CN106167808A - A kind of method eliminating mecA plasmid based on CRISPR/Cas9 technology - Google Patents

Abstract

The invention discloses a method for eliminating the mecA plasmid based on CRISPR/Cas9 technology. The MRSA strain is selected to perform PCR amplification on the DNA sequence of the C-terminus of the transpeptidase encoded by the mecA gene; the mecA gene is recovered from the gel; Simple) vector connection: preparation of DH5α competent cells; T-pMD19- mecA plasmid electroporation of DH5α competent cells; extraction and sequencing verification of T-pMD19- mecA plasmid; double restriction treatment of T-pMD19- mecA plasmid and mecA gene Gel recovery; ligation of pET‑21a(+) plasmid with mecA gene; design and synthesis of oligos; construction of pCas9:: mecA plasmid; electroporation of pET‑21a(+)‑ mecA plasmid into E. coli expression strain BL21 (D3); Electroporate pCas9:: mecA plasmid and pCas9 plasmid into BL21(D3)+pET‑21a(+)‑ mecA competent bacteria and BL21(D3)+pET‑21a(+) competent bacteria, respectively. The invention has simple operation and strong specificity, and can effectively block the spread of mecA and eliminate MRSA bacterial strains.

Description

A method for eliminating mecA plasmid based on CRISPR/Cas9 technology

technical field

The invention relates to the technical field of molecular biology, in particular to a method for eliminating mecA plasmids based on CRISPR/Cas9 technology.

Background technique

Staphylococcus aureus is one of the main pathogens causing a series of human infectious diseases, referred to as Staphylococcus aureus, can cause skin infection, severe pneumonia, endocarditis, joint suppurative inflammation, and food poisoning, severe cases can cause severe Toxic shock syndrome can easily lead to death of patients. Among them, methicillin-resistant Staphylococcus aureus (methicillin-resistant Staphylococcμ s aμ reμ s , MRSA) has become an important pathogen in hospital and hospital infection and drug resistance monitoring because of its more serious drug resistance. The key reason for the drug resistance of MRSA is the mecA gene. The mecA gene exists on the mobile element cassette chromosome ( Staphyloccoccal Cassette Chromosome mec , SCC mec ) of Staphylococcus. The mobile element can also carry other special structures such as multiple drug-resistant genes, transposons, and inserts to carry out horizontal transfer between bacterial strains, thereby causing the mutual transmission of drug-resistant genes between bacterial strains and causing multi-drug resistance of bacterial strains . Therefore, mecA horizontal gene transfer (Horizontal Gene Transfer, HGT) is an important way for many sensitive S. aureus to acquire drug-resistant genes and transform into multidrug-resistant MRSA bacteria.

At present, the research of new drugs against MRSA mainly has the following methods. One is to modify the structure on the basis of the original antibiotics to explore new antibiotics. For example, telavancin (telavancin) is a vancomycin derivative formed through the transformation of vancomycin. , and has good antibacterial activity against MRSA, but it is not widely used due to its poor effect on vancomycin-resistant S. The antibacterial protein developed by the good antibacterial effect of this method has obvious effect and high specificity, but as a macromolecular protein, its metabolic half-life in vivo and its immunogenicity limit its development.

Contents of the invention

In view of this, the purpose of the present invention is to address the deficiencies of the prior art and provide a method for eliminating mecA plasmids based on CRISPR/Cas9 technology with high specificity, obvious effect and no side effects.

To achieve the above object, the present invention adopts the following technical solutions:

A method for eliminating the mecA plasmid based on CRISPR/Cas9 technology, comprising the following steps:

(1) Select the MRSA strain to perform PCR amplification on the DNA sequence encoding the C-terminal of the transpeptidase of the mecA gene;

(2) mecA gene gel recovery;

(3) Ligate the mecA gene obtained in step (2) with the T-pMD19 (simple) vector:

(4) Preparation of DH5α competent cells;

(5) Electroporate the product obtained in step (3) to DH5α competent cells

Take 100 μl of the DH5α competent cells and thaw them on ice; take 11 μl of the product obtained in step (3) and place them in a centrifuge tube, and place the centrifuge tube and electro-cup on ice for 10 minutes; put 100 μl of the thawed DH5α Transfer the competent cells to the above centrifuge tube, mix well on ice, and continue ice bathing for 10 minutes;

