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Measurement
To obtain the pili yield of our production, we first used the original method to determine the yield by measuring the concentration of purified e-pili solution with the BCA Protein Assay Kit. However pili might be lost due to the long periods of purification and multiple steps of extraction. Therefore, we established a new measurement for a quicker, clearer and more accurate determination of pili production with few hours. We uses his-tags grow on the bacteria outer membrane to combine with the primary antibodies and secondary antibodies directly without extracting and doing Western Blot.
Procedure:
1. Use 10 ul of required bacteria solution. Streak a LB broth to obtain single colony. Cultivate in 37oC incubator overnight.
2. Inoculate a single colony from the LB plate into 5 ml of liquid LB medium and add proper antibiotics and incubate overnight on a rotary shaker at 200 r.p.m. and 30oC.
3. Scrap each bacteria into 6ml of liquid M9 medium.
4. Take 300 ul of solution and spread on one M9 broth. One bacteria should be cultivated on 12 M9 plates.
5. Use parafilm to seal half of the M9 plates (6 plates) and incubate all plates in 30oC in incubator.
6. After 24 hours, take two plates unsealed and two plates sealed out. Scrap the bacteria into a solution made up of 1x M9 with 25% glycerol and 3 ml for each plate. Store at -20oC.
7. After 48 hours, take two plates unsealed and two plates sealed out. Scrap the bacteria into the solution and stored at -20oC.
8. After 72 hours, take two plates unsealed and two plates sealed out. Scrap the bacteria into the solution and stored at -20oC.
9. Take out all the solutions and use the 96 well EIA/RIA plate to measure the od of each bacteria solution. In each hole, add 90 ul of PBS solution and 10 ul of each bacteria solution.
10. Adjust all solution into same od in 1.5ml solution into 2ml ep tube.
11. Centrifuge all solutions and discard the supernatant. Add 500 ul of PBS and resuspend the bacteria to wash the buffer.
12. Centrifuge all solutions and discard the supernatant. Add 100ul of diluted primary antibodies(1:500) to each tube. Resuspend the bacteria using vertex and incubate on a rotary shaker at 4oC and 200 r.p.m.
13. After 1 hour, centrifuge all solutions and discard the supernatant. Add 500 ul of washing buffer and resuspended the bacteria.
14. Centrifuge all solutions again and discard the supernatant. Add 500 ul of washing buffer and resuspended the bacteria on vertex and incubate on a rotary shaker at 4oC and 200 r.p.m.
15. After 5 min, centrifuge all solutions again and discard the supernatant. Add 500 ul of washing buffer and resuspended the bacteria on vertex and incubate on a rotary shaker at 4oC and 200 r.p.m.
16. After 15 min, centrifuge all solutions again and discard the supernatant.
17. Add 100 ul of secondary antibodies and resuspended the bacteria on vertex and incubate on a rotary shaker at 4oC and 200 r.p.m.
18. After 1 hour, centrifuge all solutions and discard the supernatant. Add 500 ul of washing buffer and resuspended the bacteria.
19. Centrifuge all solutions again and discard the supernatant. Add 500 ul of washing buffer and resuspended the bacteria on vertex and incubate on a rotary shaker at 4oC and 200 r.p.m.
20. After 5 min, centrifuge all solutions again and discard the supernatant. Add 500 ul of washing buffer and resuspended the bacteria on vertex and incubate on a rotary shaker at 4oC and 200 r.p.m.
21. After 15 min, centrifuge all solutions again and discard the supernatant.
22. Add Diaminobenzidine DAB in the proportion of 1:1 of A and B solution and total 100 ul to each tube. Resuspend the bacteria using a vertex and keep in dark place overnight.
23. Use a 96 well EIA/RIA plate to measure the od 450 and od 600 for all solutions.
24. Analyze the datas.
25. Calculate the average value for the two same plate and use the value at od 450 divided by the value at od 600.
Datas:
Our new measurement is based on the combination of His-tags with the primary antibodies and secondary antibodies directly on the pili on E.coli without extracting and doing Western Blot.
In the experiment, we washed the bacteria with PBS solution and adjust all samples to the same od 600. Then we added primary antibodies and waited for 1 hours and washed three times. Repeat the process with secondary antibodies with HRP and then allowed the solution to react with DAB for 12 hours in darkness.
In our experiment, we measures the yield of four different generators, BBa_K3552009, BBa_K3552010, BBa_K3552011 and BBa_K3552012. We collect the data of production of 24 hours, 48 hours and 72 hours of sealed and unsealed plates. In the analyzation of the datas, we uses the 96 well EIA/RIA plate and add 100 ul of colored solution with same concentration under 12 hours of reaction in darkness to each well with two negative controls. We first measured four values for visually colored solution and negative controls from od 300 to od 800 and we discovered the DAB absorption peak at od 450. After the verification of peak value at 450 we used this value to divided by the value at od 600 which almost kept constant for all samples. Then we measured the datas of each well and subtracted the average value of negative control. This was the final optimized od 450 on our graph.
In the new measurement, we achieved the possibility of directly comparing the production of different pili which wasn't achieved before by other methods. We had carried out the experiments for a few times repeating of the optimized od 450 for four types of pilA with generators. Although for each single measurement, we couldn't conclude extremely precise results, the tendency of production for four different pili in different condition was the same. This supports that our experiments is repeatable by others. When taking the average value in one plate, the deviation were quite small and the results were precise. However, for different plates there will be large variance.
Resulting from the requirement of many labor forces to obtain accurate result but according to time insufficiency, we didn't receive absolutely precise or accurate result. In the future, we are trying to amplify our experiment to achieve better results. We will carry out large-scale sampling such as six plates for each bacteria and two detection for the same plate. Therefore the repeat in sampling could prove the reliability of our measurement and to see whether it is stable or not. We will continue to find the methods the optimize our measurements.
The most valuable meaning of this new measurement is that it is not only able to detect the production of pili for bacteria, but also provide the ability to test any protein that is expressed out of the cell membrane. Without the extraction or purification, we are able to detect the yield and the expression of protein through the new measurement.