containing polymyxin B and gentamicin is LB medium, and the amounts of polymyxin B and gentamicin added are 3 to 6mg/L and 8 to 12mg/L, respectively.

In one embodiment of the present application, in the step (8), the medium containing gentamicin and chloramphenicol is LB medium, and the addition amounts of gentamicin and chloramphenicol are 8-12 mg/L and 28-32 mg/L, respectively.

In one embodiment of the present application, in the step (7), the ligating is to culture E.coli/pSg-EN and Vibrio parahaemolyticus to OD, respectively ₆₀₀ About 0.8 to 1.2, the same volume of bacterial liquid is taken and washed for 2 to 3 times by a culture medium, and the bacterial liquid is co-suspended by the culture medium and uniformly mixed, then is planted on a flat plate and is cultivated overnight.

In one embodiment of the present application, in step (8), the ligating is culturing E.coli/pBAD 33T-d9 and Vibrio parahaemolyticus ATCC33846: pSg132-EN to OD, respectively ₆₀₀ About 0.8 to 1.2, the same volume of bacterial liquid is taken and washed for 2 to 3 times by a culture medium, and the bacterial liquid is co-suspended by the culture medium and uniformly mixed, then is planted on a flat plate and is cultivated overnight.

In one embodiment of the present application, in the step (9), the medium containing arabinose is an LB medium, and the amount of arabinose added is 20 to 40mmol/L.

In one embodiment of the application, the vibrio parahaemolyticus includes, but is not limited to, ATCC33846.

Drawings

Fig. 1: agarose gel electrophoresis verification of pSg-EN. Verification is carried out by using a primer sgRNA-test +/-and the correct fragment length is 782bp;

fig. 2: agarose gel electrophoresis verification of pBAD33T-d9. Verification was performed using primer d9-test +/-4537 bp of correct fragment length;

fig. 3: growth of wild type ATCC33846, ΔrpoE, ATCC33846+pBAD33T-d9, ATCC33846: pSg132-EN+pBAD33T-d9 on LB plates with or without arabinose;

fig. 4: the transcript levels of rpoE, rpoN in ATCC33846: pSg132-EN+pBAD33T-d9 were measured using RT-qPCR with or without arabinose. The control bacteria were ATCC33846+pBAD33T-d9 with or without arabinose. * Represents a P value <0.05;

Detailed Description

The application is further illustrated in the following drawings and specific examples, which are not intended to limit the application in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present application are those conventional in the art.

Unless otherwise indicated, the reagents and materials used in the following examples are commercially available or may be prepared by known methods.

The following examples relate to the following media:

ddH was used for the medium ₂ And (3) preparing O, and sterilizing for 15-20 min at 121 ℃ after the preparation is finished.

LB liquid medium (g/L): yeast powder 5, peptone 10, nacl10.

LB plate (g/L): yeast powder 5.0, tryptone 10.0, nacl10.0 and agar powder 15.

TABLE 1 primer sequences according to the following examples

TABLE 2 strains and plasmids involved in the examples below

EXAMPLE 1 construction of pSg132 plasmid

Vibrio parahaemolyticus itself has a homologous recombination system. With this, the pSg132 plasmid containing the homology arm was integrated into the genome after entry into the cell. The targeting sequence of sgRNA was designed by the website CHOPCHOP (uib.no) and was compared using the genome of Vibrio natriurus since there was no Vibrio parahaemolyticus genome. The rpoE (NCBI accession number: VP_RS 12550) and rpoN (NCBI accession number: VP_RS 13100) of the present application are exemplified as target genes, but the scope of the present application is not limited thereto.

(1) Fragments of genes upstream and downstream of 16s rRNA were amplified from the Vibrio parahaemolyticus genome using 16s rRNA primers 16s-U +/-and 16s-D +/-. The PCR product was recovered using a PCR product recovery kit.

(2) Gentamicin resistance gene Gm was amplified from the pWJW101 plasmid using primers Gm+ and Gm-.

(3) The sgRNA-rpoE, sgRNA-rpoN gene fragments were obtained by template-free overlap PCR using the pair of sgRNA primers CZ-sgRNA-rpoE+ and CZ-sgRNA-Gm-, CZ-sgRNA-rpoN+ and CZ-sgRNA-Gm-, and PCR product recovery was performed.

(4) When constructing a plasmid containing double sgrnas, overlapping PCR was performed on the 16srRNA upstream fragment in step (1) and the sgRNA-rpoE gene fragment in step (3) using primers 16s-u+ and LX-sgRNA-rpoN-, and PCR product recovery was performed to obtain 16sU-sgRNArpoE-rpoN.

(5) Overlapping PCR was performed on the 16srRNA downstream fragment obtained in step (1) and the Gm gene fragment in step (2) using primers Gm+ and 16s-D-, and PCR product recovery was performed.

(6) Overlapping PCR of the sgRNA-rpoN in step (3) with the 16sU-sgRNArpoE-rpoN in step (4) and the product of step (5) using primers 16s-U+, 16s-D to obtain the complete rpoE, rpoN double sgRNA-containing gene fragment, and recovering the PCR product.

