Experiments

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Prepare 200ml medium, weigh 2g of tryptone, 1g of yeast extract, 2g of NaCl, 3g of agar powder in a beaker, add 200ml of purified water, and stir with a glass rod until the solutes are dissolved. Adjust the pH to 7.0~7.4 with 5mol/L NaOH (usually 40μl), and then dispense the mixture into a 250ml conical flask. Seal the flask with sealing film, and then wrap it with double-layered kraft paper. Then steam-sterilize it under high pressure for 20 min at 121℃, 103.4kPa. Take it out and put it on the ultra-clean bench and cool it down to 50~55℃, and then add the antibiotic (kanamycin) to reach the final working concentration of 50μg/ml.

1. Take an ice box, put dNTP, prime star enzyme, 5X prime star buffer, upstream and downstream primers, template plasmid, double distilled water, into the ice box and wait for melting. Take the corresponding number of microtubules and put them into the ice box.

2. Configure the reaction system as required by primestar. Note the amount of template used (concentration determines volume)

Total volume: 50μL

Volume Final concentration
5 X PrimeSTAR Buffer (Mg2+Plus) 10μl 1 X
dNTP Mixture (2.5mM each) 4μl 200μM each
Primer1 10-15pmol 0.2-0.3μM
Primer2 10-15pmol 0.2-0.3μM
Template <200ng
PrimeSTAR HS DNA Polymerase (2.5U/μl) 0.5ul 1.25units/50μl
DdH2O Up to 50 μl

3. Turn on the PCR instrument and set up the program according to the reaction conditions.

Gut-sweeper

1. Gel dispensing: weigh 0.35g agar powder, add 30ml 1X TAE into the flask, microwave heat for 2.5 min, then cool it down to 50℃. Take gelred from 4 degrees refrigerator, aspirate 3ul with a special gun for electrophoresis and add it with the gun tip sticking out under the liquid surface, rotate it slightly to mix well. Pour the liquid into the grid for dispensing gel, insert the comb and wait for solidification.

2. Sample preparation: After PCR, take out each reaction tube, take 5μl from each tube, add each into a new microtube, label it well, add 1ul 6X loading buffer to each tube, mix well.

3. Run the gel: take out the Marker from the -20℃ refrigerator, pull out the comb from the solidified gel, put it into the electrophoresis tank full of 1X TAE according to the direction, add the prepared samples into each gel well, and add 5ul of melted Marker, record the order of the samples in the wells. Run the gel at 150V constant voltage.

4. Observe under UV light and compare the position of the fluorescent bands with the Marker instructions.

5. Take Photographs.

1. Column equilibration: Add 500μl of equilibrium solution BL to the adsorption column CB2 (adsorption column in the collection tube) and centrifuge for 1 min at 12,000 rpm.

Centrifuge the column for 1 min, pour out the waste liquid from the collection tube and put the column back into the collection tube.

2. Cut a single target DNA strip from the agarose gel (remove as much excess as possible) into a clean centrifuge tube

3. Add an equal volume of solution PC to the gel block (if the gel weighs 0.1g and its volume can be considered as 100pl, add 100μl of PC solution. Use a gel cutter to cut 1% agarose gel, the weight of a single block is about 0.06g, the actual weight of the gel block is related to the concentration and thickness of the gel), water bath at 50℃ for about 10 min, during which time the centrifuge tube is constantly and gently up and down to ensure that the gel block is fully dissolved.

4. Add the solution obtained in the previous step to an adsorbent column CB2 (adsorbent column in a collection tube), centrifuge at 12,000rpm (~13,400xg) for 1 min, pour off the waste liquid in the collection tube, and place the adsorbent column CB2 in a collection tube.

5. Add 600μl of rinse solution PW to the adsorbent column CB2, centrifuge at 12,000rpm for 1 min, pour off the waste liquid in the collection tube, and place the adsorbent column CB2 into the collection tube.

