1.1 Reagents:
Tryptone, yeast extract, sodium chloride, water, Prime Mix, p15A-GEx-A/B, Primer-R, Primer-F, Template, TAE buffer, Marker, NEB dye, Wash solution, Elution buffer, Buffer SP1 / Buffer SP2 / Buffer SP3 / Buffer S / Buffer DW1 / Wash Solution, Recombinase, Mold label plate, Detection antibody, Standard diluent, Streptavidin, Detection buffer solution, Chromogenic substrate, Wash solution, LB/SB medium, A bacterial liquid, B bacterial liquid1.2 Experimental tools:
Beaker, Pipette, Medicine spoon, Weighing paper, PCR thermal cycler, Centrifuge tube, pipette, Pipette tip, Centrifuge, Electronic balance, Bain-marie, Microwave oven, UV transilluminator, Electrophoresis chamber, Thermostat, Clean bench, Automatic ice maker.2.1Disposition of LB Culture Base
Goal: To complete the LB culture medium with 500ml water, which can be used for pre-culturing bacterial strains in experiments, to multiply the bacterial strains and meet the requirements for use. It can also be used for culturing Escherichia coli. Experimental steps:1 .Weigh using an electronic balance and a medicine spoon, paying attention to the weight of the weighing paper and keeping the balance closed.
2. Pour the weighed solid into a conical flask
3. Constant volume: Measure 500ml of water with a measuring cylinder
4. Mix the measured solid and water together
5. Finally, plug the prepared liquid culture medium and keep it closed and flowing
6. Sterilize, if not possible, temporarily store in the refrigerator.
2.2 Construction of p15A-GEx-A/B
2.2.1 Obtaining target gene fragments by PCR
Goal: PCR (polymerase chain reaction), which rapidly replicates DNA through a very simple method, can rapidly increase the quantity of trace amounts of DNA in a short period of time. PCR has a great role in medical research and forensic fields. During DNA replication, the two strands of the parent DNA are used as templates to synthesize two new strands based on the principle of complementary bases, resulting in the formation of DNA with the same sequence of bases as the parent. PCR is the process of denaturing DNA in a high-temperature environment, allowing it to unwind. Then cool down and allow the primer (containing approximately 20 bases) to bind to complementary DNA. DNA without a primer does not bind. Finally, heat up and extend. Taq enzyme pairs with the primer and synthesizes it into offspring DNA, and then repeats this process.Experimental steps:
1. Adjust the numbers with a pipette first, and then mix the materials
2. Inactivate at a high temperature of 98 °C Celsius, causing DNA to change from double stranded to single stranded
3. Deactivate at 56 °C and primer binding to DNA
4. 72 °C extended DNA pairing
5. Repeat about 30 times to obtain a large amount of DNA
2.2.2 Agarose gel construction and deployment
1. Dissolve 0.3g Agarose in 30mL Erlenmeyer flask
2. Add TAE buffer (Running buffer)
3. Heat the material with microwave
4. Melt the mixture at 30 sec interval
5. See whether bubbles are forming, if did successfully mixed
6. Add 3μl nucleic acid gel stain (10000x)
7. Let it cool for 5~10 minutes
8. Pour the agarose mixture into the casting tray
9. Place the appropriate well comb to create the wells
10. Wait to solidify 7~10 minutes
11. Remove the comb and place the gel in the gel box
2.2.3 Agarose gel electrophoresis
Purpose: To separate, identify, and purify DNA fragments.
Experimental steps:
1. Prepare the gel: Mix 200 ml of TAE buffer with 2 g of agarose, and heat in a microwave for 5 minutes.
2. Wait for the solution to cool until it is no longer hot to the touch, then add NEB dye at a volume of 1/10,000 of the solution.
3. Pour the solution into the gel tray.
4. Allow the gel to solidify (approximately 33 minutes), then use a pipette to add PCR products and marker to each well.
5. Perform electrophoresis at 180 V for 20 minutes.
6. Capture the image under UV light
2.2.4 Recovery of DNA
Purpose: Recover DNA and determine its concentration
Experimental steps:
- Cut the gel and place it into a tube.