(7) The pDS132 plasmid is subjected to enzyme digestion by using a fast-cutting enzyme SmaI, recovered by an RCP product recovery kit, subjected to one-step cloning with the product obtained in the step (6) to obtain a pSg-EN plasmid containing rpoE and rpoN double sgRNA, and subjected to transformation into escherichia coli CC118 (lambda pir), and the colony PCR verification result is shown in figure 1. The correctly sequenced plasmid was electrotransferred to E.coli S17 (λpir) and 800. Mu.L of the bacterial liquid was added to a glycerol tube and kept at-70 ℃. The nucleotide sequence of the pSg-EN plasmid is shown in SEQ ID NO. 5.

EXAMPLE 2 construction of pBAD33T-d9 plasmid containing dCAS9 Gene

(1) The oriT gene fragment was amplified from template pDS132 using primers 33-oriT+, 33-oriT-. pBAD33 was linearized using the fast cutting enzyme BstZ 17I. The oriT gene fragment was cloned in one step with linearized pBAD33 and transformed into E.coli CC118 (λpir). The plasmid with correct sequencing was designated pBAD33T, extracted and stored at-20 ℃.

(2) PCR amplification was performed using the template pCas plasmid with the dCAS9 point mutation primer pair dCAS9-1+/dCAS9-1+ -dCAS 9-2+/dCAS9-2+ -dCAS 9-3+/dCAS9-3+ -in Table 1 to obtain three gene fragments. These three genes were subjected to overlap PCR using primers 33-dCAS9-1+, dCAS9-3 to obtain dCAS9 gene fragments.

(3) The pBAD33T plasmid in step (1) was linearized using the fast cutting enzymes SmaI and BamHI, and after recovery of the PCR product, the plasmid was cloned in one step with the dCS 9 gene fragment obtained in step (2) and transformed into E.coli CC118 (λpir), and the colony PCR verification results are shown in FIG. 2. The correctly sequenced plasmid was electrotransferred to E.coli S17 (λpir) and 800. Mu.L of the bacterial liquid was added to a glycerol tube and kept at-70 ℃. The nucleotide sequence of the pBAD33T-d9 plasmid is shown as SEQ ID NO. 6.

EXAMPLE 3 construction of Gene-inhibiting Strain

(1) The final E.coli S17 (λpir) obtained in example 1 containing the pSg-EN plasmid was subjected to conjugation transduction with Vibrio parahaemolyticus ATCC33846: respectively culturing to OD ₆₀₀ About 1.0, 1mL of each bacterial liquid was washed twice with LB liquid medium, and 100. Mu.L of LB liquid medium was used to co-suspend and mix E.coli S17 (λpir) containing pSg-EN plasmid with Vibrio parahaemolyticus ATCC33846, and then inoculated on LB plates, followed by culturing overnight. The lawn was suspended in 1mLLB by scraping with a cotton swab and spread on LB plates containing 5mg/L polymyxin B and 10mg/L gentamicin. The colony PCR was used to verify whether the plasmid was successfully introduced into Vibrio parahaemolyticus. This step will give ATCC33846: pSg132-EN.

(2) The strain ATCC33846 in step (1): pSg-EN was subjected to conjugation transduction with E.coli S17 (λpir) containing the pBAD33T-d9 plasmid obtained in example 2, and the resultant was plated on a dual-resistance LB plate of 10mg/L gentamicin and 30mg/L chloramphenicol, and the colonies were maintained at-70℃after verification of correctness. This step will give ATCC33846: pSg132-EN+pBAD33T-d9.

Example 4 detection of phenotype of Gene-inhibited Strain and RT-qPCR

(1) The strain ATCC33846 obtained in example 3, pSg-EN+pBAD 33T-d9 was inoculated into LB liquid medium and cultured overnight at 37℃and 5. Mu.L of the bacterial liquid was inoculated onto LB plates and LB plates containing 30mmol/L arabinose, respectively. After overnight incubation at 37℃the growth of the lawn was observed.

(2) After inoculating pSg-EN+pBAD 33T-d9 of the strain ATCC33846 obtained in example 3 in LB liquid medium for overnight culture at 37 ℃, the bacterial liquid is transferred to a new LB medium to adjust the OD ₆₀₀ 0.02, cultured to OD ₆₀₀ To about 0.5. 1mL of the bacterial liquid is taken and added with 30mmol/L arabinose with the final concentration for treatment for 1h. The supernatant was centrifuged to extract RNA. RNA extraction, reverse transcription, RT-qPCR procedures were obtained from literature (Tan X, qiao J, zhou Q, et al identification of a phosphoethanolamine transferase for lipid A modification in Vibrio parahaemolyticus [ J ]]Food Control,2021, 125:108033.). Three sets of biological replicates. And (3) data processing: fold change = 2 (- ΔΔct); the error value is the standard deviation.