6. Repeat step 5

7. Place the column CB2 in a collection tube and centrifuge at 12,000rpm for 2 min to remove as much rinse solution as possible. Leave the column at room temperature for a few minutes and dry thoroughly.

Note: Ethanol residue in the rinse solution will affect subsequent enzyme reaction (digestion, PCR, etc.) experiments.

8. Place the column CB2 into a clean centrifuge tube and add an appropriate amount of Elution Buffer EB to the center of the adsorbent membrane (if the recovered target fragment is >4kb, the Elution Buffer EB should be warmed up in a water bath at 65-70°C) and leave it at room temperature for 2 min. Centrifuge the column at 12,000rpm for 2 min and collect the DNA solution.

1. The enzymatic reaction system is as follows:

Reaction System Composition Volumes
DdH2O Up to 50μl
10x NE Buffer(cutsmart) 5μl
HindIII 1μl(Ensure that the glycerol concentration does not exceed 5% of the total reaction solution)
EcoRI 1μl
Plasmid or PCR recovery product 1μg (Adjust the volume according to the concentration)

2. Put into the water bath and react at 37℃ for 15 min

3. Inactivate at 65℃ for 20 min, or use DNA Purification Recovery Kit for the recovery of the digested product.

1. The molar ratio of carrier to fragment is approximately 1:3~4. Calculate the desired volume ratio based on concentration and fragment length.

2. Prepare the following reaction system on ice:

Reaction System Composition Volumes
Solution l The sum of the volumes used for the carrier and the fragment, the 4~5X
Plasmid gel recovery products X
PCR digestion of recovered products 3~4X

3. The reaction solution is collected to the bottom of the tube by brief centrifugation. React for 120 min at 16°C and may be repeat overnight at 4°C.

1. Take the cloned receptor cells from the -80°C refrigerator and thaw them on ice.

2. Take 2-5μl of recombinant product and add it to 50μl of receptor cells, flick a few times to mix, ice bath for 30 min.

3. Heat-excite in 42℃ water bath for 30-45s.

4. Immediately place on ice to cool for 2-3 min.

5. Add 500μl LB medium (without antibiotics) and shake the bacteria for 1h at 37°C, 180rpm.

6. Preheat Amp-resistant LB plate solid medium in a 37°C incubator.

7. Centrifuge at 12000rpm for 1 min and discard 200-400μl of supernatant. Resuspend the bacteria with the remaining medium and spread it gently on the plate with a pasteurized tube.

8. Invert the culture in 37℃ incubator for 12-16h (first positive for 5 min to evaporate the water, then invert the culture).

1. Turn on the UV light of the aseptic table 10 minutes in advance. (Place the alcohol-wiped guns to be used into the table together).

2. Turn off the UV light and turn on the fluorescent light, wipe the table with alcohol and spray alcohol in the air, light the alcohol lamp and turn on the first level of air.

3. Put all items into the table and sterilize hands with alcohol.

4. Aspirate 8.3μl of kanamycin with a 10ul gun. Hold the gun in the right hand, take the test tube of LB liquid in the left hand, bake the mouth of the tube with the alcohol lamp fire for 2-3 seconds, remove the stopper with the right little finger and palm, tilt the test tube, hit the Amp into the inner wall of the mouth of the tube, and parallel the test tube so that the kanamycin will be immersed in the LB. Alcohol lamp flame grilled the mouth of the tube and plugged back the stopper.

5. Do the same for the other tubes.

6. Burn tweezers, take a white tip, open the lid of the plate, pick a single larger colony, the tip of the gun can be stained with bacteria.

7. Hold the forceps in the right hand and hold the tip of the gun with bacteria, take the test tube of LB liquid in the left hand, bake the mouth of the tube with alcohol lamp for 2-3 seconds, use the little finger and palm of the right hand to pull out the stopper and put the tip of the gun into the test tube. Alcohol lamp fire grilled the mouth of the tube and stuffed back the stopper.

