The purpose of our experiment is to verify that the proteins we chose (CA125 and IL-6) are able to react with the antibodies.
To achieve this aim, we started by building plasmids. We built two plasmids based on fragments of the two antigens, CA125 and IL-6. After the construction is complete, the plasmid is injected into the E. coli and therefore, the plasmids are going to replicate.
After adding IPTG, and the plasmid expresses more protein, we will then break the E. col to make the protein flow out. The protein electrophoresis experiment will be conducted to verify whether the molecular content of the protein was what we needed (25Kda).
If the result of the protein electrophoresis experiment shows the molecular content of the protein is what we need, we will know that the protein we chose can react with the antibodies. It will tell us the feasibility of our product.
Extracting plasmids
1. Using 1000µl micropipette added 1ml of various bacteria solution into EP tube (total 4 types of bacteria, each type 3 tubes, total 12 tubes) 2. Place all 12 EP tubes into centrifuge for 3 minutes 3. Extract supernatant 4. Repeat 3 times of above steps 5. Added 250µl of K1 reagent, using micropipette to aspirate and dispense until bacteria pallet has disappeared 6. Added 250µl of K2 reagent in all EP tubes then slightly shake 3 times 7. Added 350µl of K3 reagent in all EP tubes then aggressively shake 8. Put all EP tubes inside centrifuge for 10 minutes 9. Extract supernatant (around 750µl) into a collection tube and place into centrifuge for 30 seconds 10. Pour out supernatant inside collection tube 11. Add 750µl of KW reagent, centrifuge for 1 minute, pour out supernatant in collection tube 12. Step 11 repeat 2 times 13. Place into centrifuge for 1 minute 14. Pour out the liquid inside the collection tube, place collection column into new EP tube, add 50µl of KE reagent 15. Place into centrifuge for 1 minute 16. Throw out collection column, low temperature storage of liquid inside EP tube
Nanodrop Test:
- Take 1µl sample plasmid from each tube and place on Nanodrop to test the plasmid concentration and purity.
Nanodrop test results:
Picking Colony:
1. Use small pipette head to pick out a small amount of bacteria from each tray, place in 15ml centrifuge tube, place on shaking incubator and shake for 1 night. Shaking incubator criteria 37°C, 180 rpm, 4 hours
2. Add 1ml iptg(concentration 50mg/ml) into four of the tubes (one tube for each strain) and mark the tubes.
Bacteria Preservation:
In the laminar flow hood add 500µl of bacteria solution and 500µl of 50% glycerine in all 11 tubes and shake slightly
Competent cell preparation and maintenance:
- Operate on a super clean table - Draw approximately 1.5ml of bacterial fluid from each tube and add it to two 2ml centrifuge tubes (1.5ml each) - Centrifuge for two minutes at 12000rpm - Remove the supernatant and add the remaining bacteria (about 1.5ml per tube) - Centrifuge for two minutes at 120,00rpm - remove the supernatant - Add 400µl of 0.1M CaCl, blow until the fungus disappears, and place in ice for 30 minutes - Take 2ml bacterial solution from a large ice bath centrifuge and put it into a 2ml centrifuge tube at 4℃,12000rpm, for 2 minutes. - Remove the supernatant and add 2ml of bacterial solution - Centrifuge, remove supernatant - Blot out residual culture fluid - Add 400ml of Cacl2 of ice to each small centrifugal tube and repeatedly suck until the bacteria are suspended - Ice bath for 30 minutes -4 ℃,12000rpm,2 minutes, remove the supernatant, put the centrifugal tube upside down on the absorbent paper for 1 minute - Add 100-200ul ice CaCl2, repeatedly suck until bacteria are suspended, and put into a 4°C ice bath
Transformation
- Take the centrifuge tube containing the competent cells and place it in an ice bath - Add 10µl of target DNA into the competent cell suspension, mix it with a whisk, label it, and leave it in an ice bath for 30 minutes. - Place the centrifuge tube in a 42 ° C water bath for 90 seconds, then quickly transfer the tube to the ice bath and cool it for 3 minutes, keeping the centrifuge tube stable during the process. - 900µl sterile SOC or LB medium (without antibiotics) was added to each centrifuge tube, mixed, and oscillated in a 37℃ shaker for 45 minutes (150 rpm).
