Method for carrying out gene knockout and complementation on staphylococcus aureus srap gene

Abstract

The invention discloses a method for carrying out gene knockout and complementation on a staphylococcus aureus srap gene. The gene knockout method comprises the following steps: 1) extracting genome DNA of staphylococcus aureus; 2) designing primers; 3) amplifying upstream and downstream homologous arms of the srap gene and knocking out resistance gene segments; (4) carrying out enzyme digestion on an empty vector and constructing a recombinant plasmid; 5) transforming the recombinant plasmid; 6) screening positive transformants; 7) knocking out the inserted DNA fragment and amplifying; 8) electrotransformation of staphylococcus aureus; 9, positive transformants are screened, and the staphylococcus aureus srap gene knockout strain is obtained.The invention further provides a method for conducting knockout gene back-supplement on the staphylococcus aureus knockout strain. Knockout of the srap gene and back-supplement operation of the knockout strain can be successfully achieved, and research on the important staphylococcus aureus virulence factor srap can be helped.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and particularly relates to a method for gene knockout of Staphylococcus aureus and complementation of the knockout strain. Background Art

[0002] Staphylococcus aureus is an important human pathogen that can cause various severe infections, such as pneumonia, sepsis, endocarditis, etc. In recent years, the ST59 type of Staphylococcus aureus strain has gradually replaced ST239 as the most dominant ST type in China, and the srap gene is considered to be one of the main contributing factors for the prevalence of the ST59 type strain, but the current understanding is still limited. Summary of the Invention

[0003] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a method for gene knockout and complementation of the srap gene of Staphylococcus aureus.

[0004] The technical solution for achieving the purpose of the present invention is as follows: A method for gene knockout of Staphylococcus aureus srap, comprising the following steps: 1) Extract the genomic DNA of Staphylococcus aureus; 2) Primer design: Use the upstream and downstream homologous arms of the target gene srap to be knocked out as two fragments, and design primer pairs for amplifying the upstream homologous arm and the downstream homologous arm respectively: Srap-UP-F shown in SEQ ID NO.1 and Srap-UP-R shown in SEQ ID NO.2; Srap-DN-F shown in SEQ ID NO.3 and Srap-DN-R shown in SEQ ID NO.4; 3) Amplify the upstream and downstream homologous arms of the target gene and the knockout resistance gene fragment: Use the genomic DNA of Staphylococcus aureus containing the target gene as a template, and perform PCR amplification with the above-mentioned primer pairs respectively to amplify the upstream and downstream homologous arms and the knockout resistance gene fragment of the target gene; 4) Empty vector digestion and recombinant plasmid construction: Digest the empty vector with a restriction endonuclease to obtain a vector plasmid fragment, and treat the vector plasmid fragment with the upstream and downstream homologous arms of the target gene in a recombinase reaction system to construct a recombinant plasmid; 5) Transformation of the recombinant plasmid: 6) Screening of positive transformants: The recombinant plasmid after the treatment in step 5) was subjected to plate screening to obtain positive transformants; single colonies of positive transformants were selected, colony PCR was performed with the said primer pair, the PCR products were detected by agarose gel electrophoresis, and the monoclonal strains with the target bands were sequenced. Strains with correct sequences were selected to extract plasmids to obtain gene knockout recombinant plasmids; 7) Amplification of the knockout insertion DNA fragment: Using the gene knockout recombinant plasmid as a template, the knockout insertion DNA fragment was amplified with the said primer pair; 8) Electrotransformation of Staphylococcus aureus: The knockout insertion DNA fragment was inserted into the Staphylococcus aureus genome by electrotransformation to achieve the knockout of the target gene; 9) For the Staphylococcus aureus after the treatment in step 8), positive transformants with the knockout resistance gene correctly inserted into the target gene position of the Staphylococcus aureus genome were screened by colony PCR to obtain the Staphylococcus aureus srap gene knockout strain.