8. Do the same operation for the other tubes.

9. When all tubes are done, put them together in a shaker, at 180rpm at 37℃ for 16-18h.

(i) Protein sample preparation

1. Amplify the bacteria.

2. Discard medium and rinse.

3. Add lysis solution to lysate for 10 min (operated on ice), transfer to Ep tube and continue lysis for 20 min.

4. Centrifuge at 4℃ at 14000rpm for 15 min and remove the supernatant.

(ii) SDS-PAGE electrophoresis

1. Washing the glass plate

2. Filling and Sampling

a) The glass panes are aligned and placed in the clamps and stuck tightly. After leak checking, clamp the glass plate vertically on the rack and prepare for filling.

b) Dispense 10% Separation Gel, add TEMED and shake well immediately before filling. When filling, use a 10ml gun to draw 5ml of glue along the glass, and wait until the surface of the glue rises to the height of the center line of the green band. Then add a layer of water to the glue.

c) When there is a line of refraction between the water and the glue, it means the glue has solidified. Wait for another 3 minutes for the glue to fully solidify, pour off the water on the layer of glue and absorb the water with blotting paper.

d) Prepare 5% gel concentrate, add TEMED and shake immediately to fill the gel. Fill the remaining space with concentrate and insert the comb into the concentrate. After the gel concentrate has solidified, gently pull out the comb by squeezing each side of the comb with both hands and holding it vertically upwards.

e) Rinse the gel concentrate with water and put it into the electrophoresis tank.

f) Add 1×Tris-glycine to the upper and lower tanks of the electrophoresis tank and top sample.

g) Connect the electrophoresis tank to the electrophoresis instrument. The upper tank is connected to the negative pole, the lower tank is connected to the positive pole, energized with a starting voltage of 80 V. When the bromochromophenol blue indicator reaches the junction of the separated gel and the concentrated gel, switch to a constant voltage of 120 V, and stop electrophoresis after running to the bottom layer.

(iii) Transferring the membrane

1. Immerse the gel in transfer buffer and equilibrate for 10 min.

2. According to the size of the glue cut PVDF membrane and filter paper 6 pieces, into the transfer buffer equilibrium 10 min. (PVDF membrane need to be soaked in pure methanol first 1 min).

3. Assemble the transfer sandwich: sponge 3 layers of filter paper film, 3 layers of filter paper sponge, after each layer is placed, use a test tube to drive away the air bubbles. The gel is placed on the negative side (black side).

4. Place the transfer bath in an ice bath, put sandwiches (black side to black side), add transfer buffer, insert electrodes, 100V, 2h (current 0.25A).

5. After transferring the membrane, cut off the power and remove the hybridization membrane.

(iv) Confinement and hybridization

1. Prepare 5% skimmed milk powder with TBST as solvent.

2. Soak the membrane in 5% skimmed milk powder, shake it at low speed on a shaker, seal it for 2h, discard 5% skimmed milk powder, shake it at high speed on a shaker with TBST and wash it for 5 min, repeat three times.

3. Primary antibody incubation: add primary antibody and incubate at 4℃ overnight.

4. Secondary antibody incubation: Recover primary antibody, wash with TBST for 15 min, repeat three times. Add the corresponding secondary antibody, incubate on shaking table at room temperature for 2h, wash with TBST for 15 min, repeat three times.

1. Take 1-4ml of the overnight culture, add it to a centrifuge tube, and centrifuge it for 1 min at 12,000 rpm using a conventional tabletop centrifuge, aspirating the supernatant as much as possible.

2. Add 150μl of Solution P1 to the centrifuge tube with the bacterial precipitate, and use a pipette or vortex shaker to thoroughly suspend the bacterial precipitate.

3. Add 150pl of Solution P2 to the centrifuge tube and gently turn it up and down 6-8 times to fully lyse the organisms.

Due to the use of TIANRed, the color of the solution will be a clear purple after the addition of solution P2 and thorough mixing. If there is a cloudy red color mixed with the purple color, the lysis is insufficient. Continue mixing until the color of the solution has completely changed to a clear purple.