Turn on the electroporation apparatus, transfer the above mixture to the electroporation cup, conduct electroporation at 2500V, and quickly add 1ml of resistant BHI medium into the electroporation cup, mix it evenly and transfer it to a 10ml centrifuge tube, at 37°C , centrifuge at 250r/min for 1.5h, and perform recovery treatment; take 50μl of the above mixture and 50μl of BHI medium, mix well, and then smear it on a TSA plate containing 100μg/ml ampicillin, place it in an incubator at 37°C, and culture overnight ;

(6) Extraction and sequencing verification of T-pMD19- mecA plasmid;

(7) Double digestion treatment of T-pMD19- mecA plasmid and gel recovery of mecA gene

After the T-pMD19- mecA plasmid was digested by BamHI and Hind III, it was verified by gel electrophoresis and the mecA gene gel was recovered;

(8) Double digestion treatment and gel recovery of pET-21a(+) plasmid

The pET-21a(+) plasmid was recovered by the plasmid DNA mini-extraction kit, then double-digested with BamHI and Hind III endonucleases, and then recovered by gel;

(9) The pET-21a(+) plasmid is linked to the mecA gene;

(10) Design, synthesis, phosphorylation and annealing of oligos;

(11) Construction of pCas9:: mecA plasmid

Ligation system between pCas9 gel recovery product and annealed double-stranded oligos: pCas9 gel recovery product 12 μl, diluted oligos 1.5 μl, T4 ligase 1 μl, T4 ligase buffer 2 μl, double distilled water 3.5 μl;

Connection conditions: connect overnight at 16°C, then electroporate 100 μl DH5α competent cells, select monoclonal colonies to extract pCas9:: mecA plasmid;

(12) Electroporation of pET-21a(+)- mecA plasmid into E. coli expression strain BL21(D3);

(13) Electrotransfer of pCas9∷mecA plasmid and pCas9 plasmid into BL21(D3)+pET-21a(+)- mecA competent bacteria;

(14) Electroporation of pCas9:: mecA plasmid and pCas9 plasmid into BL21(D3)+pET-21a(+) competent bacteria.

Preferably, the amplification primers required in the PCR amplification process described in step (1) are:

F: CGGGATCCACTATTGATGCTAAAGTTCAAAAG

R: CCCAAGCTTATTCATCTATATCGTATTTTTTATTA.

Preferably, the PCR amplification system includes: 12.5 μl of 2×Taq PCR Master Mix, 1 μl of amplification primer F, 1 μl of amplification primer R, 2 μl of MRSA DNA, and 8.5 μl of double distilled water.

Preferably, the process of the PCR amplification is:

<1> Pre-denaturation: heating at 94°C for 5 minutes;

<2> Denaturation: heating at 94°C for 30 seconds;

<3> Annealing: heating at 50°C for 30 seconds;

<4> Extension: heating at 72°C for 1 minute;

<5> Cycle processing: cycle <2>~<4> process 35 times;

<6> Re-extend: Continue to extend for 10 minutes at 72°C.

Preferably, the conditions for phosphorylation in step (10): 37°C water bath for 1 hour, then 65°C water bath for 20 minutes to inactivate the enzyme.

Preferably, the annealing treatment described in step (10) is: add 2.5 μl of 1M Nacl to the phosphorylated oligos pair, then heat the water bath to 95°C and put the system in, and turn off the power of the water bath after 5 minutes , when it was slowly cooled to 50°C, slowly add ice water to the water bath until the temperature dropped to room temperature.

Preferably, the extraction process of the plasmid is as follows: select a single clone colony, place it in a sterile BHI liquid medium containing 100 μg/ml ampicillin, perform bacterial enrichment, and then extract the plasmid.

Bacterial strains and plasmid sources used in this experiment: Clinical specimens collected from a general hospital in Zhengzhou City between June 2014 and December 2014 were selected, among which the MRSA mel-stap-a2014124/zz strain was selected to transpeptide the mecA gene The DNA sequence of the C-terminal of the enzyme was amplified by PCR, and the target fragment length was 1046bp; Escherichia coli DH5α strain was preserved by our laboratory; Escherichia coli BL21(D3) was purchased from Kangwei Century Co., Ltd.; T vector (PMD-19 simple) was purchased The pCas9 plasmid was purchased from Jiangsu Jirui Company; pET-21a(+) was preserved in our laboratory; BsaI endonuclease was purchased from NEB Company; T4 PNK was purchased from NEB Company; BamHI and Hind III were purchased from From NEB Company; T4 DNA ligase was purchased from NEB Company.

