Medium

LB Medium

• Yeast Extract 5 g / L

• Sodium Chloride 10 g /L

• Tryptone 10 g / L

• Agar 20g/L(solid)

▲ Dissolve in demineralized water, stir and autoclave.

MRS Medium

• Peptone 10 g / L

• beef extract 10 g / L

• yeast extract 5 g / L

• glucose 5 g / L

• sodium acetate trihydrate 5 g / L

• polysorbate 80 (also known as Tween 80) 1 g / L

• dipotassium hydrogen phosphate 2 g / L

• triammonium citrate 2 g / L

• magnesium sulfate heptahydrate 0.2 g / L

• manganese sulfate tetrahydrate 0.05 g / L

• agar 15g / L

• calcium carbonate 20g / L

▲ Dissolve in demineralized water, stir and autoclave.

Antibiotics

Kanamyicin

Working concentration: 100 μg/ml (E. coli), storage concentration: 100 mg/ml, pro-tected from light, stored at -20°C. Sterilize with filter membrane before use.

Erythromycin

working concentration: 300 μg/ml, storage concentration: 50 mg/ml erythromy-cin-ethanol solution, protected from light, stored at -20°C. Sterilize with a filter membrane before use.

Bacteria Culture

Escherichia coli culture

Solid: the preserved strains/cultured strains were coated/strewed onto the pre-prepared LB solid medium and placed in a 37℃ constant temperature incubator for aerobic static culture for 18h.

Liquid: Inoculate the preserved strain/culture liquid into a shaker tube containing 5 ml pre-prepared LB liquid medium, placed on a constant temperature shaker, and aerobically cultured at 185rpm at 37℃ for 18h.

Lactobacillus plantarum culture

Solid: the preserved strains/cultured strains were coated/strewed onto the pre-prepared MRS solid medium and placed in a 37℃ constant temperature incubator for aerobic static culture for 12h.

Liquid: Inoculate the preserved strain/culture liquid into a shaker tube containing 5 ml pre-prepared MRS liquid medium, placed on a constant temperature shaker, and aerobically cultured at 185rpm at 37℃ for 12h.

Bacteria Inoculation

Liquid to Liquid: Pipette 50μL of culture medium into a shaking tube containing 5 ml of culture medium (diluted at a ratio of 1:100), rinse the tip of the pipette, and discard it.

Bacteria Preservation

Liquid to Solid: 80% glycerol solution and cultured bacterial liquid are added to the same preservation tube at a volume of 1:4, pipetted evenly, and stored in a refrigerator at -80℃.

Bacteria Recovery

Solid to Liquid: Use a pipette tip to pick a single colony into a shaking tube containing 5 ml of culture medium, rinse the pipette tip, and discard it.

Chemical Transformation of Escherichia Coli DH5A

Preparation of competent cells

1. The bacteria to be transformed are cultured in advance to the plateau or late loga-rithmic growth stage. Then the bacterial suspension is inoculated in a 5 ml LB liquid medi-um at a ratio of 1:100-1:50 and cultured with shaking at 37°C 3 hours to OD600 = 0.5-0.7.

2. Transfer 1 ml of culture medium into a 1.5 ml centrifuge tube, place it on ice for 5 minutes and then centrifuge at 4°C and 8000 rpm for 2 minutes.

3. Discard the supernatant, gently suspend the cells in 200 μL of pre-chilled 0.05 mol/L CaCl2 solution, and then place on ice for 10 min.

4. Centrifuge for 2 minutes at 4°C and 8000 rpm. (If there is no need to preserve the seeds, this step can be omitted. )

5. Discard the supernatant, add 200 μL of pre-cooled 0.05 mol/L CaCl2 solution con-taining 15% glycerol, gently suspend the cells, and place on ice for a few minutes to form a competent cell suspension. (If there is no need to preserve the seeds, this step can be omitted) Competent cells can be used for a transformation immediately or stored at -80°C for half a year.

Transformation

1. Take 200μL of competent cell suspension from -80℃ refrigerator and thaw on ice. (If there is no need to preserve the seeds, this step can be omitted.)