Protein electrophoresis-ScFv expression verification
1) Preparation of DNA gel (25g agarose gel (1%)): - Put 0.25g agarose into the conical bottle - Add 25ml 1XTAE - Microwave until bubbling, clear and clear - Add 2.5µl nucleic acid staining solution - Pour into container and insert comb to prevent air bubbles - Let stand until the glue sets
2) Preparation of the tris buffer: - Preparation of protein electrophoresis requires 100ml 20mM tris buffer - Calculated using the formula C1*V1=C2*V2 - Add 100ml of water and 1.335ml of 1.5M tris buffer to the beaker
3) Sample preparation: - There are eight tubes of bacterial solution, each about 5ml, of which four tubes have IPTG added and four tubes have no IPTG added. (the four tubes with IPTG are the experimental group, and the four tubes without IPTG are the control group) - Remove 4ml bacterial solution from each tube with a pipette and inject into a 50ml centrifuge tube. (eight tubes in total) - Centrifuge 10000-12000rpm for 5-8 min. - Remove the supernatant and add 20mM tris buffer - Ultrasonic crusher for 10 minutes for each tube of bacterial liquid
Experimental group: - Pack the experimental group bacterial solution broken by ultrasound, take 1.5ml out of each tube, and put it into 2ml centrifuge tube - Centrifuge for 5 minutes at 12000rpm, 4˚C - Place in an ice bath -Pack the supernatant and precipitation separately, take out 1ml of supernatant and add it into a new centrifuge tube, label the tube, and add 1ml of 20mM tris buffer into the remaining precipitation for re-suspension and label the tubes
Control group: - The bacterial solution of the control group broken by ultrasound was divided into four tubes and three ep tubes were divided into each tube, each tube was 1.5ml - Centrifuge for 8 min, 10000rpm, 4˚C - Take 1ml supernatant and label it
4) Heating the protein: - Select one of each sample, a total of 12 centrifugal tubes (each bacteria has three tubes: supernatant of the control group, supernatant of the experimental group, and precipitation of the experimental group) - Remove 160µl per tube and add 40µl loading buffer - Heat in a constant temperature metal field at 95˚ C for ten minutes
5) Electrophoresis: - Take 10µl of liquid per tube, inject it into the vertical electrophoresis apparatus, start the instrument, the voltage is 70 volts (the lane on the far right is marker) - When the instrument is finished, remove the lane, scrape off the concentrated glue at the top, remove the remaining part, and put it in the petri dish
6) Dyeing the glue: - Pour Coomassie brilliant blue stain into the petri dish, put the petri dish on to the shaker and wait 15 minutes - Remove the dyed gel from the dye solution and rinse it off with deionized or distilled water to remove residual dye solution from the surface. - Place the rinsed gel in the decolorizing solution, ensuring that the decolorizing solution completely covers the surface of the gel. - Use a shaker to distribute the decolorizing liquid evenly on the gel surface and accelerate the decolorizing process. The speed and time of the oscillation or shaker should be adjusted according to the actual situation to avoid excessive decolorization or damage to the gel. - As the decoloring process progresses, the color of the decoloring solution will gradually become darker. When the color of the decolorization solution is similar to the color of the gel, a new decolorization solution should be replaced to ensure the decolorization effect. - The above decolorization steps can be repeated as needed until the blue background on the gel has largely faded and the protein bands are clearly visible.
Magnetic Agarose Beads for Protein Purification:
1) Sample preparation: - Add the bacterial solution to the centrifuge tube and centrifuge at 4ºC 4000xg for 20 minutes or at 4ºC 15000×g for 1 minute - remove the supernatant to collect the precipitation. - Re-suspension precipitation with Binding/Wash Buffer. - Ultrasonic cracking of bacteria on ice. Ultrasonic power 200-300W, each ultrasonic treatment 10 seconds, each interval 10 seconds, a total of 6 ultrasonic treatment. 2) Preparation of magnetic beads: - Gently blow the heavy suspended magnetic beads with pipette, and take the appropriate amount of magnetic beads into a clean centrifuge tube according to the dosage of 1ml magnetic bead suspension for every 5mg of target protein (molecular weight of about 60kD), and magnetic separation removes the supernant; Add Binding/Wash Buffer with the same volume as the original magnetic bead suspension or appropriately larger volume to wash the magnetic bead. - Gently blow the suspended magnetic bead with the pipette. Place on magnetic rack and separate for 10 seconds to remove supernatant. - Repeat the above steps twice. 3) The target protein binds to the magnetic bead: - Add magnetic beads and incubation: add magnetic beads at the ratio of 1ml magnetic bead suspension per 5mg target protein (molecular weight of about 60kD) sample (usually about 10ml), place in a shaker, incubate at room temperature for 20-30 minutes. - Magnetic separation: After incubation, place on magnetic rack for 10 seconds to remove supernatant. Part of the supernatant was retained for testing the effect of purification. - Wash: Add 2ml Binding/Wash Buffer and gently blow the resuspended magnetic bead with the pipette. Place on magnetic rack and separate for 10 seconds to remove supernatant. Repeat washing three times. 4) Elution: - According to the concentration of the target protein and the requirements of subsequent experiments, 0.2-1ml Elution Buffer can be added, the centrifuge tube can be turned over gently several times, and the eluent can be shaken in the shaking table for 10 minutes for elution, magnetic separation, and the eluent can be collected into the new centrifuge tube, which is the purified His-Tag protein. 5) Cleaning and regeneration of magnetic beads: - Add 2ml Elution Buffer to the centrifuge tube, turn the centrifuge tube up and down several times, make the magnetic bead re-suspended, vortex for 10 seconds, magnetic separation, remove the supernatant. Repeat 3 times. - Add 2ml 20% ethanol to the centrifuge tube, turn the centrifuge tube up and down several times, so that the magnetic bead is re-suspended, the vortex is rotated for 10 seconds, the magnetic separation is done, and the supernant is removed. Repeat 3 times. - The magnetic beads are stored in 20% ethanol so that the total volume is equal to the initial suspension volume at 2-30ºC (long-term storage at 2-8ºC), which can be used for the next purification of the same protein.