Sources of main reagents used in this experiment: Ethidium Bromide (EB), chloramphenicol and TAE were purchased from Shanghai Solarbio Company, agarose was purchased from Spain BIOWEST Company, and Loading buffer was purchased from Biyuntian Biotechnology Co., Ltd. , PH precision test paper was purchased from Tianjin Jinda Chemical Reagent Factory, yeast extract powder and tryptone were purchased from British Oxoid Company, TSA medium was purchased from Qingdao Haibo Biotechnology Co., Ltd., brain heart infusion medium (BHI) was purchased from Beijing Luqiao Technology Co., Ltd., bacterial plasmid DNA mini-extraction kit, micro DNA recovery kit, polymerase chain reaction (PCR) related reagents, DL2000 DNA maker (DL2000 and DL15000) and ampicillin were purchased from Shanghai Laifeng Biotechnology Co., Ltd. Company, sodium hydroxide and sodium chloride were purchased from Luoyang Haohua Chemical Reagent Co., Ltd., agar powder was purchased from Sigma Company, glycerol was purchased from Tianjin Kaitong Chemical Co., Ltd., bacterial genome DNA extraction kit was purchased from Beijing Tiangen Biochemical Technology Co., Ltd., lysostaphin was purchased from SIGMA-ALDRICH.

The preparation of the main reagents used in this test process:

Lysostaphin buffer: add 2mM EDTA and 20mM Tris-HCl to 1.2% Triton, adjust the pH value to 8.0 with NaOH, sterilize at 121°C for 20min, cool to room temperature, then add lysostaphin to The concentration is 200μg/mL, then aliquoted into 180μl aliquots, and stored at -20°C for future use.

1.5% agarose gel: Weigh 1.5g of agarose and add it to 100mL 1×TAE working liquid, stir evenly and heat on an electric stove until the liquid turns transparent after boiling, cool to 56°C, then add 5μl EB, Mix well and set aside.

25mg/ml chloramphenicol solution: Weigh 250mg chloramphenicol powder, add to 10ml absolute ethanol and mix well, filter with 22μm filter membrane to get chloramphenicol solution, aliquot into 1ml small portions and store at -20℃ Store at 100°C for later use; when in use, sterilize 100ml of LB, BHI, or BSA medium at 121°C for 20min, cool down to 60°C, and then add 100µl of the chloramphenicol solution obtained above to make the final working concentration 25µg/ml.

100mg/ml ampicillin aqueous solution: Weigh 1000 mg chloramphenicol powder, add it to 10ml deionized water, mix well, filter with a 22μm filter membrane to obtain ampicillin aqueous solution, divide into 1ml small portions and store at -20°C Standby; when in use, sterilize 100ml of LB, BHI, or BSA medium at 121°C for 20min, and cool down to 60°C, then add 100μl of the ampicillin aqueous solution obtained above to make the final concentration 100ug/ml.

LB liquid medium: Weigh 1.0g tryptone, 0.5g yeast extract powder and 1.0g sodium chloride and dissolve in 100mL distilled water, adjust the pH value to 7.2 with 10mol/L NaOH, and sterilize at 121°C for 20min, then Store at 4°C for later use; add 100 μl of the chloramphenicol or ampicillin antibiotic solution prepared above according to the resistance of the electroporated plasmid contained in the cultured strain, and mix well before use.

LB solid medium: Weigh 1.0g tryptone, 0.5g yeast extract powder, 1.0g sodium chloride and 1.5g agar powder, dissolve in 100mL distilled water, adjust the pH value to 7.2 with 10mol/L NaOH, and incubate at 121℃ Sterilize under high temperature for 20 minutes, cool to 50 ℃ ~ 60 ℃, add 100 ul of the prepared chloramphenicol or ampicillin antibiotic solution according to the resistance of the electroporation plasmid contained in the cultured strain and pour it into the plate for later use.

TSA medium: Weigh 4g of the purchased TSA medium and add it to 100mL deionized water, sterilize at 121°C for 20min, and cool down to 50°C~60°C, add 100ul of chlorine prepared above according to the resistance of the electroporated plasmid. Ampicillin or ampicillin antibiotic solution, mixed evenly and poured into medium plate, 15ml per plate, after drying, store at 4°C for later use.