2. Add plasmid DNA solution (the volume should not exceed 5 μL, depending on the concentration), shake gently, and place on ice for 20 min.

3. Heat in a 42℃ water bath for 60 seconds, and quickly place on ice to cool for 3 minutes after heat shock.

4. Add 800 μL of LB liquid medium (without antibiotics) to the tube, place it in a shak-er at 37°C, and incubate for 10-30 minutes.

5. Centrifuge at 3000 rpm for 2 minutes, discard part of the supernatant (100-200 μL of the supernatant is best), gently pipette the cells to resuspend them in the supernatant solu-tion.

6. Spread the above-mentioned bacterial liquid on a screening plate containing antibi-otics (place it face up for half an hour, and invert the culture dish after the medium absorbs the bacterial liquid completely ). Then incubate at 37°C for 16-24 hours.

Electric Transformation of Escherichia Coli Nissle 1917

Preparation of competent cells

1. Day 1: Remove the preserved strains from the refrigerator at -80℃, thaw them on ice, streak them on the GM17 solid medium, and cultivate them overnight at 30℃.

2. Day 2: Take a single colony from the plate on Day 1, place it in 100 ml G-SGM17 medium, and cultivate it anaerobic at 30 ℃ until the OD600 is 0.6-0.8.

3. Aliquot into 50 ml centrifuge tubes, pre-cool on ice for 20-30 minutes, and centrifuge at 4°C and 4000 rpm for 10 minutes.

4. Discard the supernatant, add 3 ml of pre-chilled ddH2O, gently blow to suspend the bacterial pellet, dispense it in a 1.5 ml centrifuge tube, and centrifuge at 4°C and 4000 rpm for 10 min.

5. Repeat step (4)

6. Change ddH2O to 10% glycerol and repeat step (4)

7. Repeat step (6)

8. Finally, add 500 μL of 10% glycerol to each centrifuge tube, which can be used for a transformation immediately or stored at -80°C.

Electric Transformation

1. Set the electro-transfer parameters as follows: voltage 2000 V, capacitance 25 μF, re-sistance 200 Ω.

2. After electroporation, immediately add 1 ml of resuscitation medium SGM17MC, mix well and place on ice for 10 minutes.

3. Transfer the solution from the electroshock cup to a 1.5 ml centrifuge tube and incu-bate at 30°C for 2 h anaerobic.

4. Centrifuge at 4000 rpm for 10 minutes, discard part of the supernatant，and spread the plate. Place it on the front side for half an hour.

5. After the bacterial solution is entirely absorbed by the culture medium, place the petri dish upside down. Anaerobic culture at 30°C for 2-3 days.

Electric Transformation of Lactobacillus Plantarum L168

Preparation of competent cells

1. Day 1: Remove the preserved strains from the refrigerator at -80℃, thaw them on ice, streak them on the GM17 solid medium, and cultivate them overnight at 30℃.

2. Day 2: Take a single colony from the plate on Day 1, place it in 100 ml G-SGM17 medium, and cultivate it anaerobic at 30 ℃ until the OD600 is 0.6-0.8.

3. Aliquot into 50 ml centrifuge tubes, pre-cool on ice for 20-30 minutes, and centrifuge at 4°C and 4000 rpm for 10 minutes.

4. Discard the supernatant, add 3 ml of pre-chilled ddH2O, gently blow to suspend the bacterial pellet, dispense it in a 1.5 ml centrifuge tube, and centrifuge at 4°C and 4000 rpm for 10 min.

5. Repeat step (4)

6. Change ddH2O to 10% glycerol and repeat step (4)

7. Repeat step (6)

8. Finally, add 500 μL of 10% glycerol to each centrifuge tube, which can be used for a transformation immediately or stored at -80°C.

Electric Transformation

experimental group: Put 100 μL of competent cells into a pre-cooled 2 mm electric shock cup containing 10 μL of plasmid DNA solution, and place them on ice for 10 min.

control group: Put 100 μL of competent cells into a pre-cooled 2 mm electric shock cup containing 10 μL ddH2O, and place them on ice for 10 minutes.