- Add gel dissolution solution (3 times the volume of the gel weight, like 100ml Buffer for 0.1g gel).
- Heat at 50°C in a water bath for 10 minutes, shaking continuously.
- Transfer 700µl of the solution to the combined adsorption column and collection tube.
- Centrifuge to separate DNA at 12,000 rpm for 1 minute.
- Repeat this step until all DNA is filtered through the binding column.
- Add 600µl of wash solution and centrifuge at 12,000 rpm for 1 minute. Discard the waste liquid and place the adsorption column into a new collection tube. Repeat this washing step once.
- Heat the elution buffer at 65°C for 2 minutes. Then, suspend and add the elution buffer to the adsorption column and a new centrifuge tube. Centrifuge at 12,000 rpm for 1 minute.
- Measure the concentration: first measure the water, then measure the elution buffer separately. Record the A260 and concentration data.
2.3 Transformation of plasmid into E. coli
2.3.1 Homologous RecombinationPurpose: ligate target DNA fragment with vector backbone fragment
Experimental steps:
- Transfer the DNA recovery product to a PCR tube.
- Add 5µl of recombinase to each tube.
- Heat at 50°C in a PCR machine for 30 minutes.
- Store the finished product at -20°C.
2.3.2 Transformation of p15A-GEx-A/B into E. coli DH5α via heat-shock
Purpose: Cultivate E. coli
Experimental steps:
1. Take out E. coli DH5a competent cells from -80 °C refrigerator;
2. Add 10μl of p15A-GEx-A/B and 50μl of DH5α cells into an EP tube.
3. Mix the EP tubes with the mini-centrifuge for 5 seconds.
4. Put the EP tubes on ice for 30 minutes.
5. Heat shock the samples at 42 °C for 45 seconds in the water bath.
6. Put the EP tubes in ice for 5 minutes.
7. Transfer the bacteria samples from the EP tubes into liquid LB medium. This step is performed in the clean bench.
8. Shake the cultures for 2 hours at 37 °C, at 220rpm.
9. Spread coat 50μl on the petri dishes with LB solid culture medium containing Chloramphenicol, respectively.
10. Inverted culture at 37 °C and/or 16 °C for 12-16 hours.
2.3.3 Plasmid Extraction
purpose: To isolate plasmid DNA from bacteria for use as a vector.
Experimental steps:
1. Check if RNase A has been added to Buffer SP1. Verify if absolute ethanol has been added to the Wash Solution. Check for any precipitation in Buffer SP2 and SP3.
2. Column Equilibration: Add 500 µL of Buffer S to the column (place the column in a collection tube), centrifuge at 12,000 g for 1 minute, discard the waste liquid from the collection tube, and place the column back in the collection tube.
3. Take 1.5-5 mL of overnight bacterial culture, centrifuge at 8,000 g for 2 minutes to collect the cells, and discard the culture medium.
4. Add 250 µL of Buffer SP1 to the pellet and resuspend the cells thoroughly.
5. Add 250 µL of Buffer SP2, gently invert the tube 5-10 times to mix, and let it sit at room temperature for 2-4 minutes.
6. Add 350 µL of Buffer SP3, gently invert the tube 5-10 times to mix.
7. Centrifuge at 12,000 x g for 5-10 minutes. Transfer the supernatant to the column, centrifuge at 8,000 g for 30 seconds, and discard the liquid from the collection tube.
8. Add 500 µL of Wash Solution, centrifuge at 9,000 g for 30 seconds, and discard the liquid from the collection tube.
9. Repeat Step 8 once.
10. Spin the empty column at 9,000 x g for 1 minute.
11. Place the column into a clean 1.5 mL centrifuge tube, add 50-100 µL of buffer.
12. Elution Buffer to the center of the membrane, let it sit at room temperature for 1 minute, and then centrifuge for 1 minute. Save the DNA solution in the tube.