BHI medium: Weigh 3.8g of the purchased BHI medium and add it to 100mL of deionized water, sterilize at 121°C for 20min, cool down to 50°C~60°C, add 100μl of the above-mentioned configuration according to the resistance of the electroporated plasmid when using Chloramphenicol or ampicillin antibiotic solution, mix well and set aside.

1M NaCl: Weigh 5.844g NaCl into 100mL deionized water, sterilize at 21°C for 20min, set aside.

10% glycerin: Add 10mL of glycerol to 80mL of deionized water, dilute to 100ml, sterilize at 21°C for 20min, and set aside.

Sources of main instruments and equipment used in this test:

LabcyCler basic gradient PCR instrument was purchased from SensoQuest Company of Germany, JW-2017H high-speed centrifuge was purchased from Anhui Jiawen Instrument Equipment Co., Ltd., HVE-50 automatic electric pressure steam sterilizer was purchased from Hirayama Company of Japan, and Haier vertical ultra-low temperature preservation box was purchased From Qingdao Haier Co., Ltd., the SK-1 type fast mixer was purchased from Jiangsu Jintan Medical Instrument Factory, the water-proof electric heating constant temperature incubator was purchased from Shanghai Xinmiao Medical Instrument Manufacturing Co., Ltd., and the DYC31B horizontal electrophoresis tank was purchased from Beijing City Liuyi Instrument Factory, HH-42S digital display constant temperature magnetic stirring circulating water tank was purchased from Changzhou Guohua Electric Co., Ltd., ZD-85 air bath constant temperature oscillator was purchased from Jintan Jingda Instrument Manufacturing Co., Ltd., Gene pulser XCell electrotransformation The instrument was purchased from Bio-Rad Company, the micro-adjustable pipette was purchased from Thermo Company of the United States, the 101-1B electric blast drying oven was purchased from Shanghai Experimental Instrument General Factory, and the WD-9403C ultraviolet instrument was purchased from Beijing Liuyi Instrument Factory , Gene-Genius bioimagingsystem was purchased from Gene Co., Ltd., and DYY-6C electrophoresis instrument was purchased from Beijing Liuyi Instrument Factory.

The test materials and methods that are not specifically stated are all known techniques, and can be found in commonly used reference books, including "Molecular Cloning Experiment Guide" (J. Sambrook, D.W Russell, third edition, Science Press, 2002), etc. in the search.

The beneficial effects of the present invention are:

The present invention constructs the pCas9:: mecA plasmid targeting the mecA gene and the Escherichia coli plasmid pET-21a(+)- mecA plasmid containing the mecA gene, and electrotransforms the former into the E. coli expression strain containing the latter, thereby realizing the elimination of mecA The purpose of the plasmid. This method is simple to operate and has strong specificity. It can not only eliminate MRSA strains, but also prevent sensitive strains from transforming into MRSA strains after accepting the mecA gene . It is also more flexible than traditional gene editing methods. higher efficiency.

detailed description

The features of the present invention and other relevant features are described in further detail below through the embodiments, so as to facilitate the understanding of those skilled in the art:

The method for eliminating the mecA plasmid based on CRISPR/Cas9 technology provided by the invention comprises the following steps:

(1) DNA template preparation

DNA was extracted from MRSA mel-stap-a2014124/zz strain using bacterial genomic DNA extraction kit;

(2) PCR amplification of mecA gene

Select the MRSA strain to carry out PCR amplification on the DNA sequence of the mecA gene encoding transpeptidase C-terminus;

Among them, the amplification primers are:

F: CGGGATCCACTATTGATGCTAAAGTTCAAAAG

R:CCCAAGCTTATCATCTATATCGTATTTTTTATTA;

The PCR amplification system includes: 12.5 μl of 2×Taq PCR Master Mix, 1 μl of amplification primer F, 1 μl of amplification primer R, 2 μl of MRSA DNA, and 8.5 μl of double distilled water;

The process of PCR amplification includes:

<1> Pre-denaturation: heating at 94°C for 5 minutes;

<2> Denaturation: heating at 94°C for 30 seconds;

<3> Annealing: heating at 50°C for 30 seconds;

