Agarose gel electrophoresis
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 determine 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.
Polyacrylamide gel electrophoresis
1. Prepare running buffer: Dissolve 15.1 g Tris and 94 g glycine in 900 mL deionized water, add 50 ml 10% SDS, and then dilute to 1 L with deionized water.
2. Add 5x SDS loading buffer to the protein sample and control its volume fraction in the mixed solution to 20%.
3. Take out the Meilunbio® Gel protein precast gel, and then fix the precast gel in the electrophoresis tank. Add running buffer solution to check for leaks.
4. Fill the inner tank of the electrophoresis tank with electrophoresis buffer, and add the electrophoresis solution to the corresponding level in the outer tank, and the maximum shall not overflow the inner tank. Take out the comb, use a syringe or other tools to suck the electrophoresis solution, and then gently blow the sample well to remove the remaining storage buffer and impurities in the sample well.
5. Add sample: Use conventional 1X loading buffer to process the sample. After aspirating the sample with the pipette, insert the pipette tip into the sample hole vertically to add the sample. Be careful not to pierce the gel with the pipette tip, and do not over-insert the comb hole to deform the rubber plate and cause liquid leakage.
6. Electrophoresis conditions: 110 V. When the bromophenol blue indicator is electrophoresed at the bottom of the gel or a predetermined position in the experiment, the electrophoresis can be ended.
7. After the electrophoresis is over, remove the gel. Use a blade to cut the glue on both sides along the gap between the short glass and the edge strip, open the glass plate to take out the gel. 8. After staining with Coomassie Brilliant Blue for 3 hours, elute with eluent. Eluent configuration 200 mL ethanol 300 mL acetic acid 500 mL water (volumes of ethanol and acetic acid are variable)
2. Prepare electro-transfer solution (prepared during electrophoresis, need to be placed in the refrigerator to pre-cool) or buy directly
3. Cut the fibre membrane and cut off a corner, put it in methanol for activation, carefully cut the required fragments with a rubber cutting board (compared to maker). This step needs to be soaked in electro transmission fluid.
4. Cut a small piece at the corner of the gel to recognize the order of sample addition.
5. Assemble the sandwich structure: sponge-filter paper-fibre membrane-sample glue-filter paper-sponge (black below).
6. Connect the membrane to the positive electrode and transfer it for 90 minutes at 110 A on ice (4°C).
7. Dilute the 10x blocking solution to 1x (4℃), dilute 10xTBS to 1x and add 2mL Tween (cut off the tip with scissors).
8. Take out the fibre membrane, discard the gel, soak it in the blocking solution and place it on a shaker for 15-20 minutes.
9. Wash with TBST two to three times, once for 10 minutes.
10. The primary antibody (stored in a refrigerator at 4°C) was diluted 1:10000 with TBST, put the membrane into it, and incubated overnight at 4°C on a shaker.
11. Take out the membrane and wash it three times with TBST for 10 minutes each time.
12. Dilute the secondary antibody (stored in a refrigerator at 4°C), dilute TBST 1:10000, put the membrane into it, and incubate at room temperature for 2 hours.
13. Wash three times with TBST and prepare for development.
Add 1/4 volume to get methanol when used
Adjust pH to 7.6 with HCl and dilute to 1L
3. TBST buffer
|20% Tween-20||1.65m L|
4. Blocking buffer
Dilute 10×TBS to 1×, add 5% skimmed milk, 0.1% Tween-20, and store at 4°C.
1. Dilute the cultured strains into an LB liquid medium containing the corresponding antibiotics at a ratio of 1:100,
and place them in a shaker at 37°C and 185rpm for overnight culture.
2. Divide the cultured bacteria liquid into 50 mL shake tubes and centrifuge at 4000 rpm for 10 min at 4°C.
3. Suspend the cells by pipetting with 3mL 1×PBS solution.
4. Use repeated freezing and thawing methods to lyse the cells.
5. After centrifugation at 14000 rpm for 15 minutes, collect the supernatant to obtain a protein sample.
According to Beyotime His-tag protein purification kit，Beyotime GST-tag protein purification kit， Biolab His-tag protein purification kit (inclusion body protein) kit.
Based on E. coli
Triclosan gradient settings: 0, 0.1625, 0.325, 0.625, 1.25, 2.5, 5, 10 μM/mL
1. Prepare LB is containing 20 μM/mL triclosan, LB containing 100 μg/mL Cana, LB contains 100 μg/mL ampicillin, and LB without anti-antibody. Cultivate for 24h to plateau.