13. Test concentration (range 100 to 200).
2.3.4 Monoclonal verification
Purpose: To generate a specific type of antibody to recognize and bind to a particular antigen. By cloning a single B cell, a large quantity of identical antibodies can be produced, which is very useful in research, diagnosis, and treatment.
Experimental steps:
- Prepare the gel: Mix 200 ml of TAE buffer with 2 g of agarose, and heat in a microwave for five minutes.
- Wait for the solution to cool until it is no longer hot to the touch, then add NEB dye at a volume of 1/10,000 of the solution.
- Pour the solution into the gel tray.
- Allow the gel to solidify (approximately 33 minutes), then use a pipette to add PCR products and marker to each well.
- Perform electrophoresis at 180 V for 20 minutes.
- Capture the image.
2.3.5 Amplification culture of engineered Escherichia coli
Experimental steps:
1. Add 200μL of bacterial liquid to each well of the microplate.
2. Measure the OD.
3. Dilute the four bacterial liquids to the same OD value.
4. Add 30 mL of culture medium.
5. Place in a shaker at temperatures of 37 °C and 16 °C.
2.3.6 Transformation of p15A-GEx-A/B into EcN 1917 via heat-shock
Experimental steps:
1. Take out EcN competent cells from -80 °C refrigerator;
2. Add 10μl of p15A-GEx-A/B and 50μl of EcN into an EP tube.
3. Mix the EP tubes with the mini-centrifuge for 5 seconds.
4. Put the EP tubes on ice for 30 minutes.
5. Heat shock the samples at 42 °C for 45 seconds in the water bath.
6. Put the EP tubes in ice for 5 minutes.
7. Transfer the bacteria samples from the EP tubes into liquid LB medium. This step is performed in the clean bench.
8. Shake the cultures for 2 hours at 37 °C, at 220rpm.
9. Spread coat 50μl on the petri dishes with LB solid culture medium containing Chloramphenicol, respectively.
10. Inverted culture at 37 °C and for 12-16 hours.
2.3.7 Extraction plasmid from EcN
Goal: (1) Dissolve bacteria (2) Isolate DNA (3) Purification
Check if the buffer has RNase A
Check anhydrous ethanol/precipitate
Experimental steps:
- Pour out the supernatant, add it to the adsorption column, add 500 microliters of buffer, centrifuge for 1 minute, discard the waste liquid, and then pour it back.
- Mix well and centrifuge 1.5-5ml for 2 minutes
- Add 250 microliters of buffers to the precipitate
- Centrifuge and add 250 microliters of Buffer SP2. Slowly invert 5-10 times and let it stand for 2-4 minutes
- Add 350 microliters of buffers and invert 10 times
- Centrifuge for 5-10 minutes, transfer the supernatant to the adsorption column and centrifuge for 30 seconds
- Add 500 microliters of WASH SOLUTION and centrifuge for 30 seconds, repeat once
- Transfer the centrifugal adsorption column into a centrifuge tube and add ELUTION BUFFER. Centrifuge and let it stand for 1 minute
- The concentration data is around 100-200.
2.4 Validation of nano-antibody expression by Western Blot
Boil samples: Boil the lysates at 95°C for 5 minutes to denature the proteins and allow them to bind to the SDS.
Step 2: Electrophoresis (SDS-PAGE)
Run the gel: Apply a constant voltage (e.g., 80V for stacking gel, then increase to 120V for resolving gel) until the tracking dye reaches the bottom of the gel.
Step 3: Transfer (Blotting)
Perform transfer: Use either a semi-dry transfer apparatus or a wet transfer tank. Apply a constant current (e.g., 300mA for 90 minutes) to transfer the proteins from the gel to the membrane.
Step 4: Blocking
Block non-specific binding sites: Incubate the membrane in blocking buffer for 1 hour at room temperature to prevent non-specific antibody binding.