<4> Extension: heating at 72°C for 1 minute;

<5> Cycle processing: cycle <2>~<4> process 35 times;

<6> Re-extend: Continue to extend for 10 minutes at 72°C;

(3) mecA gene gel recovery

Add 40 μL of PCR system to the sample well of gel electrophoresis, then add DL2000 DNA marker, treat at 90V for 40 minutes, and then put it into WD-9403C ultraviolet imager, and the strips corresponding to about 1000bp of the above obtained Cut off the band quickly, cut off the excess gel block, recover it with a micro DNA rapid recovery kit, and elute it with double distilled MILLI-Q water, measure the concentration of the recovered DNA, so as to calculate the ratio of the carrier and the target fragment in the next ligation experiment;

(4) Ligate the mecA gene obtained in step (3) with the T-pMD19 (simple) vector

The connection system includes: T-pMD19 (simple) vector 1 μl, mecA gene 5 μl, Takara Buffer 5 μl; place the obtained connection system in a water-proof electric thermostat incubator at 16°C for overnight connection;

(5) Preparation of DH5α Competent Cells

Inoculate DH5α bacteria into non-resistant sterile LB solid medium, and culture overnight at 37°C; put 5ml of sterile BHI medium without antibiotics into a 20ml sterilized glass test tube, and select a single strain of DH5α bacteria Clone the colonies, inoculate them into the above-mentioned sterile BHI medium without antibiotics, and shake overnight at 37°C at a rate of 250r/min to enrich the bacteria;

Add the DH5α bacteria obtained above into a new sterile BHI medium without antibiotics at a ratio of 1:100, and shake at a rate of 250r/min for 2.5 hours at 37°C. When the OD value of the bacteria reaches 0.3~ Stop culturing at 0.4, place on ice for ice bath pre-cooling, until the temperature of the bacterial solution drops to 3°C~4°C;

Sterilize the 50ml centrifuge tube and 10% glycerol at 121°C for 20 minutes, and cool to 3°C~4°C for later use; Centrifuge at 6000r/min for 10min at 4°C to remove the supernatant. ;Repeat this step once;

Then take 45ml of the above-mentioned glycerol and place it in a centrifuge tube, centrifuge at 4°C for 10 minutes at a rate of 6000r/min, and discard the supernatant;

Operation on ice: Add 500 μl of the above-mentioned glycerol to the centrifuge tube, mix well to obtain DH5α competent cells, and store them in a -80°C refrigerator for later use;

(6) Electroporate the product obtained in step (4) to DH5α competent cells

Take 100 μl of DH5α competent cells and thaw on ice; take 11 μl of the product obtained in step (4) and place it in a 1.5ml centrifuge tube, and place the centrifuge tube and a 0.2cm electro-cup on ice for 10 minutes; put 100 μl of the thawed Transfer the competent cells to the above-mentioned 1.5ml centrifuge tube, mix well on ice, and continue ice bathing for 10 minutes;

Turn on the electroporation apparatus, transfer the above mixture to the electroporation cup, conduct electroporation at 2500V, and quickly add 1ml of resistant BHI medium into the electroporation cup, mix it evenly and transfer it to a 10ml centrifuge tube, at 37°C , centrifuge at 250r/min for 1.5h, and perform recovery treatment; take 50μl of the above mixture and 50μl of BHI medium, mix well, and then smear it on a TSA plate containing 100μg/ml ampicillin, place it in an incubator at 37°C, and culture overnight ;

(7) Extraction and sequencing verification of T-pMD19- mecA plasmid

Select the monoclonal colony in the product obtained in step (6), place it in a sterile BHI liquid medium containing 100 μg/ml ampicillin, carry out bacterial enrichment, and then extract the T-pMD19- mecA plasmid;

Use the plasmid DNA mini-extraction kit, collect 1ml of the bacterial solution twice each time, centrifuge each time for 1min, discard the supernatant, suck out the residual bacterial solution with the tip of the pipette, follow the instructions in the intermediate steps, and elute with MILLI-Q water in the last step and dissolve, add water volume to 60 μl per column, add the first eluent to the column again for the second elution, in order to increase the concentration of plasmid DNA;

(8) Send the extracted T-pMD19- mecA plasmid to Shanghai Sangon Biotechnology Company for sequencing verification;