2. Re-inoculate bacteria, press 1:100 volume, culture until od is between 0.7 and 0.8.
3. Prepare 10 shaking tubes for each type of bacteria, add 1 mL of anti-LB to the first eight tubes, and then add 1 mL of LB containing 20 μM/mL triclosan to the first tube, and mix thoroughly by pipetting and aspirating. , Suck 1 mL of LB liquid in the tube, add it to the second tube, suck and mix thoroughly, then aspirate the LB liquid in the second tube, add it to the third tube, and so on to the seventh tube. Finally, there should be 2 mL LB in the seventh tube, 1 mL must be discarded, and the 7 is 1 mL. Add 1 mL of LB containing 100 mg/mL ampicillin to the ninth tube, 1 mL of LB containing 100 μg/mL Kana to the tenth tube, and 2 mL of LB without anti-antibody to the eleventh tube.
4. Add 1 mL of the bacterial solution to the first to tenth tubes. Finally, it is guaranteed that all eleven tubes contain 2 mL LB. Measure the OD of each tube immediately, measure the OD once every 2 hours, and measure continuously for 12 hours. Using a microspectrophotometer, only take 2 μL for each measurement. Take the first OD as 100% and subtract the OD of the blank control to draw a growth curve.
5. According to the set triclosan gradient, configure different concentrations of triclosan-resistant solid LB medium, coat and cultivate the bacteria, and observe the growth of the bacteria after 24 hours.
1. Prepare LB plates under non-resistant, 0.625μM/mL triclosan concentration, 1.25μM/mL triclosan concentration.
2. Spread the bacterial liquid in the early stage of logarithmic growth on the culture medium, and control the bacterial concentration of the two groups to be about 0.4 to keep the same.
3. Place it in a constant temperature incubator at 37°C and incubate for 18 hours, and observe the plate results.
HSP stress resistance test
1. Temperature: Connect the engineered bacteria containing the HSP gene and the control bacteria to each of 15 tubes of medium containing 5ml LB,
and divide them into 5 groups, each with 3 tubes. All the groups were placed in a static culture at 37°C for 5 h, the expected OD was 1.0,
and then the different groups were placed at different temperatures, respectively: 37°C, 42°C, 47°C, 52°C, and 57°C. The OD600 of the bacterial solution was measured every 2 hours,
and the measurement was continued for 12 hours. Taking the first OD600 as 100%, calculate the survival rate.
2. Ethanol: Connect the engineered bacteria containing the HSP gene and the control bacteria to each of 6 tubes of liquid medium containing 5 mL of LB, and divide them into two groups, each with three tubes, one group with 10% (v/v) ethanol ( 0.5mL ethanol plus 4.5mL LB), one group with the same volume of water (0.5mL water plus 4.5mL LB), placed in a static culture at 37°C for 8h, the OD600 of the bacterial solution was measured every 1 hour for the first 4 hours, and the last 4 Measure the OD every 2 hours every hour. Taking the first OD600 as 100%, calculate the survival rate.
3. Peroxide: The engineered bacteria containing HSP gene and the control bacteria were connected to each of 6 tubes of 5mL LB liquid medium, divided into two groups, each group of three tubes, one group with 1 mM hydrogen peroxide, one group with The same volume of water was placed in a static culture at 37°C for 8 hours, and the OD600 of the bacterial solution was measured every 8 hours for two consecutive days. Taking the first OD600 as 100%, calculate the survival rate.
Detection of butyric acid by high-performance liquid chromatography
1. Crush the cells of wild-type strains (Nissle 1917, DH5α) and engineered strains and take the supernatant for testing.
2. Prepare 100 mg C/L of different short-chain fatty acid samples (acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid).
1. Preparations before using the instrument
(1) Processing of sample and mobile phase
The prepared solution needs to be filtered with a 0.45 μm disposable filter membrane. For pure organic phase or a certain proportion of organic phase, use organic filter membrane, and for water phase or buffer salt, use water filter membrane. Water, methanol, can be used after filtration; the water needs to be re-filtered or replaced with freshwater if stored for more than one day. The mobile phase containing stabilizer needs to be specially treated or use a chromatographically pure mobile phase.
(2) Replace the cleaning fluid in the cleaning bottle in the pump head
The mobile phase is different, and the cleaning solution is different. 50% methanol can be used; if the mobile phase contains electrolytes, 95% deionized water or even high-purity water is usually used if the mobile phase is a methanol-water system. If the instrument is used frequently, it is recommended to replace it twice a week. If the instrument is rarely used, it must be replaced before each use.
(3) Replace the lotion in the needle washing bottle in the tray （the lotion is generally） 50% methanol.
2. Eliminate bubbles in the pump
The pump must be turned off when opening and closing the exhaust valve. The specific operations are as follows:
(1) With the pump closed, open the exhaust valve.