Step 5: Primary Antibody Incubation
Prepare primary antibody: Dilute the anti-His tag antibody in blocking buffer according to the manufacturer's instructions (1:2000).
Incubate: Incubate the membrane with the diluted primary antibody overnight at 4°C.
Step 6: Wash
Step 7: Secondary Antibody Incubation
Prepare secondary antibody: Dilute the HRP-conjugated secondary antibody in blocking buffer.
Incubate: Incubate the membrane with the diluted secondary antibody for 1 hour at room temperature.
Step 8: Final Wash
Wash the membrane: Wash the membrane three times with TBS-T followed by a final wash with TBS alone.
Step 9: Detection
Apply ECL substrate: Apply the chemiluminescent substrate to the membrane.
Develop the signal: Place the membrane in a cassette and expose it to a digital imaging system to visualize the bands.
2.5 Validation of nano-antibody expression by ELISA
Experimental steps:
1. Prepare 1x wash solution by diluting 20x wash solution with distilled water at a ratio of 2:38.
2. Prepare 1x assay buffer by diluting 10x buffer with distilled water at a ratio of 1:10.
3. Prepare the detection antibody working solution by diluting 1x antibody working solution with distilled water at a ratio of 1:10.
4. Dilute the sample with 200 µl of 1x assay buffer.
5. Centrifuge at 10,000g for 1 minute and let it stand for 10 minutes.
6. Take 230 µl of reconstituted standard, add 230 µl of standard diluent, and perform a 1:1 dilution using the high concentration standard.
7. Take 230 µl of reconstituted standard, add 230 µl of cell culture medium, and perform a 1:1 dilution using the high concentration standard.
8. Detach any unnecessary strips from the plate and reseal them to avoid contact with the inner surface of the microwells.
9. Add 300 µl of 1x wash solution to each well on the ELISA plate and let it soak for 30 seconds.
10. Discard the wash solution and blot the microwell plate dry on absorbent paper. After washing, do not let the microwell plate dry out.
11. Add 100 µl of 2-fold serially diluted standard to the standard wells.
12. Add 50 µl of 1x assay buffer and 50 µl of the sample to the sample wells.
13. Add 100 µl of cell culture supernatant to the sample wells.
14. Add detection antibody: Add 50 µl of detection antibody working solution to each well to be tested.
15. Incubate at 100-300 rpm at room temperature (25°C ± 3°C) for 2 hours.
16. Discard the liquid, add 300 µl of wash solution to each well, and wash the plate 6 times. Blot the plate dry on absorbent paper, ensuring all residual liquid is removed.
17. Add enzyme: Add 100 µl of streptavidin working solution to each well.
18. For non-acidic liquid waste, add 1.0% sodium hypochlorite and soak for 30 minutes. For acidic liquid waste, neutralize it first, then add sodium hypochlorite.
19. Incubate: Seal the plate with a new adhesive cover. Incubate at 100-300 rpm at room temperature (25°C ± 3°C).
20. Repeat Step 16.
21. Add 100 µl of the substrate solution to each well, incubate in the dark at room temperature (25°C ± 3°C) for 5-30 minutes.
22. Add 100 µl of stop solution to each well; the color will change from blue to yellow.
2.6 Validation of nano-antibody function
Experimental steps:
1. Add 100μl of cells (30,000 cells) (U937 and mouse primary CD8-positive inguinal lymph node T cells) to a 96-well plate and incubate it overnight in a cell incubator.
2. Create a control group by adding TNFα+PBS. Create an experimental group by adding TNFα and 5 different antibodies, each with three concentration gradients, with the concentration halved sequentially. Then incubate the 96-well plate at 37°C for 6 hours.
3. Prepare CellTiter-Lumi solution and bring it to room temperature.
4. Take out the 96-well plate and balance it to room temperature.
5. Add 100μl of CellTiter-Lumi solution to each well, cover the plate, oscillate for 2 minutes, and incubate for 20 minutes.
6. Measure chemiluminescence using a microplate reader.