CN117025651B

CN117025651B - Laccase gene knockout method in Erwinia

Info

Publication number: CN117025651B
Application number: CN202311287810.5A
Authority: CN
Inventors: 卫亚红; 黄丽丽; 谭涛; 赵姝婷; 邓东涛; 邓磊; 田乾易; 冯洁
Original assignee: Shenzhen Research Institute Of Northwest University Of Agriculture And Forestry Science And Technology
Current assignee: Shenzhen Research Institute Of Northwest University Of Agriculture And Forestry Science And Technology
Priority date: 2023-10-08
Filing date: 2023-10-08
Publication date: 2023-12-19
Anticipated expiration: 2043-10-08
Also published as: CN117025651A

Abstract

The invention belongs to the technical field of genetic engineering, and relates to a laccase gene knockout method in Erwinia. The invention constructs a CRISPR-Cas9 gene editing system consisting of a plasmid pCas, sgRNA with a target gene and a recombinant plasmid pTarget with homology arms at the upstream and downstream of a fragment to be knocked out, and provides a method for constructing a gene knockout mutant strain by using the gene knockout system under the induction of IPTG, so as to directionally knock out laccase ELAC_205 genes of QL-Z3 strains. The gene knockout method mediated by the CRISPR-Cas9 gene editing system provided by the invention has the knockout rate reaching 82.22%, which is far higher than that of the traditional homologous recombination method.

Description

Laccase gene knockout method in Erwinia

Technical Field

The invention belongs to the technical field of genetic engineering, and relates to a laccase gene knockout method in Erwinia.

Background

CRISPR-Cas systems are adaptive immune systems obtained by long-term evolution of bacteria and archaea, and some bacteria store a small piece of DNA of a viral gene into their own DNA sequence called CRISPR after they have been invaded by a virus, if the bacteria do not die. If subsequently invaded again by this virus, the bacteria are able to recognize it on the basis of the spacer sequences stored in the CRISPR array, after which transcription of the relevant gene is initiated to cleave the foreign DNA. The CRISPR-Cas system has a plurality of kinds, and the CRISPR-Cas9 (CRISPR-Cas ii) technology is one of the most widely used at present, and is developed from a bacterial CRISPR-Cas system, and is mainly used for targeted knockout or knock-in of almost any gene, and the system mainly adopts Cas9 nuclease, and uses sgrnas directly in eukaryotes and prokaryotes to guide a gene editing system.

When CRISPR-Cas cleaves double-stranded DNA, the gRNA forms a gRNA-DNA hybrid double-strand with a perfectly complementary target DNA single-strand. One important limitation of using CRISPR-Cas RNA-guided nucleases: excess DNA mutations may be generated at sites other than the intended target. Subsequent studies directly demonstrate that CRISPR/Cas9 has severe off-targeting, i.e., the technology can undergo non-specific cleavage, causing mutations in non-targeted sites of the genome, which can lead to uncertainty in the results of the study and a substantial increase in the study effort, which severely limits the application of Cas 9.

Because of the limited base of the sgRNA pairing-binding region, there may be several DNA fragments in the genome that can pair, but if the location of the sgRNA is designed too long, a portion will be cut off and will not function. And in case of local unpaired, the localization region of the sgRNA may also bind to similar DNA sequences, resulting in a wrong gene for cleavage. The sgRNA (crRNA) has a seed sequence near PAM, which can lead to a significant drop in target cleavage efficiency or even an absence of any mismatch in the seed sequence. The more bulky the genome the more dangerous, the consequences of off-target effects may be false phenotypes, and more troublesome may be false phenotypes-deletion of the wrong gene, but the same phenotype as the deletion of the gene of interest. Therefore, how to design appropriate sgrnas and upper and lower homology arm primers for specific genomes and target genes is a technical point for achieving gene knockout.

Disclosure of Invention

At present, a method for directionally knocking out genes by using a CRISPR-Cas9 gene editing system exists, but proper sgRNA and upper and lower homology arm primers are designed aiming at different genomes and target genes, so that higher knocking-out rate is ensured, and the method is still very difficult.

To solve the above problems, the present invention provides a method for knocking out laccase genes in erwinia to meet the need in the art. The laccase gene knockout method in the Erwinia provided by the invention has the laccase gene knockout rate of 82.22% on the Erwinia.