(2) Select the channel to discharge bubbles and turn on the pump.
(3) Press the "Purge" key at the bottom right of the front panel of the pump, the instrument will automatically and quickly clean the remaining bubbles in the pump at a rate of 6.0 mL/min, and it will stop automatically in 5 minutes. If you want to stop manually, press the "Purge" key again to stop the cleaning.
(4) Change to other channels to discharge air bubbles. Note Only one channel can be used to discharge air bubbles When using the quick cleaning valve. Several channels must not be discharged proportionally at the same time. The rapid switching of the proportional valve may easily cause damage.
(5) When there are no bubbles in the flow path, turn off the pump and close the exhaust valve.
Note: The exhaust valve cannot be over-tightened or over-tightened. If it is over-tightened, the mobile phase will easily flow into the pump head from the cleaning valve and cause an alarm.
3. Set the temperature of the column thermostat
Press and hold the "+" or "-" key on the column oven until the number starts to flash to set the temperature.
4. System preparation
(1) Flush the flow path with methanol or acetonitrile for about 20 minutes before analyzing the sample, balance and activate the chromatographic column, and drive away impurities and water in the pipeline.
(2) If the mobile phase is a mixture of the organic phase and water phase, after the first step is completed, adjust the ratio of the valve according to the needs of analyzing the sample, and then flush the flow path for about 20 minutes. After the baseline becomes flat, the sample can be injected.
(3) If the flow phase contains buffer salt solutions, organic/inorganic acids or other electrolytes, after step 1 is completed, rinse the flow path with 95% deionized water for about 20 minutes, and then adjust the proportional valve according to the needs of the analysis sample After flushing the flow path for about 20 minutes, the sample can be injected after the baseline becomes flat.
5. Wash the needle
Before making a sample, press the "wash" button on the panel of the autosampler to wash the needle and remove the remaining air bubbles in the needle. If the bubbles in the needle are still not clear, press the "wash" key again until the bubbles are cleared.
6. Sample injection
(1) Establishment of program files
The pump's flow rate, the ratio of each channel; the autosampler's injection volume, the temperature of the column oven; the detector's wavelength, and the time required to measure each sample must be specified in the program file.
(2) Establishment of method documents
After sample injection, the software will automatically collect chromatograms, and a method file is needed to process these spectra, such as integration, qualitative, quantitative.
(3) Establishment of the sample sequence
standard samples there are, samples there are, volumes are required to be fed, must be specified in the sequence file. (4) Sampling.
(5) Data processing and report printing.
1. 0.54 mL of HCl (12.1 M), 0.86 mL of pyridine and 8.6 mL of water, ensure the pH is at 5.0.
2. 50 μL of 1 M EDC and 1 M O-BHA were dissolved in pyridine-HCl solution and centrifuged at 23°C and 1000 rpm for 10 min.
3. After centrifugation, add 250 μL H2O and mix well.
4. Add 2 mL of ethyl acetate solution for extraction, mix well, and centrifuge at 23°C and 1000 rpm for 5 min.
5. After the extraction is completed, use a disposable syringe to suck up the lower layer of solution to avoid sucking up the upper layer of organic solvents.
6. Take out the solution and dilute it 800 times with mobile phase (95% A-5% B), draw 1 mL and put it into a sample bottle.
7. Chromatographic conditions:
Column: Kinetex C18 2.6 um 100×2.1mm
Mobile phase: A: 0.1% phosphoric acid-water, B: 0.1% phosphoric acid-acetonitrile
Flow rate: 0.5 mL/min
Injection volume: 1 μL
Column temperature: 35℃
Mobile phase gradient
Bile hydrolase is to decompose the combined bile acid to produce bile acid and glycine/taurine. We mix the crude enzyme solution with 0.5mM sodium glycocholate/sodium taurocholate and react at 37℃ for 30 minutes after testing its amino acid content to indicate enzyme activity. BSH enzyme activity is defined as the amount of the substance that produces amino acids by hydrolysis of bound bile salt by crude enzyme per unit time and unit volume, unit nmol/(minml);
BSH specific enzyme activity is defined as unit time, unit mass. The crude enzyme in the total protein hydrolyzes the bound bile salt to produce amino acids in nmol/(ming).
Take different concentrations of glycine working solution to measure its absorbance at 570 nm, and draw a standard curve with the concentration of glycine as the abscissa and the absorbance as the ordinate.
|double distilled water/μL||750||600||450||300||150||0|
1. Take three 0.1 mL of the broken bacterial supernatant, and add the following reagents to each:
1 mL 5mM sodium glycocholate
1 mL 5mM sodium taurocholate
1 mL of PB buffer (pH=6.8)
After mixing, incubate at 37 constant temperature for 30 min
2. Take 0.3mL of the sample after the reaction, add 0.45mL of ninhydrin working solution and 0.15mL of vitamin C working solution to mix well, heat at 80°C for 15min, and cool quickly.