(9) Double digestion treatment of T-pMD19- mecA plasmid and gel recovery of mecA gene

After the T-pMD19- mecA plasmid was digested by BamHI and Hind III, it was verified by gel electrophoresis and the mecA gene gel was recovered;

20 μl enzyme digestion system: 5 μl plasmid DNA, 1 μl each of BamHI and Hind III endonucleases, 2 μl Cutsmart buffer, 11 μl double distilled water;

Place the above enzyme digestion system in water bath at 37°C for 4 hours to complete the double enzyme digestion reaction of the T-pMD19- mecA plasmid, then put it into a DYY-6C electrophoresis apparatus, and perform electrophoresis at 90V for 40 minutes, in which the electrophoresis medium is 1.5% agarose gel Gel; The gel recovery process of mecA gene is consistent with step (3);

(10) Double digestion treatment and gel recovery of pET-21a(+) plasmid

The pET-21a(+) plasmid was recovered by the plasmid DNA mini-extraction kit, then double-digested with BamHI and Hind III endonucleases, and then recovered by gel;

Enzyme digestion system (20 μl): pET-21a(+) plasmid DNA 15 μl, BamHI and Hind III endonucleases 1.5 μl each, Cutsmart buffer 2 μl;

The enzyme digestion process is consistent with step (10), and the gel recovery process is consistent with step (3);

(11) The pET-21a(+) plasmid is linked to the mecA gene

The gel-recovered fragment (10ng/μl) of the pET-21a(+) plasmid was double-enzymatically digested and ligated with the mecA gene (12ng/μl) recovered from the gel in step (9);

Ligation system (32 μl): pET-21a(+) plasmid double-digested and gel-recovered fragment 20 μl, gel-recovered mecA gene fragment 8 μl, T4 ligase 1 μl, T4 ligase buffer 3 μl;

Ligation reaction conditions: The ligation system was ligated overnight in a water bath at 16°C. After that, 300 μl of DH5α competent cells were electroporated, and the pET-21a(+)- mecA plasmid was extracted from monoclonal colonies and sent to Shanghai Sangong Biotechnology Company for sequencing.

(12) Design, synthesis, phosphorylation and annealing of oligos

Using sgRNAcas9 software to design, select 6 oligos sequences with the lowest off-target efficiency, and refer to two pairs of oligos sequences in the literature, and add BsaI restriction site according to the oligos design principle, and then synthesized by Shanghai Sangong Biotechnology Co., Ltd.

Oligos phosphorylation system (50 μl): oligo I (100 μM) 1 μl, oligo II (100 μM) 1 μl, 10X T4 Ligase buffer (NEB) 5 μl, T4 PNK (NEB) 1 μl, ddH ₂ O 42 μl;

Dephosphorylation conditions: 37°C water bath for 1 hour, then 65°C water bath for 20 minutes to inactivate the enzyme;

Oligos annealing steps:

Add 2.5μl 1M Nacl to the phosphorylated oligos, then heat the water bath to 95°C and put the system in. After 5 minutes, turn off the power of the water bath, let it cool down to 50°C slowly, and slowly pour it into the water bath. Add ice water until the temperature drops to room temperature;

Oligos sequences are shown in Table 1:

Table 1 Oligos sequence

Note: "F" stands for sense strand, "R" stands for antisense strand.

(13) Construction of pCas9:: mecA plasmid

Enzyme digestion system of pCas9 plasmid Bsal endonuclease: pCas9 plasmid 15 μl, Bsal endonuclease 1 μl, cutsmart buffer 2 μl, double distilled water 2 μl;

Digestion conditions: 37°C water bath for 3 hours, 1.5% agarose gel electrophoresis and gel recovery;

Ligation system of pCas9 gel recovery product and annealed double-stranded oligos: pCas9 gel recovery product 12 μl, diluted oligos 1.5 μl, T4 ligase 1 μl, T4 ligase buffer 2 μl, double-distilled water 3.5 μl;

Connection conditions: connect overnight at 16°C, after that, electroporate 100 μl DH5α competent cells, select monoclonal colonies to extract plasmids, and send them to Shanghai Sangon Biotechnology Company for sequencing;

(14) Electroporation of pET-21a(+)- mecA plasmid into E. coli expression strain BL21(D3)