In one aspect, the invention relates to a method for erwinia gene knockout comprising: knocking out an upstream homology arm of the ELAC_205 gene, knocking out a downstream homology arm of the ELAC_205 gene and sgRNA;

the primers for knocking out the upstream homology arm of the ELAC_205 gene comprise: the nucleotide sequence is shown in SEQ ID NO:1 and the nucleotide sequence of the F1 end primer of the upstream homology arm is shown as SEQ ID NO:2, an upstream homology arm R1 end primer;

the primers for knocking out the downstream homology arm of the ELAC_205 gene comprise: the nucleotide sequence is shown in SEQ ID NO:3, and the F2 end primer and the nucleotide sequence of the downstream homology arm are shown as SEQ ID NO:4, a downstream homology arm R2 end primer;

the nucleotide sequence of the sgRNA is shown as SEQ ID NO:5 is shown in the figure;

erwinia speciesErwiniaDirected knockout of the paintase ELAC-205 gene in QL-Z3.

Further, in the Erwinia gene knockout method provided by the invention, plasmid pCas and plasmid pTargetF are adopted in ErwiniaErwiniaDirected knockout of the paintase ELAC-205 gene in QL-Z3.

Further, in the Erwinia gene knockout method provided by the invention, the plasmid pCas is electrochemically transformed into ErwiniaErwiniaIn QL-Z3;

transformation of recombinant plasmid pTargetF into pCas-containing Erwinia with the insertion of primer for knocking out upstream homology arm of ELAC_205 gene, primer for knocking out downstream homology arm of ELAC_205 gene and sgRNAErwiniaIn sp.QL-Z3, gene knockout is completed to obtain Erwinia-pCas-pTargetF strain with double plasmids introduced;

the Erwinia-pCas-pTargetF strain was subjected to the elimination of the plasmid pTargetF in sequenceRemoval and plasmid pCas elimination to yield directed knockdown of ErwiniaErwiniastrain of laccase elac_205 gene in ql-Z3.

Further, in the erwinia gene knockout method provided by the invention, the electrochemical conversion of the plasmid pCas comprises: preparation of ErwiniaErwiniaAdding 3-5 mu L of pCas plasmid into competent cells of sp.QL-Z3, standing on ice for 20min, carrying out electric excitation at 1.5 KV, carrying out ice bath for 3min, adding 700 mu L of LB liquid culture medium, and culturing at 30 ℃ for 1-2 h at 180 rpm.

Further, in the Erwinia gene knockout method provided by the invention, the elimination of the plasmid pTargetF comprises: the erwinia-pCas-pTargetF strain into which the double plasmid was introduced was cultured in a Km-resistant LB solid medium to a cloud form, and then IPTG was added to the culture at a final concentration of 0.5 mmol/L.

Further, in the erwinia gene knockout method provided by the invention, the elimination of the pCas plasmid comprises: culturing the strain with pTargetF plasmid removed in LB liquid medium containing Amp resistance, shaking at 30deg.C at 180rpm to OD ₆₀₀ The value was 0.5, which was then transferred to a 180rpm shaker at 37℃and incubated to OD ₆₀₀ The value is 0.8-1.2.

Further, in the erwinia gene knockout method provided by the invention, the LB liquid medium comprises: typtone 10g/L, naCl g/L and yeast powder 5g/L.

In the Erwinia gene knockout method provided by the invention, the LB solid medium is Typtone 10g/L, naCl g/L, yeast powder 5g/L and agar powder 18g/L.

In another aspect, the present invention relates to an erwinia CRISPR-Cas9 gene editing system for directional knockout of erwiniaErwiniaThe laccase ELAC_205 gene of sp.QL-Z3,

it comprises the following steps: a primer for knocking out an upstream homology arm of the ELAC_205 gene, a primer for knocking out a downstream homology arm of the ELAC_205 gene, and sgRNA;

the nucleotide sequence of the sgRNA is shown as SEQ ID NO: shown at 5.

On the other hand, the invention relates to the Erwinia gene knockout method or the Erwinia CRISPR-Cas9 gene editing system in ErwiniaErwiniaUse of laccase elac_205 gene knockout in ql-Z3.