1. Set the electro-transfer parameters as follows: voltage 2000 V, capacitance 25 μF, re-sistance 200 Ω.

2. After electroporation, immediately add 1 ml of resuscitation medium SGM17MC, mix well and place on ice for 10 minutes.

3. Transfer the solution from the electroshock cup to a 1.5 ml centrifuge tube and incu-bate at 30°C for 2 h anaerobic.

4. Centrifuge at 4000 rpm for 10 minutes, discard part of the supernatant，and spread the plate. Place it on the front side for half an hour.

5. After the bacterial solution is entirely absorbed by the culture medium, place the petri dish upside down. Anaerobic culture at 30°C for 2-3 days.

PCR

System (20μL in total):

r Taq Buffer（10x	2 μL
dNTPs	1.6μl
Pre primer	0.5μL
Back primer	0.5μl
rTap	0.5μl
Template sequence	1.0μl
ddH2O	14.8μl

Reaction procedure:

94℃	5min
90℃	30 s
55℃	30 s
72℃	x min
72℃	8 min
16℃	∞ min

Step2 to Step4 30cycles

Plasmid Amplification

Materials:

• Sterile pipet tips.

• Sterile 1.5 ml Eppendorf tubes.

• Plasmids to amplify.

• DH5alpha, transformation competent. Stored at -80oC. Thaw rapidly immediately before use and keep on ice.

• Agar plates, with appropriate antibiotics. Leave on room temperature bench for 1-2days to get a semi-dry surface. Protect from light.

• LB medium

• SOC medium

Procedure

1. Mix 0.5 - 1 ul plasmids with 0.1 ml competent E. coli in a sterile Eppendorf tube on ice. Mix gently by tapping. Also prepare a no DNA control tube.

2. Incubate on ice for 30 min.

3. Incubate at 42oC for exactly 45 seconds. Use floater in a circulating water bath.

4. Incubate on ice for 2 minutes.

5. Add 900 ul SOC medium, mix by gentle tumbling (optional, for high efficiency transformation to increase the number of transformants).

6. Incubate for 30-60 min at 37oC.

7. Pipet 10 - 300 ul onto an agar plate. For larger volumes, break them into multiple small puddles. Tilt the plate around to make even coverage.

8. Smear the E. coli gently with sterile glass rod (ethanol soaked and flamed) or pipets across the agar surface. Hold the lid over the surface with one hand and rotate the plate to smear in multiple directions.

9. Incubate the plate overnight at 37oC, place upside down to avoid condensation prob-lem.

10. Add 3 ml of LB with antibiotics to a 15 ml conical tube.

11. Select a well-formed, smooth-edged, isolated colony on the agar plate. Do not use colonies with tiny dots around (these may be non-transformed E. coli growing in regions of depleted antibiotics).

12. Pick E. coli from the colony with a sterile toothpick. Dip the tip into the LB medi-um.

13. Place on a shaking incubator at 37o C, 200-250 rpm for 12 hours. The medium should turn cloudy. Store the tube overnight at 4o C if necessary.

14. Plasmid extraction

Plasmid Extraction

Small extraction: See TIANGEN plasmid small extraction kit.

Large-scale extraction based on TIANGEN plasmid large-scale extraction kit.

1. Take 100 mL (choose the appropriate amount according to the concentration of the cultured cells, 200 mL is recommended for a soft copy) overnight cultured bacteria liquid into the centrifuge tube, room temperature 8000 rpm (~8228×g) centrifugation for 3 minutes to collect the bacteria liquid, try to suck Except the supernatant. Note When there is much bacterial liquid, the bacterial pellet can be collected in a centrifuge tube by centrifugation. The amount of bacterial liquid should be sufficiently lysed. Too much bacterial liquid will lead to insufficient lysis and reduce the efficiency of plasmid extraction.

2. Try to absorb the supernatant as much as possible. To ensure that the supernatant is completely absorbed, please use clean absorbent paper to absorb the water droplets on the bottle wall. 3. Add 8 mL of solution P1 (please check whether RNaseA has been added) to the centrifuge tube with bacterial pellet, and use a pipette or vortex to lyse the suspended bacterial cell pellet completely.