[0005] A method for constructing a complementation strain of MRSA ST59 △srap, and the method for obtaining the Staphylococcus aureus srap gene knockout strain is as described above; The construction of the complementation strain includes the following steps: 1) Using the genomic DNA of MRSA ST59 as a template, the full length of the srap gene plus its promoter region and the related binding sites of ribosomes were amplified; 2) Construct the complementation plasmid pLI50-srap, and at the same time, transfer the empty plasmid pLI50 as a negative control.

[0006] An application of the said Staphylococcus aureus srap gene knockout strain.

[0007] The beneficial effects of the present invention are as follows: The method for knocking out the srap gene provided by the present invention can specifically target the srap gene of Staphylococcus aureus to obtain strains with the srap gene cluster knocked out, thereby providing technical support for clarifying the specific function of the important virulence factor - srap of Staphylococcus aureus. Description of the Drawings

[0008] Figure 1 This is the vector plasmid pKOR1 used in the examples of the present invention.

[0009] Figure 2 This is the RT-qPCR verification result graph of the srap gene knockout in the examples of the present invention. Detailed Embodiments

[0010] The present invention will be further described below in conjunction with the drawings and examples.

[0011] 1. Extraction of genomic DNA from Staphylococcus aureus 1) Resuscitate the strain from the -80 °C refrigerator and activate it by streaking in four zones on a Columbia blood agar plate.

[0012] 2) Pick single colonies from the blood agar plate, suspend them separately in 4 mL of TSB for activation, and culture them overnight with shaking at 37 °C.

[0013] 3) Centrifuge at 12000 g for 2 min and discard the supernatant. Meanwhile, wash the bacterial cells with sterile deionized water and repeat 3 times to collect the bacterial cells.

[0014] 4) Resuspend the bacteria in 200 μL of sterile deionized water, then add 5 μL of lysostaphin (10 mg / mL), mix well, and incubate in a water bath at 37 °C for more than 2 h until the bacteria are lysed and become clear and transparent.

[0015] 5) Add 20 μL of proteinase K (20 mg / mL), mix well, and incubate in a water bath at 56 °C for 30 min.

[0016] 6) Add 200 μL of absolute ethanol and 200 μL of Buffer BD solution, and mix by shaking.

[0017] 7) Discard the waste liquid, put the adsorption column back into the collection tube, add 600 μL of detergent PW Buffer, and centrifuge at 12000 rpm for 1 min.

[0018] 8) Discard the waste liquid. Add 500 μL of detergent Wash Buffer and centrifuge at 12000 rpm for 1 min.

[0019] 9) Place the adsorption column into a centrifuge tube and centrifuge at 12000 rpm for 2 min.

[0020] 10) Take out the adsorption column and air-dry the residual detergent at room temperature.

[0021] 11) Place the adsorption column into another sterile 1.5 mL EP tube, add 100 μL of sterile deionized water preheated at 65 °C to the center of the adsorption membrane, let it stand at room temperature for 2 min, and then centrifuge at 12000 rpm for 1 min to elute the DNA.

[0022] 12) Detect the concentration of DNA with NanoDrop2000, label it, and store it at -20 °C in the refrigerator.

[0023] 2. Primer design First, according to the genomic sequences in the sequenced strains, specific primers targeting approximately 1000bp fragments upstream and downstream of srap were designed respectively. Using the genomic DNA of Staphylococcus aureus as a template, the upstream and downstream homologous arm fragments of the srap gene were amplified by PCR. The homologous arm primers are represented by UP-F / UP-R and DN-F / DN-R (Srap-UP-F shown in SEQ ID NO.1 and Srap-UP-R shown in SEQ ID NO.2; Srap-DN-F shown in SEQ ID NO.3 and Srap-DN-R shown in SEQ ID NO.4), which include the attB site and restriction enzyme sites.

[0024] 3. Amplification of the upstream and downstream homologous arms of the srap gene by PCR The PCR reaction used the genomic DNA of Staphylococcus aureus as a template and was amplified sequentially using PrimeSTAR® Max DNA Polymerase.