Through the technical scheme and the combination of the embodiments, the technical scheme provided by the invention has at least the following beneficial effects or advantages:

in CRISPR-Cas system design, the off-target effect is extremely easily caused by the influence of the target gene and its localization in the genome. Off-target occurs because the gRNA recognizes the wrong base sequence and targets other homologous regions. The Erwinia CRISPR-Cas9 gene editing system mediated by the CRISPR-Cas9 gene editing system provided by the invention has the advantages that the knockout rate reaches 82.22% by designing proper sgRNA and upper and lower homology arm primers, which is far higher than that of the traditional homologous recombination method.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the map of plasmid pCas.

FIG. 2 is a map of plasmid pTargetF.

FIG. 3 shows QL-Z3-pCAS colony PCR. M is 5000bp Marker; lanes 1-4 are the QL-Z3 strain transformed with pCas.

FIG. 4 is an overlapping extension PCR electrophoresis assay. M is 5000bp Marker; lanes 1-3 verify the results for overlap extension PCR.

FIG. 5 shows colony PCR of pTargetF plasmid with upstream and downstream homology arms. M is 5000bp Marker; lanes 1-5 are the results of upstream and downstream homology arm ligation PCR validation.

FIG. 6 is a seamless clonal colony PCR. M is 5000bp Marker; lanes 1-24 are seamless clone validation results.

FIGS. 7-9 are all PCR of colonies of the gene knockout strain. M is 2000bp Marker; lanes 1-24 in FIG. 7, lanes 1-10 in FIG. 8, and lanes 1-11 in FIG. 9 are all results of PCR parallel experiments of colonies of the gene knockout strain.

FIG. 10 shows colony PCR of gene knockout strain. M is 2000bp Marker; lane 1 is QL-Z3-pCas-pTargetF; lane 2 is wild type strain QL-Z3; lane 3 is mutant QL-Z3.

FIG. 11 is a plasmid pTargetF elimination verification. M is 2000bp Marker; lane 1 is a negative control, a pTargetF plasmid unabated strain; lanes 2-10 are the QL-Z3-pCas-pTargetF strain.

FIG. 12 is a plasmid pCas elimination verification. M is 5000bp Marker; lanes 1-4 are negative controls, QL-Z3-pCas; lanes 5-9 are QL-Z3-pCas verified for plasmid elimination.

FIG. 13 is a diagram of homologous recombination knockout validation. M is 5000bp Marker; lanes 1-16 demonstrate the results of homologous recombination gene knockout.

FIG. 14 is a diagram of homologous recombination knockout validation. M is 5000bp Marker; lanes 1-16 demonstrate the results of homologous recombination gene knockout.

FIG. 15 is a diagram of homologous recombination knockout validation. M is 5000bp Marker; lanes 1-20 verify the results of homologous recombination gene knockout.

Detailed Description

The following describes the embodiments of the present invention with reference to examples, but the present invention is not limited to the examples.

The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products available on the market without the manufacturer's attention.

Example 1

The present example provides ErwiniaErwiniaTest of directed knockdown of the paintase ELAC-205 gene in QL-Z3.

The strain used in the experiment is ErwiniaErwiniasp.QL-Z3 (GenBank: MH 828331.1), E.coli used for the testE.coliDH 5. Alpha. Is a laboratory purchased competent cell.

Plasmids used for gene knockout were pCas and pTargetF, which were stored before the laboratory. Table 1 shows the elements contained in plasmid pCas, and Table 2 shows the elements contained in plasmid pTargetF.

TABLE 1 plasmid pCas contains elements

TABLE 2 elements contained in plasmid pTargetF

Erwinia QL-Z3 was cultured in LB solid and liquid media at 30℃and E.coli DH 5. Alpha. Was cultured in liquid and LB solid media at 37 ℃. The media used are specifically shown in tables 3 and 4.

Table 3 LB solid Medium

The pH was adjusted to 7.0 and sterilized at 121℃for 30 min.

Table 4 LB liquid Medium

The pH was adjusted to 7.0 and sterilized at 121℃for 30 min.

And (3) glycerol preservation: adding 2% bacterial liquid into LB liquid culture medium, shake culturing for a period of time to OD ₆₀₀ The value is 0.8-1.2, and then 500 mu L of bacterial liquid and 500 mu L of 50% glycerol is added into a centrifuge tube and placed in a refrigerator at-80 ℃ for preservation.