3. Take 0.09 mL of the reaction solution, add 0.21 mL of 60% ethanol, mix well, and measure the absorbance at 570 nm.
Leagene Amino Acid (AA) Detection Kit (Ninhydrin Copper Colorimetry)
Interact with cells with LL37 standard products and engineered bacterial protein samples. See 5 for details.
SOD enzyme activity determination
According to Nanjing built superoxide dismutase (SOD) test kit. The total SOD activity detection kit (WST method) of Nanjing Jiancheng Company is a colour reaction based on WST-1. WST-1 can react with the superoxide anion catalyzed by xanthine oxidase to produce water-soluble formazan dye. (formazan dye), this reaction step can be inhibited by SOD. The enzyme activity of SOD can be calculated by colourimetric analysis of the WST-1 product. When the SOD inhibition rate reaches 50% in this reaction system, the corresponding enzyme amount is one SOD activity unit (U). The corresponding formula can calculate the SOD inhibition rate (here omitted) and then the SOD activity (U/ mL).
1. Collection of cultured cells:
The cells in suspension culture can be collected directly by centrifugation (4000 r/min, centrifugation for 10 minutes, discard the supernatant and leave the precipitated cells).
2. The fragmentation of cultured cells:
Choose to use repeated freezing and thawing method to break the cells: add a certain amount of PBS buffer to the EP tube (depending on the number of bacteria, usually 0.3~0.5mL for 10^6 cells) to entirely suspend the cells by pipetting. Put it directly in liquid nitrogen for 3～5s, immediately transfer it to -20°C refrigerator (20～30s), then take out the water bath to thaw at 37°C, and repeat the previous 3 times after thawing. (Be careful not to take it out of the liquid nitrogen and place it directly in a water bath 37°C to thaw so that the EP tube will easily burst and cause sample loss, so you must use freezing for gradient thawing).
3. Determination of protein concentration:
Use Biosharp company's BCA protein concentration determination kit.
|Control well||Control blank hole||Measure well||Measure blank well|
|Sample to be tested(uL)||-||-||20||20|
|Enzyme working solution(uL)||20||-||20||-|
|Substrate application solution(uL)||200||200||200||200|
Mix well, incubate at 37°C for 20 minutes, read on the microplate reader at 450nm
In this reaction system, the enzyme corresponding to the SOD inhibition rate reaches 50% is one SOD activity unit (U).
2. Calculation formula in cell sample
Inhibition percentage = [(A control-A control blank)-(A measurement-A measurement blank)] / (A control-A control blank) × 100% Calculate the vitality of SOD according to the formula: SOD activity (U / mgprot)=SOD inhibition rate/50% × (reaction system 0.24mL/dilution multiple 0.02mL)/protein concentration of the sample to be tested (mgprot/mL)
Glucose suicide switch
1. Add 50 μL of ddH2O to wells 2 to 8 of a 96-well plate.
2. Add 100 μL of the prepared glucose solution (concentration 0.5 g/mL) to the first hole, and then double-dilute the glucose solution, that is, add the drug solution to the first hole, and then use a pipette to fully pipette (at least three times) Above), and then pipet 50 μL from the first well to the second well, and then fully pipette to mix it with water, and repeat until the seventh well. At this time, the drug concentration in each hole is from left to right: 0.5, 0.25, 0.125, 0.675… (unit g/mL).
3. Add 200 μl of bacteria in logarithmic phase after adding the culture to the plateau phase and then dilute it at 1:100 to form an experiment to determine the antibacterial rate of the drug; add 200 μL of bacteria to the 8th well Add 200 μL of broth to the 9th well. At this time, the drug concentration in each hole, the final drug concentration from left to right is 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625 (unit %); total liquid volume in each hole: 250 μL.
4. Put the 96-well plate in a 37 ℃ constant temperature incubator for 12 h. Measure the OD600 value within 12 hours, and measure it every 0.5h. Survival rate = OD600 of the hole/value of the first measurement*100%
Bacteria treatment process
1. Place the Nissle 1917 strain stored in a glycerin preservation tube in a refrigerator at -80°C in a water bath to quickly thaw,
inoculate it in liquid LB medium for recovery, and culture it on a shaker at 140 rpm overnight for activation.