4. Add 350μl of Solution P5 to the centrifuge tube and immediately and rapidly mix up and down 12-20 times, after mixing well enough, a flocclent precipitate will appear. Centrifuge at 12,000rpm (~13,400xg) for 2 min.

5. Transfer the supernatant collected in the previous step into the adsorbent column CP3 with a pipette (the adsorbent column is placed into the collection tube), taking care to try not to aspirate the precipitate. centrifuge at 12,000 rpm (~13,400xg) for 30 sec, pour off the waste liquid in the collection tube, and place the adsorbent column CP3 into the collection tube.

6. Add 300μl of rinse solution PWT to the adsorbent column CP3, centrifuge at 12,000rpm (~13,400xg) for 30 sec, pour off the waste liquid in the collection tube.

7. Place the adsorbent column CP3 into the collection tube and centrifuge at 12,000rpm for 1 min, in order to remove the residual rinse solution from the adsorbent column.

8. Place the adsorbent column CP3 in a clean centrifuge tube, add 50-100μl of elution buffer TB dropwise to the middle part of the adsorbent membrane, and centrifuge at 12,000rpm for 30 sec to collect the plasmid solution into the centrifuge tube.

(i) Preparation of initial liquid culture of bacteria

1. Prepare liquid LB medium, then autoclave (120°C, 41 min) and cool to room temperature.

2. Inoculate individual colonies of bacterial strains into 5ml of LB medium in culture tubes with kanamycin.

3. Note: If more replicates are needed, several colonies in separate culture tubes can be used.

(ii) Pretreatment of 96-well plate and lid

1. Prepare 10% Triton X-100 in ethanol by mixing 1ml of Triton X-100 with 9ml of ethanol.

2. Pour 10ml of the diluted Triton X-100 (10% Triton X-100) to a new microplate lid.

3. Incubate the lid with diluted Triton X-100 for 15 seconds at room temperature.

4. Take a new 96-well plate and fill each well with 100% ethanol (400μl per well). Incubate for 15 minutes at room temperature.

5. Discard ethanol from the microplate wells and diluted Triton X-100 from the microplate lid.

6. Air-dry the microplate wells and microplate lid in a biosafety cabinet for 30 minutes.

7. Turn on the UV light of the biosafety cabinet to further sterilize the microplate wells and lid for 15 minutes.

(iii) Sample Testing

Plasmids Sodium butyrate (mM) Bacteria solution(μl) Total volume (μl) Replicates Resistance
Pet-28a (sensor1) 20 1 200 3 KN+CM
Pet-28a (sensor1) 10 1 200 3 KN+CM
Pet-28a (sensor1) 0 1 200 3 KN+CM
Gut-sweeper
Plasmids Sodium butyrate (mM) Bacteria solution(μl) Total volume (μl) Replicates Resistance
Pet-28a (sensor2) 20 1 200 3 KN+CM
Pet-28a (sensor2) 10 1 200 3 KN+CM
Pet-28a (sensor2) 5 1 200 3 KN+CM
Pet-28a (sensor2) 0 1 200 3 KN+CM
Gut-sweeper

(iv) Enzyme marker and software setup

1. Turn on the enzyme marker and open the Gen5 software.

2. Adjust the parameters and measure the fluorescence intensity and bacterial growth curve.

a. Plate type: 96-well plate.

b. Lid using: checked.

c. Temperature: 37°C.

d. Shake: orbital for 0:10.

e. Kinetic measurement for a desired total time (e.g., 16h, 24h, or 48h) and interval of 10 min with continuous orbital shaking.

f. Reading: absorption at 600 nm, fluorescence at excitation = 488 nm and emission = 525 nm.

(v) Collating results

1. After completing data collection, remove the 96-well plate from the enzyme labeler.

2. Sterilize the sample and discard the microplate properly.

3. Save the results in the Gen5 software and export the data to an Excel spreadsheet file.

4. Close the Gen5 software and then turn off the enzyme marker.

1. Configure different concentrations of L-malic acid solution according to the effect concentration from previous literature: 25μM, 50μM, 75μM.