(1) Competent cell preparation

Preparation of heat shock transformed competent cells:

inoculating the bacterial liquid into blank liquid LB culture medium, shake culturing at 180rpm at proper temperature until logarithmic phase (OD ₆₀₀ =0.4), 1.5mL of bacterial liquid is sucked, the bacterial liquid is placed in a centrifuge for centrifugation at 4000g for 3min at 4 ℃, and 1mL of precooled 0.1M CaCl is added after the supernatant is discarded ₂ Lightly blowing and suspending the thalli. Centrifuge at 4000g for 3min at 4 ℃. Placing the centrifuged bacterial liquid in an ultra-clean bench, removing supernatant, adding 100 μL of 0.1M CaCl ₂ The cells were gently suspended. The prepared competence should be stored at 4 ℃.

Electrochemical conversion competent cell preparation:

inoculating the bacterial liquid into blank liquid LB culture medium, shake culturing at 180rpm at proper temperature until logarithmic phase (OD ₆₀₀ =0.4), ice-bath for 10min, then 1.5mL of bacterial liquid was centrifuged (4500 rpm,3 min) in a centrifuge at 4 ℃, and the supernatant was discarded. Sterile ddH ₂ The cells were resuspended in O1 mL, centrifuged at 4℃and the supernatant discarded, and the procedure was repeated. The cells were then resuspended in 1mL of 10% glycerol, centrifuged (4500 rpm,3 min) at 4℃and the supernatant discarded and the procedure repeated. Finally, the cells were resuspended in 100. Mu.L of 10% glycerol and the prepared competence was stored at 4 ℃.

(2) Construction of plasmids

The pCas plasmid of the laboratory already includes all the elements required for gene knockout. The map of plasmid pCas is shown in fig. 1.

The laboratory plasmid pTargetF requires an insertion element to be able to function in gene knockout. FIG. 2 is a map of plasmid pTargetF.

Designing primers of upstream and downstream homology arms of a gene to be knocked out (upstream homology arm primers: F1/R1; downstream homology arm primers: F2/R2), wherein the primers are primers of overlap PCR, and adding an enzyme digestion site for connecting with a vector plasmid pTargetF and an overlap fragment for the overlap PCR at the 5' end of the primers F1/R2, wherein the overlap length is about 20bp. Amplifying the upstream and downstream homology arms, detecting the products by electrophoresis, cutting glue, recovering and purifying to obtain the upstream and downstream homology arms; overlapping PCR is carried out by taking the upstream and downstream homology arms as templates, so as to obtain a DNA sequence which is connected with the upstream and downstream homology arms and does not contain fragments to be knocked out, the overlapping PCR products are subjected to electrophoresis detection, then cut, recovered and purified, and then stored in a refrigerator at the temperature of minus 20 ℃, and the DNA purification and recovery kit of Tiangen reagent company is adopted for the recovery and purification of the gel. The PCR system and reaction conditions are shown in tables 5 and 6.

TABLE 5 PCR reaction System

TABLE 6 PCR reaction conditions

The plasmid pTargetF and the homology arm fragment of the target gene ELAC_205 were digested simultaneously, and water-bath was carried out overnight at 37 ℃. The cleavage system is shown in Table 7.

TABLE 7 enzyme digestion system

The digested product was purified and recovered (using the Tiangen kit), and the digested plasmid vector and digested homology arm were ligated by T4 DNA ligase at 22℃for 1h in a PCR apparatus. The T4 DNA ligase system is shown in Table 8.

Table 8 connection reaction System

Plasmid pTargetF was transformed into E.coli DH 5. Alpha. By adding pTargetF to competent cells, incubating on ice for 30min under the number of flicks, and rapidly placing on ice for 5min after heat shock 90s in a water bath at 42 ℃. 500. Mu.L of LB liquid medium was added thereto, and the mixture was shake-cultured at 37℃and 180rpm for 45 minutes. 4000 The cells were collected by centrifugation at rpm for 3min, and 20. Mu.L of the bacterial liquid was uniformly spread on a chloramphenicol-containing plate. Single colonies were picked for colony PCR, and whether the plasmid was successfully transferred into E.coli was identified, and the PCR system was as shown in Table 9.

TABLE 9 colony PCR reaction System

Inoculating positive bacteria into LB liquid medium containing chloramphenicol resistance, shake culturing for a period of time to OD ₆₀₀ The value is 0.8, and the strain is preserved. pTargetF plasmid extraction was performed using OMEGA plasmid extraction kit, and the extracted plasmid was stored in a-20deg.C refrigerator.

The plasmid pTargetF with the homology arms attached thereto was then singly digested and the digestion system was as shown in Table 10.