2. After overnight, inoculate it in liquid LB medium at a volume ratio of 10%. When the OD value is about 0.5, take the cultured bacterial solution for 16s gene identification. The experimental steps are as follows - Take 5 μL of bacterial solution and 45 μL of ALP to mix well by pipetting, 90℃ for 10 min, and record as a template after reaction; - Prepare the PCR reaction system as follows:
|PCR reaction system|
|Pre primer (27F) 10 μM||2 μL|
|Back primer (1492R) 10 μM||2 μL|
|PCR Enzyme||12.5 μL|
3. The PCR reaction program is as follows: 98°C, 5 min; 98°C, 15 s， 55°C, 15 s， 72°C, 15 s， 32 ×， 72°C, 5 min， 4°C, forever.
4. After the reaction is over, take 5 μL for agarose gel electrophoresis to verify the product's molecular weight and send the remaining samples to BGI for sequencing and two-way testing.
5. After the sequencing results are returned, perform a Blast comparison with the gene bank (NCBI) and perform follow-up experiments after verifying that they are correct. If there is contamination, it is necessary to pick out a single clone from the solid medium, culture it, and re-identify it.
Exploring the conditions of interaction
In order to further confirm the interaction time between Nissle 1917 strain and THP-1 cell line, wild-type Nissle 1917 strain was used to complete growth curve drawing, dilution plate counting, and determine the best interaction time.
The cultured Nissle 1917 wild-type bacterial solution was inoculated into the liquid medium according to the volume ratio of 10%, and the initial OD 600 was about 0.1. The OD600 was measured with a microplate reader at different culture time points, and the growth curve was drawn.
When E. coli enters the plateau phase, take the medium at different time points and dilute the medium gradually (repeat 3 times in parallel, 100μL+900μL), and take the dilution multiples of 10^-3, 10^-4, 10^-5, 10^-6 Carry out coating, the coating volume is 100 μL. Aerobic culture at 37°C for 48h, count the plates with a colony number of about 150 and calculate the CFU.
Live bacteria amplification and sample preparation
1. The verified Nissle 1917 bacteria and Nissle 1917 WT containing the four target genes are inoculated in a liquid LB medium at a volume ratio of 10%.
(When the interaction research object is bacteria, the logarithmic growth phase should be selected Appropriate, at this time, the bacterial activity is relatively vigorous，
when the interaction study object is the supernatant, the plateau period should be selected)
2. Measure the OD value of the bacteria in the appropriate period, centrifuge at 8000 rpm for 5 min to separate the supernatant from the bacteria. After the bacteria were washed and resuspended in PBS once. They were concentrated according to the requirements of the experimental design and recorded as live bacteria samples.
Collection of bacterial products
After cultivation, the related products are produced and purified from the DH5α and Nissle 1917 engineered bacteria containing four target genes.
Antibiotics' inhibitory effect on bacteria
1. Use gentamicin sulfate solid powder configuration, set 100μg/mL, 200μg/mL, 300μg/mL, 400μg/mL, 500μg/mL gentamicin gradient,
and complete negative control (LB liquid medium) and complete Positive control (no antibiotics).
2. Connect the 10% bacterial solution, culture in a shaker at 140rpm 37℃, measure the OD600 absorbance value, and draw the growth curve， and at the dilution factor of 10^-5, coat the plate to count.
Cell processing process
The conserved THP-1 (P6 generation) was taken out of liquid nitrogen, thawed at room temperature, and directly inoculated in a T175 culture flask containing 40 mL of RPMI medium.
Conventional cell culture
THP-1 cells are subcultured (change medium) once every 2 days, and half of the medium is changed during subculture (change medium). That is, the same amount of medium is added and divided into two cultures.
Cell PMA polarization
Phorbol ester (PMA) induces THP-1 cells to differentiate into macrophages and establishes an inflammation model through LPS stimulation.
1. Centrifuge at 1000 rpm for 6 minutes (you can aliquot the cell culture solution into four 15ml centrifuge tubes).
2. Add PBS to mix well, centrifuge again at 1000 rpm for 6 min, and wash the cells once.
3. Add RPMI1640 complete medium to beat the cells, count and adjust the cell concentration to 1×106 cell/mL.
4. Add PMA reagent (50ng/mL) for polarization induction and shake well.
5. Connect to a 96-well cell culture plate, 200μL/well per well, and polarize for 48h to induce M0 macrophages.
Cells are exchanged at rest
1. Aspirate the medium in the 96-well plate, wash it once with PBS, and add a complete fresh medium at a concentration of 200 μL/well.
2. Leave it to stand for 72 hours, then the cell interaction experiment can be carried out.
1. Change to a brand new cell culture medium (complete cell culture medium with and without LPS), add live bacteria and place them in a cell incubator to interact
with the cells for 2 hours (if cell product interaction is performed, the interaction experiment can be performed Prolonged)
Note: LPS (lipopolysaccharide): increase the expression of pro-inflammatory factors.
