Liquid LB medium (take 1000 mL as an example)

1. Prepare the following mixture in an Erlenmeyer flask.

2. Sterilize in an autoclave.

Solid LB medium (take 1000 mL as an example)

1.Prepare the following mixture in an Erlenmeyer flask.

2.Sterilize in an autoclave.

Preparation of reagents

1. Coomassie brilliant blue (500mL): 37.5mL acetic acid. 50mL ethanol. 10mL Coomassie brilliant blue dye solution. 402.5mL water.

Agarose gel electrophoresis (take 30mL as an example)

  1. Prepare a suitable gel plate and comb.

  2. Weigh 0.3g（1%） agarose.

  3. Add 30mL 1×TAE.

  4. Heat in the microwave oven until the agarose is completely dissolved.

  5. Add 3 microliters of nucleic acid staining dye and mix well.

  6. Pour the solution on the flat plate with the comb inserted, and use the pipette tip to suck out all bubbles.

  7. After it solidifies, carefully pull out the comb.

  8. Mix the sample with 6×DNA loading Buffer at a ratio of 5:1. Load an appropriate amount of DNA weight makers and samples into the wells(lanes).

  9. Turn on the power supply, set the voltage to 120V, run the gel until the makers move to the bottom.

10. Remove the gel assembly from the electrophoresis apparatus. Remove the gel from the glass plates using a spatula, and prepare for subsequent analysis.

Polyacrylamide gel electrophoresis

1. Assemble the device and perform leak detection.

2. Prepare separation gel.

Note: Add modified ammonium persulfate at the end and shake it immediately before pour into the glass plate.

3. Seal the liquid surface with water or isopropanol.

4. Prepare concentration gel.

Note: Add modified ammonium persulfate at the end and shake it immediately before pour into the glass plate.

5. Insert the comb and wait for the glue to completely solidify.

6. Preparation of the sample: add a certain amount 5× loading Buffer to the sample .and mix well, heat the sample at 98°C for 12 minutes in theymocycler.

7. After the gel has solidified, pull out the comb, add 1×SDS buffer, and then load them to the wells.

8. Turn on the power supply,  voltage is 120V , run the makers until the makers move to the bottom.

9.Take out the gel, stain with Coomassie Brilliant Blue for 30 minutes, destaining with distilled water washed three times, the gel was used for subsequent analysis.

Transformation of bacteria

1. Thaw a tube of 100μL DH5a Competent cells solution on ice，then add 10ul constructed vectors solution. Carefully flick the tube 4-5 times to mix cells and DNA.

2. Place the mixture on ice for 30 minutes.

3. Heat shock at 42℃ for 90 seconds.

4. Place the mixture on ice again for 2 minutes.

5. Add 800μl LB culture medium to the mixture and incubate at 37℃ in the shaking incubator for 1h.

6. Centrifuge the medium at 12，000rpm for 5 min to concentrate the cells, abandon the unnecessary supernatant to leave 100μL solution. mix the cells thoroughly.

7. Take a Erlenmeyer flask containing 100mL LB solid medium which has been sub packed and Sterilized by autoclaving。Heat it in microwave oven for about 5-8min until the medium is transparent and free of colloidal precipitation.

8. Place it on the clean bench. When the medium is cooled to about 50℃, add 100μL Kanamycin（or 10μL chloramphenicol or 100μL ampicillin）with a concentration of 100ng / mL and shake it well. Immediately pour it into the sterilized dish. Each Petri dish add about 15-20mL medium. Place them around the alcohol lamp to accelerate the solidification for about 20min.

9. Spread 50-100 μL competent cells onto each sterilized dish and incubate overnight at 37℃.

Extract DNA from bacterial culture

We used omega Plasmid Mini Kit I  to extract DNA from bacterial culture.

Application object: used to extract 1-5 mL of Escherichia coli cultured overnight.

Preparation:

        1. Heat Elution Buffer to 70℃ if plasmid DNA is>10kb.

        2. Add the vial of RNase A to the bottle of SolutionⅠand store at 2-8℃

        3.Dilute DNA Wash Buffer with 100% ethanol as follows and store at room temperature.

Steps

1. Isolate a single colony from a freshly streaked selective plate, and inoculate a culture of 1- 5 mL LB medium containing the appropriate selective antibiotic. Incubate for 12-16 hours at 37°C with vigorous shaking (12000 rpm). Use a 10-20 mL culture tube or a flask.

      2.Take 1-5mL of bacterial liquid Centrifuge at 12,000 rpm for 1 minute at room temperature.

3. Decant or aspirate and discard the culture media.

4. Add 250 µL Solution I/RNase A. Vortex or pipet up and down to mix thoroughly.

Note: RNase A must be added to Solution I before use.

5. Transfer suspension into a new 1.5 mL microcentrifuge tube.

6. Add 250 µL Solution II. Invert and gently rotate the tube several times to obtain a clear lysate Incubate for two or three minutes and avoid vigorous mixing.