(3) As can be seen from FIGS. 3 and 4, after the expression of dCAS9 induced by arabinose, ATCC33846: pSg-ES+pBAD 33T-d9 had poor growth of Vibrio parahaemolyticus, probably due to toxicity of dCAS9, vibrio parahaemolyticus ΔrpoE after the rpoE gene is knocked out had poor growth compared with the wild type, and Vibrio parahaemolyticus introduced with two plasmids to inhibit rpoE and rpoN had poor growth on an arabinose plate compared with that of Vibrio parahaemolyticus introduced with pBAD33T-d9 only, which indicates that pSg132-EN normally transcribes sgRNA to inhibit the gene. Subsequent verification of the transcript level (RT-qPCR) was carried out, based on ATCC33846+pBAD33T-d9, ATCC33846: pSg132-EN+pBAD33T-d9 showed a significant decrease in the transcript level of rpoE, which was 1.75-fold down-regulated and 8.17-fold down-regulated compared to the wild type, after induction with arabinose. The results prove that the system has the function of interfering gene transcription in vibrio parahaemolyticus.

While the application has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the application as defined in the appended claims.

SEQUENCE LISTING

<110> university of Jiangnan

<120> method for constructing double plasmids of Vibrio parahaemolyticus CRISPRi

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<160> 4

<170> PatentIn version 3.3

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caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt 3720

cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag 3780

cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt 3840

attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa 3900

ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct 3960

cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa 4020

gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt 4080

gatttgagtc agctaggagg tgactga 4107

<210> 4

<211> 915

<212> DNA

<213> artificial sequence

<400> 4

gttgtcggtg tcgtgatgca cggcactgac gcgctgatgc tcggcgaagc caagcccagc 60

gcagatgcgg tcctcaatcg cgcgcaacgt ctccgcgtcg ggcttctctc ccgcgcggaa 120

gctaaccagc aggtgatagg tcttgtcggc ctcggaacgg gtgttgccgt gctgggtcgc 180

catcacctcg gccatgacag cgggcagggt gtttgcctcg cagttcgtga cgcgcacgtg 240

acccaggcgc tcggtcttgc cttgctcgtc ggtgatgtac ttcaccagct ccgcgaagtc 300

gctcttcttg atggagcgca tggggacgtg cttggcaatc acgcgcaccc cccggccgtt 360

ttagcggcta aaaaagtcat ggctctgccc tcgggcggac cacgcccatc atgaccttgc 420

caagctcgtc ctgcttctct tcgatcttcg ccagcagggc gaggatcgtg gcatcaccga 480

accgcgccgt gcgcgggtcg tcggtgagcc agagtttcag caggccgccc aggcggccca 540

ggtcgccatt gatgcgggcc agctcgcgga cgtgctcata gtccacgacg cccgtgattt 600

tgtagccctg gccgacggcc agcaggtagg ccgacaggct catgccggcc gccgccgcct 660

tttcctcaat cgctcttcgt tcgtctggaa ggcagtacac cttgataggt gggctgccct 720

tcctggttgg cttggtttca tcagccatcc gcttgccctc atctgttacg ccggcggtag 780

ccggccagcc tcgcagagca ggattcccgt tgagcaccgc caggtgcgaa taagggacag 840

tgaagaagga acacccgctc gcgggtgggc ctacttcacc tatcctgccc ggctgacgcc 900

gttggataca ccaag 915

Claims

1. A CRISPRi system, comprising a plasmid carrying an sgRNA expression cassette and a dCas9 expression vector; the sgRNA expression cassette sequentially comprises a vibrio parahaemolyticus 16s rRNA upstream homology arm, sgRNA and a vibrio parahaemolyticus 16s rRNA downstream homology arm; the dCas9 expression vector comprisesdCas9Genes and origin of replication elements;

the nucleotide sequence of the upstream homology arm of the vibrio parahaemolyticus 16s rRNA is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream homology arm of the vibrio parahaemolyticus 16s rRNA is shown as SEQ ID NO. 2.

2. The CRISPRi system of claim 1, wherein the plasmid carrying the sgRNA expression cassette is pDS 132; the dCAS9 expression vector takes pBAD33 as a vector.

3. The CRISPRi system of claim 1, wherein thedCas9The nucleotide sequence of the gene is shown as SEQ ID NO. 3.

4. Use of the CRISPRi system according to claim 1 for interfering with a target gene in vibrio parahaemolyticus, characterized in that the specific steps of the use are as follows:

(5) Will bedCas9Gene ligation to the recombinant plasmid pBAD33T of (4) to obtain dCS 9 expression vector pBAD33T-d9;

(9) Inoculating pSg132-EN+pBAD33T-d9 of Vibrio parahaemolyticus ATCC33846 in (8) to a culture medium containing arabinose for culture to obtain Vibrio parahaemolyticus with reduced target gene expression level; the nucleotide sequence of the upstream homology arm of the vibrio parahaemolyticus 16s rRNA is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream homology arm of the vibrio parahaemolyticus 16s rRNA is shown as SEQ ID NO. 2.

5. The use according to claim 4, wherein in step (4) and step (5), thedCas9The nucleotide sequence of the gene is shown as SEQ ID NO. 3.

6. The use according to claim 4, wherein in steps (3) and (6), the E.coli comprises but is not limited to S17 @λpir) Or CC 118%λpir）。

7. The use according to claim 4, wherein in step (9), the amount of arabinose added is 20 to 40mmol/L.