[0025] The PCR amplification conditions are as follows: Step 1 (1 cycle) Step 2 98°C, 10 min 98°C, 10 s 55°C, 5 s 72°C, 5 s / kb (30 cycles) Step 3 (1 cycle) Step 4 72°C, 10 min End at 10°C.

[0026] 4. After electrophoresis separation of the PCR products, they were recovered 1) Add an appropriate amount of Loading Buffer to the PCR products, add all the products to the wells, and select a suitable DNA marker.

[0027] 2) Set the voltage to 100V and the time to 40 min. Start running the electrophoresis.

[0028] 3) After electrophoresis, quickly cut the complete target gel strip under the ultraviolet gel cutter, turn off the ultraviolet gel cutter, crush the gel strip, and put it into a clean 1.5 mL EPP tube. Record the weight, and add 200 μL of NTI sol solution to every 0.1 g of the gel block. Then place it in a 50°C metal bath for 10 min to fully dissolve the gel block.

[0029] 4) Add it to the adsorption column. After standing for 2 min, centrifuge at 12,000 rpm for 1 min and discard the waste liquid.

[0030] 5) Add 700 mL of NT3 solution to the adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid.

[0031] 6) Repeat step 5.

[0032] 7) Centrifuge again for 2 min and discard the waste liquid.

[0033] 8) Let it stand to dry.

[0034] 9) Transfer the adsorption column to a new sterile 1.5 mL EP tube. Add 30 μL of sterile deionized water at 65 °C to the center of the adsorption membrane and centrifuge at 12,000 rpm for 1 min to elute the DNA.

[0035] 10) Detect the purity and concentration of the DNA with NanoDrop2000, write a label and store it at -20 °C in the refrigerator.

[0036] 5. Digestion of the PCR recovery product Use EcoR I and HindIII (rapid restriction endonucleases) to perform digestion reactions on the purified PCR recovery product respectively.

[0037] 6. Purification after digestion of the PCR recovery product 1) Mix the digested reaction product (240 μL) evenly with 5 volumes (1.2 mL) of the binding solution PB Buffer.

[0038] 2) Add it to the adsorption column in batches. After standing for 2 min, centrifuge at 12,000 rpm for 1 min and discard the waste liquid.

[0039] 3) Add 500 μL of PW Buffer to the adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid.

[0040] 4) Repeat step 3).

[0041] 5) Centrifuge again for 2 min and discard the waste liquid.

[0042] 6) Let it stand to dry at room temperature for 3 min.

[0043] 7) Transfer the adsorption column to a new sterile 1.5 mL EP tube. Add 40 μL of sterile deionized water at 70 °C to the center of the adsorption membrane and centrifuge at 12,000 rpm for 1 min to elute the DNA.

[0044] Detect the purity and concentration of DNA using NanoDrop 2000, label it, and store it at -20 °C in the refrigerator.

[0045] 7. Ligation of the upstream and downstream of the PCR recovery product after digestion The digested PCR product is ligated with T4 DNA ligase.

[0046] 8. PCR amplification verification of the ligation product Since it is a single digestion, the ligated product needs to be verified by PCR, and then the target product is purified. The purified product is the upstream and downstream homologous arms of the gene to be knocked out.

[0047] Using UP-F and DN-R as primers, perform PCR amplification reaction with PrimeSTARR Max DNA Polymerase.

[0048] For the amplified product, after electrophoresis, cut the target band and recover the PCR product. The purified product is sent to the company for first-generation sequencing identification.

[0049] 9. Extraction of plasmid pKOR1 Plasmid pKOR1 is a temperature-sensitive shuttle plasmid (as Figure 1 shown), showing chloramphenicol resistance in positive bacteria and ampicillin resistance in negative bacteria, and usually stored in Escherichia coli.