Use the method of step (7) to obtain the enrichment solution, divide the enrichment solution into two parts, and directly extract the plasmid from one part of the solution for single enzyme digestion, and verify whether the bacteria have the plasmid pET-21a(+) by gel electrophoresis - mecA , another direct production of competent cells;

(15) Electroporation of pCas9∷mecA plasmid and pCas9 plasmid into BL21(D3)+pET-21a(+)- mecA competent bacteria

Electroporation plasmid pCas9:: mecA and control plasmid pCas9 each 2μl into 100μl BL21(D3)+pET-21a(+)- mecA competent bacteria;

Take 100 μl of the above-mentioned bacterial solution and dilute it 10 times, then take 10 μl of the diluted bacterial solution and spread it on a TSA plate containing 25 μg/ml chloramphenicol, incubate overnight at 37°C, and count and number all the colonies on the plate;

Randomly select 10 colonies on each plate for liquid enrichment and extract plasmids;

After the extracted plasmid was digested with hind III, agarose gel electrophoresis was performed to determine the effect of the Cas9 plasmid on pET-21a(+)- mecA ;

(16) Electrotransfer of pCas9∷mecA plasmid and pCas9 plasmid into BL21(D3)+pET-21a(+) competent bacteria

Take 100 μl of the bacterial solution and dilute it 10 times, then take 10 μl of the diluted bacterial solution and spread it on a TSA plate containing 25 μg/ml of chloramphenicol, incubate overnight at 37°C, and count and number all the colonies on the plate;

Randomly select 10 colonies on each plate for liquid enrichment and extract plasmids;

After the extracted plasmid was digested with BamHI, agarose gel electrophoresis was performed to determine the effect of the Cas9 plasmid on pET-21a(+).

The clearance efficiency of 9 different Cas9 plasmids on pET-21a(+)- mecA plasmid and pET-21a(+) plasmid, as shown in Table 2 and Table 3:

It can be seen from Table 2 that there are differences in the clearing effect of the nine Cas9 plasmids on the pET-21a(+)- mecA plasmid as a whole. After comparing the effects of each group and the control group plasmid pCas9, it is found that the P value of each comparison is 0.00 is less than the adjusted test level α=0.006, so except for the pCas9 plasmid in the control group, the remaining 8 groups of pCas9- mecA plasmids all have a clear effect on the plasmid pET-21a(+)- mecA . It can be seen from Table 3 that the detection results of 90 colonies including the control group pCas9 all show that the pET-21a (+) plasmid that does not contain the mecA gene has no elimination effect on the pET-21a (+) plasmid that does not contain the mecA gene.

Table 2 Elimination efficiency of 9 kinds of Cas9 plasmids on pET-21a(+)- mecA plasmid

Note: "*" is the two-sided test level α=0.05 by Fisher's exact probability method, and "^" uses the 2×2 four-table Fisher's exact probability method because the sample size is less than 40. After two-sided test, the corrected test level α=0.006.

Table 3 Elimination efficiency of nine Cas9 plasmids on plasmid pET-21a(+)

The present invention has been described in detail through the examples above, but the content described is only a preferred embodiment of the present invention and cannot be considered as limiting the implementation scope of the present invention. All equivalent changes and improvements made according to the application scope of the present invention shall still belong to the scope covered by the patent of the present invention.

Claims (7)

1. one kind eliminates based on CRISPR/Cas9 technologymecAThe method of plasmid, it is characterised in that comprise the following steps:

Select MRSA bacterial strain pairmecAThe DNA sequence of gene code transpeptidase C-terminal carries out PCR amplification；

mecAGene gel reclaims；

By step (2) gainedmecAGene and T-pMD19(simple) carrier is connected:

Preparation DH5 α competent cell；

Step (3) products therefrom electricity is turned DH5 α competent cell

Take DH5 α competent cell described in 100 μ l to be placed in and thaw on ice；Take 11 μ l step (3) products therefroms to be placed in centrifuge tube, Centrifuge tube is placed in pre-cooling 10min on ice together with electricity revolving cup；The described DH5 α competent cell that 100 μ l thaw is transferred to In above-mentioned centrifuge tube, on ice after mixing, continue ice bath 10min；