7. Add 350 µL Solution III. Immediately invert several times until a flocculent white precipitate forms.

8. Centrifuge at speed (12,000 rpm) for 10 minutes.

9. Transfer the supernatant to a HiBind Miniprep DNA binding column with a 2mL collection tube, centrifuge at the maximum speed for 1 min at room temperature, and pour the filtrate in the collection tube.

10. Reinstall the column into the collection tube, add 500μL HBC Buffer, centrifuge at the maximum speed for 1 min at room temperature, and discard the filtrate.

Note: HBC Buffer must be diluted with isopropanol according to the instructions before use .

11. Reinstall the column into the collection tube, add 700μL DNA Wash Buffer, centrifuge at the maximum speed for 1 min at room temperature, and discard the filtrate.

Note: DNA Wash Buffer must be diluted with absolute ethanol according to the instructions before use.

12.Discard the filtrate and repeat step 10 once .

      13. Discard the filtrate, reinstall the column back into the collection tube, and centrifuge the empty column at maximum speed for 2 minutes to spin dry the column matrix. Put the column in a clean 1.5mL centrifuge tube, add 30-100μl Elution Buffer to the column matrix, let it stand for 1 min, and centrifuge at 13,000xg for 1 min to elute the DNA.

14. Store the eluted DNA at -20°C.

Recover and purify DNA fragments from agarose gel

We successively used two kits to Recover and purify DNA fragments from agarose gel.

Preceding Kit: TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0

Preparation before Use

1. add 56 ml of 100% ethanol to Buffer WB.

2. Confirm the Buffer GM is still yellow.

Steps

1. Excise the agarose gel slice containing the DNA fragment with a clean, sharp scalpel under ultraviolet illumination. Briefly place the excised gel slice on absorbent toweling to remove residual buffer.

2. Cut the gel into small pieces by cutting the gel.

3. Weigh the gel pieces and calculate the volume of gel. 1 mg of gel is equivalent to 1μL volume.

    Add Buffer GM to gel for melting. The amount of Buffer GM is shown in the table below.

4. Mix well and melt the gel . warm at 37℃. accelerate gel solubilization.

Note: Gel must be completely dissolved.

5. After the gel has been completely molten, check the color of the solution. when the color becomes orange or pink from yellow, add 10 μL of 3M sodium acetate (pH 5.2) to the solution and vortex well untill the solution return to yellow.

6. Set a Spin Column into Collection Tube.

7. Transfer the solubilized agarose from Step 7 into the column. Centrifuge at 12,000 rpm for 1 minute. Discard the flow-through. For improvement the recovery rate of DNA, transfer the flow-through to Spin. Column again and centrifuge again.

8. Add 700 μL of Buffer WB into the Spin Column. Centrifuge at 12,000 rpm for 30 seconds. Discard the flow-through.

9. Repeat Step 10.

10. Place the Spin Column back into Collection Tube. Centrifuge at 12,000 rpm for 1 minute.

11. Place the Spin Column back into Collection Tube. Centrifuge at 12,000 rpm for 1 minute.

12. Centrifuge at 12,000 rpm for 1 minute to elute the DNA.

Subsequent Kit : TIANprep Mini Plasmid Kit

Steps

1. Column equilibration steps: put the adsorption column into the collection tube, add 500μL of the balance solution BL to the adsorption column CB2, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

2. Cut a single target DNA band from the agarose gel into a clean centrifuge tube, weigh it.

3. Add an equal volume of PC solution to the gel block, place it in a 50℃ water bath for about 10 minutes, and turn the centrifuge tube up and down gently.

4. Add the solution obtained in the previous step to an adsorption column CB2, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube.

5. Add 600μL rinsing solution PW (preliminarily added absolute ethanol) to the adsorption column CB2, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube.

6. Repeat step 5.

7. Put the adsorption column CB2 into the collection tube and centrifuge at 12,000 rpm for 2 minutes to remove the rinse solution. Put the adsorption column at room temperature for a few minutes and let it dry thoroughly.

8. Put the adsorption column CB2 into a clean centrifuge tube, and drop an appropriate amount of sterile water into the middle of the adsorption membrane, and leave it at room temperature for 2 minutes. Centrifuge at 12,000 rpm for 2 minutes to collect the DNA solution.

Infusion

Steps

1. PCR: Use PCR method to amplify HrtOand pSB1C3-cjBlue, and use TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 to recover and purify.

2. Reaction sy stem.

HrtO : pSB1C3-cjBlue  ===  2 : 1

3. Incubate at 50 degrees Celsius for 15 minutes before placing on ice.

4. Transformation.

4.1 Thaw the connected product on ice.

4.2 Add 2.5μL for conversion.

4.3 Place on ice for 30 minutes.

4.4 42℃ heat shock 45S.

4.5 Place 90s on ice.

4.6 Add 500μL SOC medium (heated to 37℃ in advance).

4.7 Shake at 37℃ for 1h.

4.8 Centrifuge the remainder of each conversion reaction at 6000 rpm for 5 minutes. Discard the supernatant and re suspend each particle at 100 μ L In fresh SOC medium.

4.9 Disperse each sample on a separate LB plate containing appropriate antibiotics.

4.10 Incubate all dishes overnight at 37 ° C. 

4.11 The next day, single colony was selected from each experimental plate and plasmid DNA was isolated. To determine the presence of the insert, DNA was analyzed by restriction enzyme digestion or PCR screening.