2. Remove the supernatant and wash twice with PBS (collect the washed liquid and centrifuge the coated plate to see the number of bacteria, and initially estimate the number of phagocytosed bacteria. Note: It needs to be added after mixing on another plate).
3. Replace with 90% RPMI1640+10% FBS+100ng/mL gentamicin or double antibodies (streptomycin and penicillin) (with LBS and without LPS) cell complete medium, and put in the cells Take pictures after 22 hours in the incubator, observe whether there are live bacteria, and collect the cell supernatant by centrifugation. The CCK8 group needs to add 20μL CCK-8 reagent for detection after 22h (test OD450 value every 1 h, and select the optimal value until 4h is complete).
Determination of CCK-8 cell viability
In order to verify the effect of Nissle 1917 WT on the cell viability of THP-1, CCK-8 reagent was used for testing. In the CCK8 group, CCK8 was added after 22h (OD450 was measured once at 1 h, 2 h, and 3 h). If more viable bacteria are observed, select two wells of the cells in the CCK8 group for trypsin digestion, collect all the liquid and sonicate for 15 seconds, then dilute and plate the plate (you can choose only one gradient plate to see the specific bacterial number); The data measured at the appropriate interval is used as the result.
1. Preparation of cell suspension
2. Inoculate a 96-well plate According to the appropriate number of plated cells, about 100μL of cell suspension per well, the same sample can be repeated 3 times.
3. Culturing in a 37°C incubator takes about 2-4 hours to culture the cells to adhere to the wall after inoculation. If you do not need to adhere to the wall, this step can be omitted. v 4. Add different concentrations of toxic substances. v 5. Cultivation in a 37℃ incubator: The incubation time for adding toxic substances depends on the nature of the toxic substances and the sensitivity of the cells. It is generally determined according to the cell cycle, at least one generation time.
6. Add 10μLCCK8 Because the amount of CCK8 added to each well is relatively small, it may cause errors due to reagents sticking to the wall of the well. It is recommended to tap the culture plate gently after adding the reagents to help to mix.
7. Cultivate for 1-4 hours: The formazan (Formazan) formed varies depending on the cell type. If the colour is not enough, you can continue to cultivate to confirm the best conditions. Especially formazan (Formazan) formed by blood cells is very small, and it takes a long time for colour development (5-6 hours).
8. Determination of absorbance at 450nm: It is recommended to use dual wavelengths for measurement, with detection wavelengths of 450-490nm and reference wavelengths of 600-650nm.
- When using a standard 96-well plate, the minimum seeding amount of adherent cells is at least 1,000 cells/well (100 μL medium). The sensitivity for detecting white blood cells is relatively low,
so the recommended inoculation volume is not less than 2,500 cells/well (100 μL medium).
- Phenol red and serum will not interfere with the detection of the CCK8 method and can be eliminated by subtracting the background absorbance in the blank well.
- CCK8 can detect E. coli but not yeast cells. It is necessary to avoid bacterial contamination during each determination of the cell proliferation experiment not to affect the results.
- CCK-8 can be stored at 0-5°C for at least 6 months, and at -20°C in the dark can be stored for 1 year.
- When culturing in an incubator, the holes in the outermost circle of the culture plate are most likely to dry and volatilize, which increases errors due to inaccurate volumes. Under normal circumstances, only add medium to the outer circle of the wells, not as a measurement well.
- Add CCK8 to the culture medium, incubate for a specific period of time, and measure the absorbance at 450 nm as a blank control. When doing the dosing experiment, the absorption of the drug should also be considered. CCK8 can be added to the medium with the drug, incubated for a particular period of time, and the absorbance at 450 nm is measured as a blank control.
- Metal affects the colour development of CCK-8: when the final concentration is 1 mM, lead chloride, ferric chloride, and copper sulfate will inhibit 5%, 15%, and 90% of the colour reaction, degrading the sensitivity. If the final concentration is 10 mM, it will be 100% inhibited.
- Suspension cells are difficult to stain, so it is generally necessary to increase the number of cells and prolong the culture time.
- If you do not want to determine the OD value temporarily, you can add 10 μL of 0.1M HCL solution or 1% w/v SDS solution to each well, cover the culture plate and store it at room temperature away from light. Measure within 24 hours. The absorbance will not change.
- If the substance to be tested is oxidizing or reducing, replace the fresh medium before adding CCK8 (remove the medium, wash the cells twice with the medium, and then add the new medium) to remove the influence of the drug. Of course, the medium does not need to be replaced when the drug has a relatively small effect, and the blank absorption after the drug is added to the medium can be directly deducted.