Table 10 enzyme digestion System

The cleavage product was treated with alkaline phosphatase after single cleavage, and the alkaline phosphatase system is shown in Table 11.

Table 11 enzyme digestion System

The reaction was stopped by incubation at 37℃for 30min followed by heat inactivation at 80℃for 2 min. Purifying and recovering the enzyme-digested product by using a root kit, measuring the concentration of DNA, and storing the DNA in a refrigerator at the temperature of-20 ℃.

Seamless cloning was performed and the system of seamless cloning is shown in Table 12. The insert was an sgRNA designed according to the gene to be knocked out, and was synthesized by primer company.

Table 12 seamless cloning reaction System

The reaction was carried out at 50℃for 20min. Immediately after the reaction, the reaction mixture was separatedThe tube was cooled on ice for 2min and allowed to transform. Then the ligation product of the seamless clone was transformed into competent E.coli DH 5. Alpha. By the same method as above. Then 500. Mu.L of LB liquid medium was added thereto, followed by shaking at 180rpm at 37℃for 45min.4000 The cells were collected by centrifugation at rpm for 3min, and 20. Mu.L of the bacterial liquid was uniformly spread on a chloramphenicol-containing plate. Single colonies were picked for colony PCR, and electrophoretically tested for successful ligation of sgRNA to plasmid pTargetF, followed by sequencing of the positive cloned gene. The positive clone with correct sequencing result is inoculated into LB liquid culture medium to be shake-cultured at 37 ℃ and 180rpm until OD ₆₀₀ 0.8, performing strain preservation, extracting plasmid pTargetF with OMEGA plasmid extraction kit, and storing the extracted plasmid in a refrigerator at-20deg.C. The pTargetF plasmid thus obtained was a plasmid which could be used for gene knockout.

(3) CRISPR-Cas9 gene knockout

Electrochemical conversion of pCas plasmid:

preparing competent cells of QL-Z3, adding 3 mu L pCas plasmid into the prepared competent cells of QL-Z3, and standing on ice for 20min. The electrotransport apparatus was electrically excited at 1.5. 1.5 KV, then ice-bathed for 3min, and then 700. Mu.L of LB liquid medium was added thereto for 1.5 hours at 30℃and 180 rpm. The cells were collected by centrifugation at 4000rpm for 3min, 20. Mu.L of the bacterial liquid was spread on LB solid medium containing Km resistance, and the culture was inverted at 30℃for 16h. After a single colony grows on the culture dish, picking a colony, and carrying out colony PCR by using pCas universal primers, wherein if the PCR result shows a band, the transformation is successful.

Transformation and gene knockout of recombinant plasmid pTargetF:

inoculating QL-Z3 containing pCas into LB liquid medium, adding Km resistance and 20% 10mmol/L arabinose to induce culture to OD ₆₀₀ After reaching 0.3, competent cells of QL-Z3 were prepared according to the method described above, 3. Mu.L of pTargetF plasmid with homologous arms on both sides of the gene to be knocked out of sgRNA was added to the prepared competent cells of QL-Z3, and the mixture was allowed to stand on ice for 20min. Respectively performing electrochemical conversion and heat shock conversion, wherein the electrochemical conversion is carried out according to the method, the heat shock conversion is carried out by carrying out heat shock for 90s in a water bath at 42 ℃, then carrying out ice bath for 3min, adding 700 mu L of LB liquid medium, and culturing for 1h at 30 ℃ and 180 rpm. Electrochemical methodBacteria obtained by transformation and heat shock transformation were centrifuged at 4000rpm for 3min to collect the cells, and then 20. Mu.L of the bacterial liquid was spread on LB solid medium containing Km resistance and Cm resistance, and cultured upside down at 30℃for 16 hours. After a single colony grows on the culture dish, picking a colony, and using primers of the ELAC_205 gene to be knocked out, primers of the upstream and downstream homology arms and a universal primer on the QL-Z3 chromosome to perform colony PCR, if the gene is knocked out successfully, a band (the length of the homology arm is 442 bp) appears at about 500bp, if no knocking out is successful, two bands appear, one is above the band of 1000bp (the length of the gene to be knocked out plus the homology arm is about 1200 bp), and the other is about 500 bp. The PCR system is shown in Table 8. Shaking the positive clone obtained by electrophoresis detection at 30 ℃ and 180rpm to OD ₆₀₀ 0.8 strain preservation was performed.