[0050] 1) Take out the Escherichia coli carrying plasmid pKOR1 from the -80 °C refrigerator, streak it on an LB plate containing 100 ug / mL, and culture it at 30 °C for 24 h.

[0051] 2) Pick a single colony, resuspend it in 4 mL of LB liquid medium containing 100 ug / mL, and culture it overnight at 30 °C with shaking at 220 rpm.

[0052] 3) Centrifuge at 5000g for 10 min at 4 °C, and discard the supernatant.

[0053] 4) Add 250 uL of P1 solution in the kit to completely suspend the bacteria.

[0054] 5) Then add 250 uL of P2 solution and gently invert it up and down 6 times.

[0055] 6) Add 350 uL of P3 solution and immediately gently invert it 6 times.

[0056] 7) Centrifuge at 13800g for 10 min, aspirate the supernatant into the adsorption column, let it stand for 3 min, and centrifuge again for 1 min, then discard the supernatant.

[0057] 8) Add 500 μL of PW buffer to the adsorption column and centrifuge at 13,800 g for 30 s.

[0058] 9) Repeat step 8).

[0059] 10) Centrifuge again for 2 min and discard the waste liquid.

[0060] 11) Air dry at room temperature for 2 min and air dry the washing solution as much as possible.

[0061] 12) Add 100 μL of preheated sterile deionized water to the membrane in the adsorption column and centrifuge at 12,000 g for 2 min.

[0062] 13) Detect the DNA purity and concentration with NanoDrop 2000, label it and store it at -20 °C in the refrigerator.

[0063] 10. B-P reaction between the upstream and downstream homologous arm fragments and plasmid pKOR1.

[0064] 11. Preparation of Escherichia coli competent DH5α cells 1) Take out the Escherichia coli DH5α strain from the -80 °C refrigerator and activate it on an LB plate.

[0065] 2) Pick a single colony, resuspend it in 4 mL of fresh LB liquid medium and culture it overnight at 37 °C.

[0066] 3) Take 500 μL of fresh bacterial liquid and add it to a 100 mL fresh sterile LB liquid culture bottle that has been autoclaved at a ratio of 1:200. Culture it at 37 °C with shaking at 220 rpm for about 2 h 30 min until the OD600 reaches about 0.5.

[0067] 4) Place the culture bottle with OD600 reaching 0.5 on ice and let it stand for 30 min.

[0068] 5) Aliquot it into two pre-cooled 50 mL sterile centrifuge tubes, centrifuge at 4000 g at 4 °C for 20 min, and pour out the supernatant of the bacterial liquid.

[0069] 6) Add 25 mL of pre-cooled CaCl 2 solution to the centrifuge tube, resuspend the bacterial cells, and centrifuge at 4000 g at 4 °C for 10 min.

[0070] 7) Repeat resuspension with 25 mL of pre-cooled CaCl 2 solution and centrifuge twice.

[0071] 8) Pour out the supernatant, then add 25 mL of pre-cooled sterile deionized water, resuspend the bacterial cells, and centrifuge at 4000 g at 4 °C for 10 min.

[0072] 9) Repeat the operation in step 8) twice.

[0073] 10) Pour off the supernatant, add 1 mL of 70% glycerol broth, and resuspend.

[0074] 11) Aliquot into sterile and enzyme-free 1.5 mL EP tubes at a specification of 100 μL of bacterial solution per tube, and store at -80 °C.

[0075] 12. Heat shock transformation of the B-P reaction product 1) Take out the DH5α competent cells from the -80 °C refrigerator and melt them on ice for 25 min.

[0076] 2) Add the system after the BP reaction to the melted competent cells, place it on ice, and let it stand for about 30 min to make the product and the competent cells fully fit; and turn on the water bath at 42 °C for standby.

[0077] 3) Perform heat transformation at 42 °C in a water bath for 90 s. Immediately insert it into ice for 5 min after the transformation to allow the cell wall of the competent cells to contract.