Note

Be sure to suspend the bacterial sediment thoroughly. If some bacteria are clumps that are not thoroughly mixed, it will affect the lysis effect, resulting in low extraction vol-ume and purity. For low-copy plasmids, increase the number of bacteria while increasing the proportion of P1, P2. The amount of P4.

3. Add 8 mL of solution P2 to the centrifuge tube, turn it upside down gently 6-8 times, and leave it at room temperature for 5 minutes.

Note

Mix gently, do not shake vigorously to avoid contamination of genomic DNA. At this time, the bacterial liquid should become transparent and viscous. If it does not become apparent, it may be due to too much bacteria and incomplete lysis, so fewer bacteria should be reduced.

4. Add 8 mL of solution P4 to the centrifuge tube, and immediately turn it upside down gently 6-8 times, mix well, until the solution appears white dispersed flocculent precipitate. Then leave it at room temperature for about 10 minutes. Centrifuge at 8000 rpm (~8228×g) for 5-10 min to allow the white precipitate to separate to the bottom of the tube (the centrif-ugation time can be increased appropriately), and carefully pour all the solution into the fil-ter CS1 (please avoid pouring a large amount of precipitate to block the filter ), slowly push the push handle to filter, and collect the filtrate in a clean 50 mL tube (provided by yourself).

Note

Mix the solution immediately after adding solution P4 to avoid local precipita-tion. If the solution poured into the filter CS1 after centrifugation has white precipitation, it would not affect the filtration. If there are too many bacteria (>100 mL), it is recommended to extend the centrifugation time to 20-30min.

5. Add isopropanol equal to the filtrate volume to the filtrate, mix upside down, and centrifuge at 8000 rpm (~8228×g) for 5 min.

6. Pour and discard the supernatant. Take care to prevent the precipitate from being poured out. Add 6 mL of 70% ethanol to wash the precipitate. Centrifuge at 8000 rpm (~8228×g) at room temperature for 5 min. Carefully discard the ethanol. Repeat the opera-tion once.

7. Leave the lid open in the air for 5-10 minutes, and dissolve the precipitate with an appropriate volume of ddH2O as needed.

Enzyme Digestion

Materials:

• Plasmid DNA: x μL (according to the actual concentration)

• Restriction enzyme 1: 1μL

• Restriction enzyme 2: 1μL

• 10×buffer　: 2μL

• ddH₂O: 20-x μL

Protocol:

1. The total system is 20 μL, add samples and mix well, 37℃ water bath for 1h-3h, electrophoresis verification.

Agarose Gel Electrophoresis

Materials:

• An electrophoresis chamber and power supply.

• Gel casting trays, which are available in a variety of sizes and composed of UV-transparent plastic.

• Sample combs, around which molten agarose is poured to form sample wells in the gel.

• 1X TAE solution

Protocol:

1. Dissolve 0.3 g of agarose in 30 mL of 1X TAE solution, heat it until the bubbles stop, and the solution becomes transparent, cool slightly and add 3 μL Goldview (10000x)

2. Pour the gel into the gel membrane tool inserted with the comb (the sample amount determines the specific size), and wait for it to set.

3. Add 1/2/3 μL 6× loading buffer to the 5/10/15μL sample and mix by pipetting.

4. Put the gel into the electrophoresis tank and add 1X TAE buffer to completely cover the gel and squeeze out the bubbles in the gel hole.

5. Add the mixed sample and marker to the gel hole. Specific experiments deter-mine the sample loading volume. The marker loading volume is based on the following standards， 11 wells gel corresponds to 2 μL, 8 wells correspond to 5 μL, and 6 wells corresponds to 10 μL.

6. Run electrophoresis under 180V voltage until the colour band of loading buffer is electrophoresed to the middle or two-thirds of the gel, stop electrophoresis.

7. Use ultraviolet light to image the gel. If necessary, cut rubber for recycling.

SDS-Page

Materials:

• Gels

• Electrophoresis Chambers

• Protein Samples

• Running Buffer

• Staining and Destaining Buffer

• Protein Ladder

Protocol

1. When the gel is ready to be put, add TEMED.

2. The gel used to separate is poured in the casting chamber.

3. You need to put butanol before polymerization to remove the unwanted air.

4. bubbles present.