(4) CRISPR-Cas9 gene knockout

Elimination of plasmid pTargetF:

the double-plasmid-introduced QL-Z3-pCas-pTargetF strain is cultured in Km-resistant LB solid medium to a cloudy state, namely OD ₆₀₀ The value was about 0.4, and then the culture was carried out overnight with the addition of IPTG at a final concentration of 0.5mmol/L, and the plating was diluted (1 mmol/L IPTG, km resistance). Selecting single colony as colony PCR, using pTargetF universal primer as primer, eliminating pTargetF plasmid if no band appears after electrophoresis detection, and shake culturing positive clone at 30deg.C at 180rpm to OD ₆₀₀ Strain preservation was performed after 0.8.

Elimination of pCas plasmid:

culturing the strain with pTargetF plasmid removed in LB liquid medium containing Amp resistance, shaking at 30deg.C at 180rpm to OD ₆₀₀ The value is about 0.5, and then the mixture is transferred to a shaking table at 180rpm at 37 ℃ for culturing until the OD ₆₀₀ The value was 0.8. The cells were collected by centrifugation at 4000rpm for 3min, and the appropriate amount of the bacterial liquid was diluted and spread on LB solid medium containing Amp resistance, followed by culturing at 37 ℃. After single colony is grown on the plate, selecting single colony as colony PCR, using pCas universal primer as primer, if electrophoresis detection has no band, proving that said bacterium has eliminated pCas plasmid, and shake culturing said single colony at 30 deg.C and 180rpm to OD ₆₀₀ And (5) preserving the strain for later use after the strain is 0.8.

(5) Test results

Plasmid pCas was transformed electrochemically into QL-Z3, and was detected by electrophoresis after colony PCR using pCas universal primers, as shown in FIG. 3, lane 4 shows a band at 1500bp, indicating successful transformation of plasmid pCas.

By designing a primer of a specific overlap PCR, homologous arm sequences (without gene to be knocked out) at two ends of a target fragment connected with each other are amplified by PCR, the length of an upstream homologous arm and a downstream homologous arm is 442bp, the PCR products are detected by electrophoresis, the electrophoresis result is shown as figure 4, the lanes 1-3 show a band at 500bp, and the overlap extension PCR is successful.

And (3) transforming the well-connected plasmid into competent cell escherichia coli DH5 alpha, and picking single colony for colony PCR identification. As a result of the electrophoresis test, FIG. 5 shows that lanes 1-5 show bands at 500bp, indicating successful ligation of the upstream and downstream homology arms of the gene to be knocked out on the plasmid.

Single colonies were picked for colony PCR, and primers were specific for sgRNA. As shown in FIG. 6, the presence of a band at 500bp in lane 8 indicates successful ligation of sgRNA to plasmid pTargetF and successful seamless cloning.

And selecting a single colony as a colony for PCR to verify whether the gene is knocked out, wherein the primer is a primer of an upstream and downstream homology arm of the target gene, and the electrophoresis result is shown as a graph in fig. 7-9, and the band of 37 strains appears at a 500bp position, which indicates that the ELAC_205 gene is knocked out successfully.

As shown in FIG. 10, the selected strain was subjected to a second PCR test, and lanes 1, 2, and 3 were the electrophoresis results of the QL-Z3-pCas-pTargetF strain, the wild-type strain, and the positive knockout strain, respectively. Lanes 1 and 2 show banding and lane 3 shows that elac_205 has been knocked out.

The strain incubated with IPTG was diluted and plated, single colonies were picked for PCR verification using the pTargetF plasmid universal primers, and a set of negative controls (pTargetF plasmid was not eliminated by IPTG induction) were added. Electrophoresis confirmed that the strain of lane 8 did not appear as a band at 750bp, indicating successful pTargetF plasmid elimination, as shown in FIG. 11.

The strain induced at 37 ℃ is diluted and coated, and single colony is selected for colony PCR. As shown in FIG. 12, lanes 1-4 are negative control strains that have not been induced at 37℃and appear as bands at 1500bp, indicating that pCas has not been eliminated; lanes 5-9 are strains induced at 37℃and no band appears at 1500bp, indicating successful pCas plasmid elimination and successful construction of the ELAC_205 gene-deficient mutant.