[0078] 4) Add 1 mL of fresh sterile LB culture medium, and culture at 30 °C and 220 rpm for 1 h.

[0079] 5) Centrifuge at 4 °C and 12000 rpm for 2 min.

[0080] 6) Discard the supernatant, add 100 μg of LB liquid medium with 100 μg / mL ampicillin resistance, and resuspend.

[0081] 7) Aspirate all the bacterial solution and evenly spread it on an LB plate with 100 μg / mL ampicillin resistance, and culture at 30 °C.

[0082] 13. PCR identification of the positive clone plasmid with successful heat transformation Use the successfully ligated PCR product as the DNA template for the positive control, and sterile deionized water as the DNA template for the negative control. Pick the suspected positive clones grown on the LB plate with 100 μg / mL ampicillin resistance and culture them in LB liquid medium with 100 μg / mL ampicillin resistance for 16 h. The obtained bacterial solution is used as the sample to be tested. According to the gene, select the corresponding primers and conditions for the PCR amplification reaction. After preliminary verification by electrophoresis, then perform bidirectional confirmation of the first-generation sequencing on the suspected positive PCR products. Confirm the true DH5α-pKOR1-Δsrap positive clones and then extract the positive homologous arm plasmids.

[0083] 14. Extraction of the positive clone plasmid Extract the plasmid of DH5α-pKOR1-Δsrap and store it in the -20 °C refrigerator.

[0084] 15. Heat transformation of pKOR1-△srap into competent DC10B Heat transform pKOR1-△srap into DC10B for modification. Only the modified plasmid can be electrotransformed into competent Staphylococcus aureus.

[0085] 16. Midiprep of the positive plasmid Since the amount of plasmid required for electrotransformation is 5-10 μg and the concentration of the positive plasmid is relatively high, a plasmid mini and midiprep kit is used for extraction. The method is as follows: 1) Streak the identified positive clone strain on an LB plate with 100 μg / mL ampicillin resistance and incubate at 30 °C for 24 h.

[0086] 2) Pick a single colony and suspend it in 100 mL of LB broth with 100 μg / mL ampicillin resistance. Incubate at 30 °C for 16-18 h.

[0087] Transfer the culture into two 50 mL centrifuge tubes. Centrifuge at 4000 g for 20 min at 4 °C. Discard the supernatant, wash the pellet with sterile saline once, centrifuge at 4000 g for 20 min at 4 °C, and discard the supernatant, retaining the cell pellet.

[0088] 3) Add 500 μL of P1 Buffer and mix well to lyse the cells.

[0089] 4) Add 500 μL of Buffer and mix well until the solution turns blue.

[0090] 5) Add 850 μL of P3 Buffer until the lysate turns white after lysis.

[0091] 6) Centrifuge at 12800 rpm for 10 min at 4 °C.

[0092] 7) Add 500 μL of BL buffer to the adsorption column and centrifuge at 12800 rpm for 1 min. Discard the supernatant.

[0093] 8) Add the supernatant from step 6) to the adsorption column and let it stand for 5 min.

[0094] 9) Centrifuge at 12800 rpm for 2 min and discard the waste liquid.

[0095] 10) Add 600 μL of PW Buffer to the adsorption column and centrifuge at 12800 rpm for 2 min. Discard the supernatant.

[0096] 11) Add 600 μL of Wash Buffer to the adsorption column and centrifuge at 12800 rpm for 2 min. Discard the supernatant.

[0097] 12) Centrifuge again for 2 min, take out the adsorption column and place it on a clean paper, and air-dry for 2 min.

[0098] 13) Add 100 μL of sterile deionized water preheated at 65 °C to elute the plasmid DNA.

[0099] 14) Detect the DNA purity and concentration with NanoDrop2000, write a label and store it at -20 °C in the refrigerator.

[0100] 17. Preparation of Competent Cells of Staphylococcus aureus 1) Take out Staphylococcus aureus from -80 °C and streak it on a blood agar plate for activation.