Polymerase chain reaction (PCR)

1. Mix individual components prior to use .

 Q5 High-fidelity PCR kit

 Taq PCR Mix

2.Gently mix the reaction and collect the liquid at the buttom of a PCR tube with a quick spin.

3. Transfer reaction quickly to a preheated thermocycler , run with the following thermocycling conditions.

Q5 High-fidelity PCR kit

Taq PCR Mix

Restriction Enzyme Digestion

1. Mix components on ice.

2. Incubate the mixture in thermostat water bath at 37℃ for 4h.

3. Run gel electrophoresis to check the length of the digested DNA fragment.

DNA ligation

1. Set up the following reaction in a microcentrifuge tube on ice, the T4 DNA Ligase should be added last.

NOTE: The table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes. We run the gel electrophoresis to estimate the DNA moles by comparing the luminance with DNA ladder.

2. Gently mix the reaction by pipetting up and down and incubate at 16℃ for 16 hours or connect overnight at 4℃

IPTG induction

1. Pick a colony from a plate and grow in LB liquid medium with Kanamycin and chloramphenicol.

incubate overnight at 37℃.

2. Dilute the overnight culture to 100ml (1:100 dilution) and incubate the cells at 37℃ until the OD600 reaches 0.6-0.8.

3. Add IPTG (1μL/mL,) to the culture and incubate at 37℃ for 12-16 hours in a shaker.

4. Store at 4℃.

Competent Cell Preparation

1. Pick a single colony from the E. coli BL21 (DE3) plate and attach it to a shaker tube of 3 ml LB liquid medium and incubate at 37 °C for overnight concussion.

2. Take 0.5 ml of bacterial liquid and transfer it to a LB liquid medium containing 50 ml Erlenmeyer flask and incubate at 37 ℃ for 2-3 h (at this time, OD₆₀₀ reaches 0.6-0.8, the number of cells must be < 10⁸/ml, which is the key to the success of the experiment)

3. Transfer the bacterial liquid to a 1.5 ml centrifuge tube on ice; place for 10 min.

4. 4 °C 8000r/min, centrifugate for 1 min.

5. Decant the culture medium.(aspirate as much as possible with a pipette)

6. Suspend the pellet with 1.0 ml of ice-cold 0.1 mol/ml CaCl₂. Centrifuge at 4 °C (6000r/min) for 1 min, recycle cells

7. Suspend the pellet with 1.0 ml of ice-cold 0.1 mol/ml CaCl₂, immediately put on ice to act for more than 15 min.

8. Centrifuge at 4 °C (6000r/min) for 1 min

9. Aspirate 850 -900 microliters of CaCl₂ solution, and use the remaining 100-150 microliters of ice-cold 0.1mol/ml CaCl₂ to suspend cells (operate on ice). This cell is the competent cell.

Ultrasonic wave breaking cell

1. After induction, the bacterial solution is transferred to the centrifuge tube after balancing at 4000r for 5min (use a fourth-degree centrifuge because heat generation will occur during centrifugation, which will lead to protein denaturation).

2. Discard the supernatant to leave the precipitate and add the same volume of PBS buffer to wash the thalli 1-2 times (shaking can be done).

3. Centrifuge at 4000r for 5min, then discard the supernatant to retain the bacteria.

4.Suspend the thallus with 10ml PBS buffer and add the buffer according to the ratio of 10ml buffer for 1g thallus.

5. Insert the ultrasonic rod and then cover the surrounding with ice water.

6. Set the ultrasonic pulverizer to work at 100-150W for 1s and rest for 2s for 10min at a temperature control of 30°C.

7. Centrifuge at 12000r for 20min at 4°C.

8. 40ul of crude protein solution in supernatant was used as supernatant sample.

Make fecal simulators

1. Material:

Flour: We use red flour, white flour and yellow flour as basic raw materials.

Normal saline :0.9% sodium chloride (NaCl) is isotonic solution, which will not destroy the form of tangible components such as cells and bacteria.

AGAR powder: 0.1g.

Anticoagulant blood: waste blood samples after routine blood determination, 2 ml samples with increased white blood cells are generally selected.

Vegetables:A leaf of spinach，green vegetables or cabbage.

Meat: minced pork or mutton 0.1g.

Flora: Recombinant strains

Liquid medium :0.3% beef extract, 1% peptone, 0.5%NaCl, distilled water.
Mortar: One set.

2. Method:

First, mix the three kinds of flour and make a paste with distilled water, which serves as a substrate. Put the vegetables and minced meat in a mortar, add a small amount of distilled water for grinding, make it into a uniform puree, add AGAR powder, vegetable puree, meat puree in the matrix, stir with bamboo skewer, make evenly mixed in the matrix.

The anticoagulant blood was centrifuged (1 500 r/min, 10min), and an appropriate amount of red blood cell layer was absorbed into the dropper and added to the specimen.

After amplification, the bacterial solution was added to the specimen and mixed well to make the positive simulated specimen for experiment.