The data of homologous recombination knockout comes from the data before the laboratory, as shown in fig. 13-15, the occurrence of a band at 1500bp indicates that the gene knockout was successful, the total 26 strains with the calculated homologous recombination success were knocked out at 50%, and the gene of elac_205 of 37 strains was knocked out according to CRISPR-Cas9 mediated gene knockout in the above, the knocked out rate was about 82.22%. The comparison of the two shows that the gene knockout mediated by the CRISPR-Cas9 system has higher knockout rate.

Example 2

This embodiment is different from embodiment 1 in that:

the LB solid medium was adjusted to pH 7.2.

The LB liquid medium was adjusted to pH 7.2.

And (3) glycerol preservation: adding 2% bacterial liquid into LB liquid culture medium, shake culturing for a period of time to OD ₆₀₀ The value was 1.2.

(2) In the construction step of the plasmid,

500. Mu.L of LB liquid medium was added thereto, and shaking culture was performed at 37℃and 180rpm for 60 minutes.

Inoculating positive bacteria into LB liquid medium containing chloramphenicol resistance, shake culturing for a period of time to OD ₆₀₀ The value is 1.2, and the strain is preserved.

In the seamless cloning step, 500. Mu.L of LB liquid medium was then added thereto, followed by shaking at 180rpm at 37℃for 60min.

In the seamless cloning step, positive clones with correct sequencing result are inoculated into LB liquid medium and shake-cultivated at 37 ℃ and 180rpm until OD ₆₀₀ 1.2.

(3) In the CRISPR-Cas9 gene knockout step,

the electrotransport apparatus was electrically excited at 1.5. 1.5 KV, then ice-bathed for 3min, and then 700. Mu.L of LB liquid medium was added thereto for 2.5 hours at 30℃and 180 rpm. The cells were collected by centrifugation at 4000rpm for 3min, 20. Mu.L of the bacterial liquid was spread on LB solid medium containing Km resistance, and the culture was inverted at 30℃for 36 hours.

Transformation and gene knockout of recombinant plasmid pTargetF:

inoculating QL-Z3 containing pCas into LB liquid medium, adding Km resistance and 20% 10mmol/L arabinose to induce culture to OD ₆₀₀ After reaching 0.45, competent cells of QL-Z3 were prepared according to the method described above, and 5. Mu.L of pTargetF plasmid with homologous arms on both sides of the gene to be knocked out of sgRNA was added to the prepared competent cells of QL-Z3, and allowed to stand on ice for 20min. Respectively performing electrochemical conversion and heat shock conversion, wherein the electrochemical conversion is carried out according to the method, the heat shock conversion is carried out by carrying out heat shock for 90s in a water bath at 42 ℃, then carrying out ice bath for 3min, adding 700 mu L of LB liquid medium, and culturing for 1h at 30 ℃ and 180 rpm. Bacteria obtained by electrochemical conversion and heat shock conversion were centrifuged at 4000rpm for 3min to collect the bacterial cells, and then 20. Mu.L of the bacterial liquid was spread on LB solid medium containing Km resistance and Cm resistance, and cultured upside down at 30℃for 36 hours. After a single colony grows on the culture dish, picking a colony, and using primers of the ELAC_205 gene to be knocked out, primers of the upstream and downstream homology arms and a universal primer on the QL-Z3 chromosome to perform colony PCR, if the gene is knocked out successfully, a band (the length of the homology arm is 442 bp) appears at about 500bp, if no knocking out is successful, two bands appear, one is above the band of 1000bp (the length of the gene to be knocked out plus the homology arm is about 1200 bp), and the other is about 500 bp. The PCR system is shown in Table 8. Shaking the positive clone obtained by electrophoresis detection at 30 ℃ and 180rpm to OD ₆₀₀ 1.2 and then performing strain preservation.

(4) In the CRISPR-Cas9 gene knockout step,

elimination of plasmid pTargetF:

the double-plasmid-introduced QL-Z3-pCas-pTargetF strain is cultured in Km-resistant LB solid medium to a cloudy state, namely OD ₆₀₀ The value was about 0.4, and then the culture was carried out overnight with the addition of IPTG at a final concentration of 0.5mmol/L, and the plating was diluted (1 mmol/L IPTG, km resistance). Selecting single colony as colony PCR, wherein the primer is pTargetF universal primer, and if no band appears after electrophoresis detection, provingThe pTargetF plasmid was eliminated, and positive clones were shake-cultured at 30℃and 180rpm to OD ₆₀₀ 1.2 and then carrying out strain preservation.