[0101] 2) Pick a single clone and resuspend it in 4 mL of sterile TSB liquid medium, and culture it overnight at 37 °C with 220 rpm.

[0102] 3) Inoculate the overnight bacterial solution at a ratio of 1:200, culture it at 37 °C with 220 rpm for 3 h to make the OD600 about 0.6. Then place it on ice for 30 min to slow down the bacterial growth.

[0103] 4) The following operations need to be carried out on ice: Aliquot the bacterial solution into pre-cooled sterile centrifuge tubes, centrifuge at 4000 g at 4 °C for 20 min to collect the bacterial cells.

[0104] 5) Resuspend and wash with sterile deionized water (pre-cooled), and centrifuge at 4 °C and 4000 rpm for 10 min, and repeat the washing 3 times.

[0105] 6) Resuspend and wash with 10% glycerol solution (pre-cooled), centrifuge for 10 min, and repeat the washing 2 times.

[0106] 7) Add 1 mL of 50% glycerol (pre-cooled), resuspend the bacterial solution, and aliquot it into sterile 1.5 mL EPPendorf tubes at a specification of 100 μL per tube, and store it in a -80 °C refrigerator for standby.

[0107] 18. Electrotransformation of the Homologous Recombinant Plasmid pKOR1-△srap into Competent Cells of Staphylococcus aureus 1) Take out the electrotransformation-competent Staphylococcus aureus stored in the -80 °C refrigerator, and place it on ice for 25 min to slowly melt.

[0108] 2) Add 10 μg of the homologous recombinant plasmid pKOR1-△srap to the electrotransformation-competent cells and incubate for 30 min for conjugation.

[0109] 3) Take out a brand-new sterile electroporation cuvette, place it in a -80 °C refrigerator for pre-cooling for 1 h for standby.

[0110] 4) Add the homologous recombination plasmid pKOR1-△srap that has been combined with competent cells into an electroporation cuvette and perform electroporation (voltage 2.6 V) for 4 min.

[0111] 5) Add 1 mL of pre-warmed B2 resuscitation liquid medium at 30°C and mix well with the competent cells after electroporation. Quickly transfer it to a sterile 1.5 mL EP tube and incubate it with shaking at 30°C and 220 rpm for 3 h.

[0112] 6) Centrifuge at 4°C and 4000 g for 5 min, aspirate 900 uL of the supernatant, resuspend the remaining 100 uL of the bacterial solution, and spread it on a TSA plate with 10 ug / mL chloramphenicol resistance. Incubate it statically in an incubator at 30°C for 48 h.

[0113] 19. Extraction and Identification of Recombinant Plasmid pKOR1-△srap in Staphylococcus aureus Since Staphylococcus aureus is a Gram-positive bacterium with a relatively thick cell wall, it cannot be used for bacterial liquid PCR, which is likely to lead to false negatives. Therefore, for the identification of the recombinant plasmid pKOR1-△srap in Staphylococcus aureus, the plasmid needs to be extracted first and then PCR reaction is carried out. The plasmid extraction of Staphylococcus aureus requires breaking the cell wall first, and the specific method is as follows: 1) Take out the positive clone carrying the plasmid from the -80°C refrigerator and streak it on a TSA plate containing 10 ug / mL chloramphenicol resistance, and incubate it at 30°C for 48 h.

[0114] 2) Pick a single colony and resuspend it in 4 mL of TSB liquid medium containing 10 ug / mL chloramphenicol resistance, and incubate it with shaking at 30°C and 220 rpm overnight.

[0115] 3) Centrifuge at 4000 g and 4°C for 10 min.

[0116] 4) Add 250 uL of P1 solution in the kit to completely suspend the bacteria. Since the cell wall of Staphylococcus aureus is relatively thick, it needs to be treated for cell wall breaking before plasmid extraction. Add 5 uL of lysostaphin and incubate it in a water bath at 37°C for 2 h until the bacterial solution becomes completely clear.