Detection of cytokines by ELISA
1. According to the interaction between the bacterial supernatant and the cells in the SOP file, the protein sample collected after purification needs to pass through the filter membrane without dilution.
In the bacterial product and cell interaction experiment, add 20 μL/well of the protein sample.
2. End the experiment after incubating for 24 hours. Transfer the supernatant of the interaction to a new well plate and store at -80°C.
3. Replace the original 96-well plate with a complete medium, and then add CCK-8 for cell viability determination.
1. centrifuge the 96-well plate at 500g for 20 minutes at the end of the interaction. Collect the supernatant of each well, and store it at -80°C.
2. Replace the original 96-well plate with a complete medium, and then add CCK-8 for cell viability determination.
1. Take out the required slats from the aluminium foil bag that has been equilibrated at room temperature for 20 minutes, and seal the remaining slats with a ziplock bag and put it back to 4°C.
2. Set up standard wells and sample wells, add 50μL of different concentrations of a standard to each standard well;
3. Add 10μL of the sample to be tested to the sample hole to be tested, and then add 40μL of sample diluent;
4. Then add 100 μL of horseradish peroxidase (HRP)-labelled detection antibody to each standard wells and sample wells, seal the reaction wells with a sealing film, and incubate in a 37°C water bath or incubator for 60 minutes.
5. Discard the liquid, pat dry on absorbent paper, fill each hole with washing liquid, let stand for 1 min, shake off the washing liquid, pat dry on absorbent paper, and repeat the washing 5 times (the plate can also be washed with a plate washer).
6. Add 50 μL each of Substrate A and B to each well, and incubate at 37°C for 15 min in the dark.
7. Add 50 μL of stop solution to each well, and measure the OD value of each well at 450nm wavelength within 15 minutes.
8. Draw a standard curve: use the concentration of the standard product as the abscissa, and the corresponding OD value as the ordinate, draw a linear regression curve for the standard product and calculate the concentration of each sample according to the curve equation.
1. Store the kit at 2-8°C, equilibrate at room temperature for 20 minutes before use. The concentrated washing liquid taken out of the refrigerator will crystallize, which is a normal phenomenon.
The water bath is heated to dissolve the crystals before use completely.
2. The slats not used in the experiment should be immediately put back into the ziplock bag, sealed (dry at low temperature) and stored.
3. The standard diluent can be regarded as a negative control or blank; the sample after pretreatment does not need to be diluted, take 10μL and add the sample.
4. Strictly follow the time, amount of liquid addition and sequence indicated in the instruction manual for incubation operations. 5. Shake all liquid components well before use.
Production and verification of freeze-dried powder
Take 1ml of each bacteria and add 100mL ampicillin (100μL of ampicillin) LB medium conical flask. At the same time, prepare sterile ampicillin LB medium as a control group, and enter the shaker 37°C 180rpm culture.
Preparation of freeze-dried protective agent
First-generation use 57g skimmed milk powder, 12g sucrose, 12g sodium ascorbate dissolved in 400mL ddH2O, autoclave at 115°C for 30min, and store in a refrigerator at 4°C for later use. Second-generation use 28.5g skimmed milk powder, 6g fructooligosaccharides, 6g sodium ascorbate dissolved in 200mL ddH2O, autoclave at 115°C for 30 minutes, prepare it as a lyophilized protective agent, and store it in a refrigerator at 4°C for later use.
Gradient dilution coating of the stock solution before powder milling
1. In determining the gradient of the counting of the gradient dilution coating plate, first measure the OD600 of the cultured strain.
2. Take 36 solid ampicillin LB plates, aseptically, use a shake tube to dilute 100μL of bacterial solution in 900μL of ddH2O for gradient dilution, the dilution gradient is generally 10^-1, 10^-2, 10^-3, 10^-4, 10^-5, 10^-6, 10^-7, 10^-8, 10^-9 (depending on the OD value).
3. Three concentrations of 10^-5, 10^-6, and 10^-7 for each strain (make the number of bacteria at the median concentration between 30-300) are used for gradient coating of the plate, and 100μl bacterial solution per plate. Make three sets of duplicate controls for each concentration, a total of 433=36 groups of plates.
4. After coating, store in 37°C incubators for 12 hours and count.
1. Pre-freeze dryer in advance to -80°C
2. Use a 50mL centrifuge tube to take 100mL of bacteria solution (2 tubes per strain) and centrifuge at 5500rpm for 50min.
3. Pour out the supernatant after centrifugation, and add the protective agent at a ratio of 20 mL of protective agent per 100 mL of bacterial liquid.
4. Mix (resuspend) the bottom cell and defoam (if it is not defoamed, it may cause cracks on the surface of the freeze-dried powder).