Elimination of pCas plasmid:

culturing the strain with pTargetF plasmid removed in LB liquid medium containing Amp resistance, shaking at 30deg.C at 180rpm to OD ₆₀₀ The value is about 0.5, and then the mixture is transferred to a shaking table at 180rpm at 37 ℃ for culturing until the OD ₆₀₀ Value 1.2. The cells were collected by centrifugation at 4000rpm for 3min, and the appropriate amount of the bacterial liquid was diluted and spread on LB solid medium containing Amp resistance, followed by culturing at 37 ℃. After single colony is grown on the plate, selecting single colony as colony PCR, using pCas universal primer as primer, if electrophoresis detection has no band, proving that said bacterium has eliminated pCas plasmid, and shake culturing said single colony at 30 deg.C and 180rpm to OD ₆₀₀ 1.2, and then preserving the strain for later use.

The present invention may be better implemented as described above, and the above examples are merely illustrative of preferred embodiments of the present invention and not intended to limit the scope of the present invention, and various changes and modifications made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the present invention without departing from the spirit of the design of the present invention.

Claims

1. A method for knocking out laccase genes in erwinia, comprising: knocking out an upstream homology arm of the ELAC_205 gene, knocking out a downstream homology arm of the ELAC_205 gene and sgRNA;

europeanism (R)Bacteria of the genus ShewanellaErwiniaThe directed knockout of the paintase ELAC_205 gene in QL-Z3;

plasmid pCas and plasmid pTargetF are used in ErwiniaErwiniaThe directed knockout of the paintase ELAC_205 gene in QL-Z3;

electrochemical transformation of the plasmid pCas into ErwiniaErwiniaIn QL-Z3;

the recombinant plasmid pTargetF into which the primer for knocking out the upstream homology arm of the ELAC_205 gene, the primer for knocking out the downstream homology arm of the ELAC_205 gene and the sgRNA were inserted was transformed into E.Erwiniens containing pCasErwiniaIn sp.QL-Z3, gene knockout is completed to obtain Erwinia-pCas-pTargetF strain with double plasmids introduced;

the Erwinia-pCas-pTargetF strain is subjected to elimination of plasmid pTargetF and plasmid pCas in sequence to obtain directional knockout ErwiniaErwiniastrain of laccase elac_205 gene in ql-Z3.

2. The method of claim 1, wherein the electrochemical conversion of plasmid pCas comprises: preparation of ErwiniaErwiniaAdding 3-5 mu L of pCas plasmid into competent cells of sp.QL-Z3, standing on ice for 20min, carrying out electric excitation at 1.5 KV, carrying out ice bath for 3min, adding 700 mu L of LB liquid culture medium, and culturing at 30 ℃ for 1-2 h at 180 rpm.

3. The method of claim 2, wherein the elimination of plasmid pTargetF comprises: the erwinia-pCas-pTargetF strain into which the double plasmid was introduced was cultured in a Km-resistant LB solid medium to a cloud form, and then IPTG was added to the culture at a final concentration of 0.5 mmol/L.

4. A method according to claim 3, wherein the LB solid medium is Typtone 10g/L, naCl g/L, yeast powder 5g/L, agar powder 18g/L.

5. The method of claim 2, wherein the elimination of pCas plasmid comprises: LB solution containing Amp resistance of the strain from which pTargetF plasmid was deletedCulturing in culture medium, shaking at 30deg.C and 180rpm to OD ₆₀₀ The value was 0.5, which was then transferred to a 180rpm shaker at 37℃and incubated to OD ₆₀₀ The value is 0.8-1.2.

6. The method of claim 5, wherein the LB liquid medium comprises: typtone 10g/L, naCl g/L and yeast powder 5g/L.

7. The Erwinia CRISPR-Cas9 gene editing primer composition is characterized by being used for directionally knocking out ErwiniaErwiniaThe laccase ELAC_205 gene of sp.QL-Z3,

comprising the following steps: a primer for knocking out an upstream homology arm of the ELAC_205 gene, a primer for knocking out a downstream homology arm of the ELAC_205 gene, and sgRNA;

the nucleotide sequence of the sgRNA is shown as SEQ ID NO: shown at 5.

8. The method of any one of claims 1 to 6, or the erwinia CRISPR-Cas9 gene editing primer composition of claim 7, in erwiniaErwiniaUse of laccase elac_205 gene knockout in ql-Z3.