[0117] 5) Then add 250 uL of P2 solution and gently invert it up and down 6 times.

[0118] 6) Add 350 uL of P3 solution and immediately gently invert it 6 times.

[0119] 7) Centrifuge at 13800 g for 10 min, aspirate the supernatant into the adsorption column, let it stand for 3 min, and then centrifuge for 1 min again to discard the supernatant.

[0120] 8) Add 500 μL of PW buffer to the adsorption column and centrifuge at 13,800 g for 30 s.

[0121] 9) Repeat step 8).

[0122] 10) Centrifuge again for 2 min and discard the waste liquid.

[0123] 11) Air dry at room temperature for 2 min to air dry the washing solution as much as possible.

[0124] 12) Add 100 μL of preheated sterile deionized water to the membrane in the adsorption column and centrifuge at 12,000 g for 2 min.

[0125] 13) Using UP-F and DN-R as primers, the successfully ligated DNA as a positive control, and sterile deionized water as a negative control, perform PCR amplification. The obtained PCR products are identified by agarose gel electrophoresis. Those with bands consistent with the positive control are positive clones. Then send the positive PCR products for sequencing and comparison and identification again.

[0126] 20. Screening of MRSA ST59 △srap knockout mutants 1) Inoculate the identified positive clones into TSA medium with 10 μg / mL chloramphenicol resistance and culture overnight at 30 °C.

[0127] 2) Pick a single colony and inoculate it into 4 mL of TSB with 10 μg / mL chloramphenicol resistance, and culture overnight at 30 °C with shaking at 220 rpm.

[0128] 3) According to a ratio of 1:1000, pipette the bacterial solution and inoculate it into 4 mL of TSB with 10 μg / mL chloramphenicol resistance, and culture overnight at 30 °C with shaking at 220 rpm.

[0129] 4) According to a ratio of 1:1000, pipette the bacterial solution and inoculate it into 4 mL of TSB with 5 μg / mL chloramphenicol resistance, and culture overnight at 30 °C with shaking at 220 rpm.

[0130] 5) According to a ratio of 1:1000, pipette the bacterial solution and inoculate it into 4 mL of TSB with 2.5 μg / mL chloramphenicol resistance, and culture overnight at 30 °C with shaking at 220 rpm.

[0131] 6) According to a ratio of 1:1000, pipette the bacterial solution and inoculate it into 4 mL of TSB with 10 μg / mL chloramphenicol resistance, and culture overnight at 42 °C with shaking at 220 rpm.

[0132] 7) Streak the fresh bacteria grown at 42 °C in four zones on a TSA plate with 10 μg / mL chloramphenicol resistance and incubate overnight in an incubator at 42 °C.

[0133] 8) Pick monoclonal colonies on the plate and add them to the TSB liquid medium without antibiotics. Culture overnight at 37°C and passage 5 times.

[0134] 9) Dilute the overnight culture of the fifth-generation bacteria 10^6 times with sterile deionized water. Pipette 100 μL of the bacterial solution and spread it on the TSA plate containing 50 ng / mL anhydrotetracycline. Culture overnight at 37°C.

[0135] 10) Pick 400 monoclonal colonies on the plate. First, streak them on the TSA plate containing 10 μg / mL chloramphenicol resistance with a sterile pipette tip, and then streak them on the TSA plate without antibiotics. Culture overnight at 37°C.

[0136] 11) Result interpretation: Colonies that can only grow on the plate without antibiotics but not on the plate with chloramphenicol resistance are suspected mutant strains with successful knockout. First, preserve them for verification.

[0137] 21. Verification of the suspected knockout strain at the genomic level 1) Use the genomic DNA of the suspected knockout strain as a template and perform PCR amplification with UP-F and DN-R as primers.

[0138] 2) Select a suitable marker. After electrophoresis, the suspected mutant strain in the PCR result shows a band of the same size as the positive control, while the band amplified from the wild-type strain genomic reference control will be larger than the target fragments amplified by the mutant strain and the positive control. Sequence the amplified DNA fragment of the knockout strain again for confirmation.