5. Slowly pour 20 ml of the resuspended bacteria + protectant mixture into the pre-sterilized egg tart cup (defoaming), and put it in the refrigerator at -80°C for pre-freezing for 30 minutes (the sterilized disposable sterile bag can be covered On the outside, prevent impurities from falling in).
6. After pre-freezing for 30 minutes, put the egg tart cup into the freeze dryer and freeze-dry in a vacuum for 2.5 days.
Take powder from freeze-dried powder
Take out the lyophilized bacterial powder, return to room temperature, grind into powder, weigh and record.
Re-dissolve and recovere the lyophilized powder, and gradient dilution coating the lyophilized powder
1. Take 10mg of lyophilized powder, re-dissolve it in 1mL D-PBS buffer, and let it stand.
2. Take the above re-dissolved bacteria solution, take the solid ampicillin LB plate, perform the aseptic operation, use a shake tube to dilute 100μL of bacterial solution in 900μL of ddH2O, the dilution gradient is 10^-1, 10^-2, 10^-3, 10^-4, 10^-5, 10^-6, 10^-7, 10^-8, 10^-9 (the specific calculation depends on the concentration of the bacterial solution before lyophilization).
3. At the same time, add ampicillin at a concentration of 40μL ampicillin per 1ml of bacterial solution and shake well. Among the bacteria, each strain takes three concentration gradients of 10^-4, 10^-5, and 10^-6 (make the number of bacteria at the median concentration between 30-300), and each plate is coated with 100μL of the bacterial solution, and each concentration makes three groups of duplicate controls, a total of 433=36 groups of plates.
4. After coating, store in 37°C incubators for 12 hours and count.
Return the freeze-dried powder to the solution liquid-based culture
Take the lyophilized powder in the above gradient dilution back to the original solution, shake the Nissle 1917 wild-type, Nissle 1917 engineered bacteria containing BSH, LL37, SOD target genes in the tube, and perform the liquid-to-liquid operation. For each type of bacteria, 900μL of the bacterial solution was added to 100mL ampicillin (100μL of ampicillin) LB medium conical flask. At the same time, a sterile ampicillin LB medium was prepared as a control group and cultured in a shaker at 37°C at 150rpm.
Verify the corresponding target product
Determine SOD enzyme activity (see 4.4.1 for details)
Time limit inspection experiment for enteric-coated
The test methods and buffer formulations used in the following experiments are implemented according to The 2020 Edition of The Chinese Pharmacopoeia
Preparation of reagents and buffers
1. Preparation of hydrochloric acid solution (gastric juice)
Draw 27mL of concentrated hydrochloric acid, slowly add 3000mL purified water while stirring, and pour into 3 beakers of the disintegrator, each beaker about 800mL hydrochloric acid solution.
2. Preparation of buffer 1 (artificial intestinal juice, phosphate buffer, pH=6.8) 6.8g potassium dihydrogen phosphate (KH2PO4), dissolved in 500mL of water, adjusted to pH 6.8 with NaOH solid), and another 10g of pancreatin, add an appropriate amount of water to dissolve, mix the two liquids and dilute to 1000 mL with purified water.
3. Preparation of buffer 2 (phosphate buffer, pH=6.6) sodium dihydrogen phosphate (Na2PO4) 3.48g, disodium hydrogen phosphate (Na2HPO4) 1.74g, dissolved in 500mL of water, another 8g of pancreatin, and an appropriate amount of water Dissolve, mix the two liquids and dilute to 800 mL with purified water.
4. Preparation of buffer 3 (phosphate buffer, pH 5.8) potassium dihydrogen phosphate (KH2PO4) 8.34g, dipotassium hydrogen phosphate (K2HPO4) 0.87g, dissolved in 500mL of water, another 10g of pancreatin, and an appropriate amount of water Dissolve, mix the two liquids and dilute to 1000 mL with purified water.
Embedding of capsules
Take 18 No. 2 enteric-coated capsules and 2g agar powder to prepare for embedding. Use the medicine spoon to fill the capsules. Each capsule is about 100mg powder.
Disintegration time limit inspection
Enteric-coated capsules, unless otherwise specified, take 6 capsules of the test substance, according to the above device and method.
1. Take the hydrochloric acid solution into the beakers 1, 2, and 3 of the disintegrator, add 800mL hydrochloric acid solution (gastric juice) to each beaker, and check for 2 hours without a baffle in the hydrochloric acid solution. There should be no cracks in the capsule shell of each capsule. Or disintegration phenomenon.
2. Take out the hanging basket, wash it with a small amount of water, and then check it in phosphate buffer saline 1, 2, 3 according to the above method, and it should all disintegrate within 1 hour. If one capsule does not disintegrate completely, another 6 capsules should be taken for a retest.