2. Dilute the bacterial solution with LB solid medium cooled to about 55°C at a ratio of 1:10, mix thoroughly and take 15ml into a sterile plate, shake gently, solidify and make a plate

3. Gently put the sterile Oxford cups into the previously made plates respectively, and then use a sterile pipette to suck 200μl of malic acid solution of different concentrations into the Oxford cups, and add 200μl of malic acid-free solution as the blank control. Three replicates were performed for each group.

4. Incubate the plates overnight at the temperature of around 37°C and measure the diameters using a circle of inhibition meter.

(i) Preparation of initial liquid culture of bacteria

1. Prepare liquid LB medium, then autoclave (120°C, 41 min) and cool to room temperature.

2. Inoculate individual colonies of bacterial strains into 5ml of LB medium in culture tubes with kanamycin.

3. Note: If more replicates are needed, several colonies in separate culture tubes can be used.

(ii) Pretreatment of 96-well plate and lid

1. Prepare 10% Triton X-100 in ethanol by mixing 1ml of Triton X-100 with 9ml of ethanol.

2. Pour 10ml of the diluted Triton X-100 (10% Triton X-100) to a new microplate lid.

3. Incubate the lid with diluted Triton X-100 for 15 seconds at room temperature.

4. Take a new 96-well plate and fill each well with 100% ethanol (400μL per well). Incubate for 15 minutes at room temperature.

5. Discard ethanol from the microplate wells and diluted Triton X-100 from the microplate lid.

6. Air-dry the microplate wells and microplate lid in a biosafety cabinet for 30 minutes.

7. Turn on the UV light of the biosafety cabinet to further sterilize the microplate wells and lid for 15 minutes.

(iii) Sample Testing

L-malic acid(mM) Mother liquor(μl) Bacteria solution(μl) Total volume (μl) Replicates Resistance
0 0 200 200 3 KN
25 5 195 200 3 KN
50 10 190 200 3 KN
75 15 185 200 3 KN

Mother liquor: 1M L-malic acid

Rhamnose(mM) Mother liquor(μl) Bacteria solution(μl) Total volume (μl) Replicates Resistance
0 0 200 200 3 KN
5 1 199 200 3 KN
10 2 198 200 3 KN
15 3 197 200 3 KN

Mother liquor: 1M Rhamnose


(iv) Enzyme marker and software setup

1. Turn on the enzyme marker and open the Gen5 software.

2. Adjust the parameters and measure bacterial growth curves.

a. Plate type: 96-well plate.

b. Lid using: checked.

c. Temperature: 37°C.

d. Shake: orbital for 0:10.

e. Kinetic measurement for a desired total time (e.g., 16h, 24h, or 48h) and interval of 10 min with continuous orbital shaking.

f. Reading: absorption at 600 nm

(v) Collating results

1. After completing data collection, remove the 96-well plate from the enzyme labeler.

2. Sterilize the sample and discard the microplate properly.

3. Save the results in the Gen5 software and export the data to an Excel spreadsheet file.

4. Close the Gen5 software and then turn off the enzyme marker.

1. Prepare sufficient solid plate containing kanamycin.

2. Inoculate the engineering bacteria into 5ml LB liquid medium in the shaker at the temperature of around 37°C and take it out when the OD value is around 0.4-0.5.

3. Add a certain amount of malic acid into 5ml of LB liquid medium, making the concentration of malic acid to be 25mM, 50mM, 75mM. Continue to culture them in the shaker at the temperature of around 37°C.

4. Every 2h, spread the plate until the 12h, a total of 6 time points. Pre-measurement of OD value can be used to roughly determine dilution gradient. Each group contains three replicates.

5. Plate clony can be counted after 12-16h of incubation, and the counting is done by jingdian camera.

6. Analyze and process the data, and draw the killing curves using graphpad.