5. Between the glass plate, the comb is inserted.

6. The “gel cassette” is the polymerized gel.

7. Sample Preparation

8. Boil some water in a beaker.

9. Add 2-mercaptoethanol to the sample buffer.

10. Put the buffer solution in microcentrifuge tubes and add protein samples to it.

11. Take MW markers in separate tubes.

12. Bring the samples to boil for less than 5 minutes to completely denature the proteins.

13. The gel cassette is put off from the casting stand and placed separately in the electrode assembly.

14. The clamp stand is fixed to the electrode assembly.

15. a 1X electrophoresis buffer is added to the opening of the casting frame to fill the wells of the gel.

16. Fit 30ml of the denatured sample in the well.

17. The tank is covered with a lid and the unit is attached to a power supply.

18. The sample is made to run at 30mA for about 1 hour.

19. UV light is used to observe the light.

Test of The Death of Bacteria

Materials:

• Engineering bacteria

• LB broth

• Incubator of 37 °C

• LB agar plates containing 50 μg/mL antibiotics of choice

• Shaking incubator of 37 °C

• Three concentrations of Arabinose LB solution: 0umol/L, 1.25umol/L, 2.5umol/L

Protocol

1. Distribute EcN 1917-pet28a(control) & EcN 1917-pet28a-mi126a into three parts respectively, each 10 ml.

2. Check the OD600 value. Adjust it to 0.3.

3. Centrifuge at 1500 rpm at 4℃ for 10 minutes.

4. Resuspend the precipitation with three concentrations of Arabinose LB solution

5. Each concentration is distributed into 10 eppendorfs, each 1 ml.

6. Cultured in all the bacteria in a shaker at 37 ℃.

To the three concentrations of the Arabinose LB solution cultured bacteria, every round we pick out a eppendorf to coat the plate. Take one of the treatment of the example:

7. After a thorough mixing, take 100ul to 900ul ddH2O. Mix thoroughly.

8. Repeat step 1 for 8 times, that is dilute original sample for 10－9.

9. Add 200ul to solid medium, use a sterile coater for coating, to ensure the full coating plate.

10. For each concertation, repeat the same procedure 2 times.

At the end of the round, a total of 9 coating plates is done. Place them in a 37 ℃ incubator, and counting CFU after 24h.

Test of Heavy Metal Eliminate Effection

Equipment

• Atomic absorption photometer

Protocol

1. Prepare EcN 1917 bacterial solution without plasmid and 1917-pEt28a-MT bacterial solution for overnight culture in advance.

2. Prepare eight 1.5ml EP tubes. Dilute the bacterial solution in each EP tube with LB at a ratio of 1:9, add corresponding amount of Pb2+, and culture it in a shaker at 37 ℃. The composition formula and test time of each EP tube are as follows:

Tube 1: The concentration of divalent lead ion is 112mg/L, add 1917-pEt28a-MT bacterial solution, culture for 4h, and take it out

Tube 2: The concentration of divalent lead ion is 112mg/L, add 1917 bacterial solution not transferred into plasmid, culture for 4h, and take it out

Tube 3: The concentration of divalent lead ion is 112mg/L, add 1917-pEt28a-MT bacterial solution, culture for 4h, and take it out

Tube 4: The concentration of divalent lead ion is 112mg/L, add 1917 bacterial solution not transferred into plasmid, culture for 4h, and take it out

Tube 5: The concentration of divalent lead ion is 112mg/L, add 1917-pEt28a-MT bacterial solution, and take it out after 2h of culture

Tube 6: The concentration of divalent lead ion is 112mg/L, add 1917 bacterial solution not transferred into plasmid, culture for 2h and take it out

Tube 7: The concentration of divalent lead ion is 112mg/L, add 1917-pEt28a-MT bacterial solution, and take it out after 2h of culture

Tube 8: The concentration of divalent lead ion is 112mg/L, add 1917 bacterial solution not transferred into plasmid, culture for 2h and take it out

3. Centrifuge the above 8 EP pipes at 12000 rpm

4. Take 40uL of supernatant from each of the above ep tubes and add it into the corresponding glass tube, then add 1ml of 65% concentrated nitric acid for overnight digestion, and add 9ml of deionized water the next day to adjust the acidity to about 5% - 7%.