[0139] 22. Construction of the complemented strain of MRSA ST59 △srap Use the genomic DNA of MRSA ST59 as a template to amplify the full length of the srap gene plus its promoter region and the relevant ribosome binding sites, and then construct the complemented plasmid pLI50-srap. The specific steps and methods for the construction and electroporation of the shuttle plasmid pLI50 and the complemented plasmid pLI50-srap are as described above. At the same time, use the empty plasmid pLI50 as a negative control. Figure 2 This is the figure for verifying the srap gene knockout result by RT-qPCR in the embodiment of the present invention.

[0140] Those of ordinary skill in the art can understand that the above are only preferred examples of the invention and are not used to limit the invention. Although the invention has been described in detail with reference to the foregoing examples, for those skilled in the art, they can still modify the technical solutions described in the foregoing examples, or perform equivalent replacements for some of the technical features. All modifications, equivalent replacements, etc. made within the spirit and principle of the invention shall be included within the protection scope of the invention.

Claims

1. A method for knocking out the gene of Staphylococcus aureus Srap, characterized in that: The following steps are involved: 1) Extracting genomic DNA of Staphylococcus aureus; 2) Primer design: The upstream and downstream homology arms of the target gene srap to be knocked out are taken as two fragments, and primer pairs for amplifying the upstream homology arm and the downstream homology arm are designed respectively: Srap-UP-F shown in SEQ ID NO.1 and Srap-UP-R shown in SEQ ID NO.2; Srap-DN-F shown in SEQ ID NO.3 and Srap-DN-R shown in SEQ ID NO.4; 3) Amplifying the upstream and downstream homology arms of the target gene and the knockout resistance gene fragment: using the genomic DNA of Staphylococcus aureus containing the target gene as a template, and using the primer pairs to perform PCR amplification respectively, to amplify the upstream and downstream homology arms of the target gene and the knockout resistance gene fragment; 4) Enzyme digestion of empty vector and construction of recombinant plasmid: the empty vector is digested with restriction endonucleases to obtain vector plasmid fragments, and the vector plasmid fragments and the upstream and downstream homologous arms of the target gene are treated in a recombinase reaction system to construct a recombinant plasmid; 5) Transformation with recombinant plasmid: 6) Screening of positive transformants: The recombinant plasmid treated in step 5) is screened on a plate to obtain positive transformants; monoclonal colonies of positive transformants are selected, colony PCR is performed with the primer pair, and monoclonal strains with target bands are obtained after agarose gel electrophoresis of the PCR products, and the strains with correct sequences are selected to extract plasmids to obtain gene knockout recombinant plasmids; 7) Amplification of knockout insert DNA fragment: using the gene knockout recombinant plasmid as a template, amplifying the knockout insert DNA fragment with the primer pair; 8) Electrotransformation of Staphylococcus aureus: The knockout insert DNA fragment is inserted into the genome of Staphylococcus aureus using electrotransformation to achieve knockout of the target gene; 9) Using colony PCR to screen the Staphylococcus aureus treated in step 8) for positive transformants in which the knockout resistance gene is correctly inserted into the target gene position of the Staphylococcus aureus genome, thereby obtaining a Staphylococcus aureus srap gene knockout strain.

2. A method for constructing a complemented strain of MRSA ST59 Δsrap, characterized in that: The method for obtaining the srap gene knockout strain of Staphylococcus aureus as described in claim 1; The construction of the complement strain includes the following steps: 1) Using MRSA ST59 genomic DNA as a template, amplify the full length of the srap gene plus its promoter region and the relevant ribosome binding site; 2) Construct the complementing plasmid pLI50-srap and transfer the empty plasmid pLI50 as a negative control.

3. A use of the Staphylococcus aureus srap gene knockout strain as claimed in claim 1.

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