Open electroporation, said mixture is transferred in electricity revolving cup, under 2500V voltage, carries out electricity turn, and 1ml is contained The BHI culture medium of resistance is rapidly added in electricity revolving cup, transfers in 10ml centrifuge tube after being mixed, 37 DEG C, 250r/min Centrifugal 1.5h, carries out recovery process；Take 50 μ l said mixtures and 50 μ l BHI culture medium, mix homogeneously, be then coated in containing On the TSA flat board of 100 μ g/ml ampicillins, it is placed in the couveuse of 37 DEG C, incubated overnight；

T-pMD19-mecAThe extraction of plasmid and sequence verification；

T-pMD19-mecAThe double digestion of plasmid process andmecAThe gel of gene reclaims

T-pMD19-mecAPlasmid after BamH I and Hind III double digestion, carry out gel electrophoresis checking andmecAGene gel returns Receive；

PET-21a (+) double digestion of plasmid processes and gel reclaims

PET-21a(+) plasmid uses plasmid DNA Mini Kit to carry out BamH I and Hind III enzymes double zyme after reclaiming After cutting process, carry out gel recovery；

PET-21a (+) plasmid withmecAGene connects；

The design of oligos, synthesis, phosphorylation and annealing；

pCas9∷mecAThe structure of plasmid

PCas9 gel reclaims the double-strand oligos linked system of product and annealing: pCas9 gel reclaims product 12 μ l, after dilution Oligos 1.5 μ l, T4 ligase 1 μ l, T4 ligase buffer 2 μ l, distilled water 3.5 μ l；

Condition of contact: overnight connect at 16 DEG C, afterwards, electricity turns 100 μ l DH5 α competent cells, selects monoclonal bacterium colony to extract pCas9∷mecAPlasmid；

Electricity turn pET-21a (+)-mecAPlasmid is to E. coli expression strains BL21(D3) in；

Electricity turns pCas9 ∷mecAPlasmid and pCas9 plasmid are to BL21(D3)+pET-21a (+)-mecAIn competence antibacterial；

Electricity turns pCas9 ∷mecAPlasmid and pCas9 plasmid are to BL21(D3)+pET-21a (+) in competence antibacterial.

One the most according to claim 1 eliminates based on CRISPR/Cas9 technologymecAThe method of plasmid, its feature exists In, amplimer required in PCR amplification procedure described in step (1) is:

F:CGGGATCCACTATTGATGCTAAAGTTCAAAAG

R:CCCAAGCTTATTCATCTATATCGTATTTTTTATTA.

One the most according to claim 1 and 2 eliminates based on CRISPR/Cas9 technologymecAThe method of plasmid, its feature Being, the system of described PCR amplification includes: 2 × Taq PCR Master Mix 12.5 μ L, amplimer F 1 μ l, amplification is drawn Thing R 1 μ l, MRSA DNA 2 μ l, distilled water 8.5 μ l.

4. eliminate based on CRISPR/Cas9 technology according to the one described in any one of claim 1 ~ 3mecAThe method of plasmid, its Being characterised by, the process of described PCR amplification is:

<1>denaturation: heat 5 minutes at 94 DEG C；

<2>degeneration: heat 30 seconds at 94 DEG C；

<3>annealing: heat 30 seconds at 50 DEG C；

<4>extend: heat 1 minute at 72 DEG C；

<5>circular treatment: circulation<2>~<4>process 35 times；

<6>re-extend: continue at 72 DEG C to extend 10 minutes.

5. eliminate based on CRISPR/Cas9 technology according to the one described in any one of claim 1mecAThe method of plasmid, it is special Levying and be, the condition of phosphorylation described in step (10): 37 times water-bath 1h, then 65 DEG C of water-bath 20min, enzyme inactivates.

6. eliminate based on CRISPR/Cas9 technology according to the one described in any one of claim 1mecAThe method of plasmid, it is special Levying and be, the annealing described in step (10) is: add the Nacl oligos centering to phosphorylation of 2.5 μ l 1M, so After water-bath is heated to 95 DEG C after system is put into, after 5 minutes close water-bath power supply so that it is during slow cooling to 50 DEG C, In water-bath, slowly add frozen water be down to room temperature to temperature.

7. eliminate based on CRISPR/Cas9 technology according to the one described in any one of claim 1mecAThe method of plasmid, it is special Levying and be, the extraction process of described plasmid is: select monoclonal bacterium colony, and be placed in containing 100 μ g/ml ampicillins is aseptic In BHI fluid medium, carry out increasing bacterium, then extract plasmid.