5. Finally, each sample is 10ml, and a total of 18 samples are packed in a glass tube. The maximum concentration of divalent lead ion does not exceed 448ug/L

6. Use graphite furnace atomic absorption spectrometry to determine the content of Pb2+in the sample. The results are as follows:

7. According to the initial concentration of each tube, the data can be analyzed to calculate the absorption ratio

Culture of Lactobacillus plantarum

Lactobacillus plantarum strain 200μl was mixed with 15ml MRS broth and incubated at 37°C for 14 hours. Then, the tubes containing MRS broth and Lactobacillus plantarum were centrifuged at 4000 rpm/min for 15 min, the supernatant was removed, washed with PBS, centrifuged, and repeated three times.

Microencapsulation and encapsulation of Lactobacillus plantarum

In the present study, Lactobacillus plantarum was microencapsulated in sodium alginate and coated with whey protein.

1. First, solutions of sodium alginate (1% w/v), whey protein (5.5% w/v) and calcium chloride (0.1 M) were prepared separately and sterilized at 121°C for 15 min.

2. Microencapsulation of Lactobacillus acidophilus was carried out by the extrusion method. One milliliter of Lactobacillus acidophilus suspension (containing 6.21 × 108 CFU/mL bacteria) was added to 5 mL of sodium alginate solution and gently stirred at 500 RPM for 30 minutes.

3. The suspension obtained was then added to a calcium chloride solution (0.1 M) using a sterile nozzle syringe to form bacterial microcapsules. The obtained microcapsules were isolated by centrifugation (3,700 g, 5 min) and washed twice with distilled water.

4. Afterwards, the microcapsules were immersed in 5 ml of coating solution (i.e. whey protein, stirred at 500 RPM for 30 min, transferred to a sterile glass container and placed in a freezer (-80°C, 2 h).

5. Finally, dry in a vacuum freeze dryer at -47°C for 48 hours. Collect the coated microcapsule powder and store in the refrigerator (4°C).

Microencapsulation yield calculation

To determine the encapsulation rate, the number of Lactobacillus acidophilus in the encapsulated microcapsules and bacterial suspension was calculated.

1. First, 0.1 g of microencapsulated and/or coated Lactobacillus acidophilus was added to 0.9 ml of sterile sodium citrate (2% w/v, pH 7) and stirred for 5 min until the capsule was completely dissolved and the bacteria were released.

2. Next, bacterial serial dilutions were prepared and incubated by decantation on plates containing MRS agar medium, which were subsequently placed in an incubator at 37°C for 48 hours and the bacterial counts were counted.

3. Then, the microencapsulation yield was calculated as follows: microencapsulation rate (%) = (N / N$_0$ ) × 100%.

N is the number of bacteria released after microencapsulation (log CFU/g) and N$_0$ is the number of bacteria in the suspension before microencapsulation and coating (log CFU/g).

Preparation of simulated gastric and intestinal fluids

Simulated gastric juice (SGI) was prepared by adding 0.3 g of pepsin to 100 ml of NaCl solution (0.2% w/v) and adjusting the pH to 2.0 using 0.1 N HCl). Simulated intestinal fluid was prepared by suspending trypsin (0.1% w/v) and bile salts (0.45% w/v) in sodium citrate (2%) and adjusting the pH to 7.4 using 0.1 N NaOH (Fazilah). both SGJ and SIJ were sterilized by membrane filtration.

Viability of Lactobacillus plantarum lyophilized powder after exposure to simulated gastrointestinal conditions

1. First, a sample of Lactobacillus plantarum lyophilized powder 0.1 g stored in the refrigerator was placed in a tube containing 0.9 ml SGJ and incubated at 37°C for 120 min.

2. Next, 1 ml of SIJ was added and then incubated at 37°C for 300 min. Finally, the number of Lactobacillus acidophilus was counted as described above.

E. coli operates in the same way as Lactobacillus plantarum

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