Team:Hong Kong UCCKE/Experiments
We have done 4 assays, which are Assay A, Assay B, Assay C and Assay D on the cultured cells to test whether part BBa_K2197300, BBa_K2197400, BBa_K2197500 and BBa_K2197502 are valid. Protocols are included in the protocol page and we will talk about the design and the result below.
Assay A
Design
Assay A is the experiment designed to prove the validity of BBa_K2197300. By incubating engineered cells with different concentration of uric acid and measure the Green fluorescence in a plate reader, we expect that there will be a positively proportional trend as theoretically, the higher the concentration is, the more GFP will be expressed. It is because the strong repressor KRAB-HucR will stop the expression of GFP when there is no uric acid, or in very low concentration.
Steps and Result
We first pipette the cells into different concentrations of uric acid and incubate them in 96 well plate for 1.5 hours. After that, we put it into the plate reader for the measurement of green fluorescence.
The result is shown as below (Click to see raw data).
There is no significant trend shown. When higher concentrations of uric acid is added the amount of GFP measured is similar to the lower concentration ones. Therefore, we carry out another experiment, Assay B, after receiving the advice from Hong_Kong_CUHK Team.
Assay B
Design
Assay B is another experiment designed to prove the validity of BBa_K2197300. By growing engineered cells with different concentration of uric acid and measure the Green fluorescence in a plate reader, we expect that there will be a positively proportional trend as theoretically, the higher the concentration is, the more GFP will be expressed. It is because the strong repressor KRAB-HucR will stop the expression of GFP when there is no uric acid, or in very low concentration
Steps and Result
We first grow cells in LB broth overnight. Then we cultured cells from the same tube with different concentrations of uric acid-LB mixed. We then incubate the cells for 5 hours and then pipette them into the 96-well plate. After that, we put it into the plate reader for the measurement of green fluorescence.
The result is shown as below (Click to see raw data).
There is also no significant trend shown. When higher concentrations of uric acid is added the amount of GFP measured is similar to the lower concentration ones. Therefore, we tried to figure out the problem. After double checking the sequence, we found out that the DNA we ordered from IDT is different from what we designed, which the operating site HucO is missing. Referring to the mechanism of the promoter, if HucO is missing, the repressor protein mUTS can not bind to it. Thus, the amount of GFP expression cannot be restricted and no trend can be shown. However, it is possible that the GFP itself is not working or the repressor KRAB-HucR is not strong enough to repress it. And more experiments should be done to figure out the answer.
Assay C
Design
Assay C is an experiment designed to prove the validity of BBa_K2197400. By incubating engineered cells(BBa_K2197400) and Competent cells with different concentration of uric acid, and adding engineered cells (BBa_K2197300) after 1.5 hours , we measure the Green fluorescence in a plate reader. We expect that the columns with engineered cells(BBa_K2197400) will have less green fluorescence while comparing it to the column containing competent cells. As theoretically, the higher the concentration is, the more smUOX will be expressed and this will catalyse uric acid into allantoin. So by adding engineered cells (BBa_K2197300) which express more GFP when there is more uric acid, the green fluorescence of the columns with engineered cells(BBa_K2197400) will be lower than the columns with competent cells although they were in the same concentration at the beginning
Steps and Result
We first grow cells in LB broth overnight. Then we cultured cells from the same tube with different concentrations of uric acid-LB mixed. We then incubate the cells for 5 hours and then pipette them into the 96-well plate. After that, we put it into the plate reader for the measurement of green fluorescence.
The result is shown as below (Click to see raw data).
There is also no significant trend shown. When higher concentrations of uric acid is added the amount of GFP measured is similar to the lower concentration ones. Therefore, we tried to figure out the problem. After double checking the sequence, we found out that the DNA we ordered from IDT is different from what we designed, which the operating site HucO is missing. Referring to the mechanism of the promoter, if HucO is missing, the repressor protein mUTS can not bind to it. Thus, the amount of GFP expression cannot be restricted and no trend can be shown. However, it is possible that the GFP itself is not working or the repressor KRAB-HucR is not strong enough to repress it. And more experiments should be done to figure out the answer.
General Steps:
- Centrifuge 4 ml of cells and discard the LB
- Resuspend them with 200ul of PBS
- Pipette 50ul of water into A4-A12
- Pipette 50 ul of 1x10^-4 mg/dL uric acid into B4-B12
- Pipette the same volume of different concentrations of uric acid into the wells (B1-G12) as the table below:
- Add 50 ul of cultured Cells (Competent cells into Column 1-3; K2197300 into Column 4-6; K2197400 into Column 7-9 and K2197500 into Column 10-12) and let it set for 1 hour
- Add 50 ul of cultured Cells ( K2197300) into Column 7-12
- Let it set for 1-2 hours
- Place it in the plate reader to measure the Green Fluorescein
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | Concentration of Uric Acid | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A | 0 | ||||||||||||
| B | 1x10^-4 | ||||||||||||
| C | 2x10^-4 | ||||||||||||
| D | 4x10^-4 | ||||||||||||
| E | 6x10^-4 | ||||||||||||
| F | 8x10^-4 | ||||||||||||
| G | 1x10^-3 | ||||||||||||
| H | 1x10^-3 |
Results
The result is shown as above, which there is no significant trend when higher concentrations of uric acid is added. After double checking the sequence, we found out that the DNA we ordered is different from what we designed, which the operating site HucO is missing. Therefore, mUTS cannot bind to it, resulting a negative feedback in terms of GFP expression. (project design: https://2017.igem.org/Team:Hong_Kong_UCCKE/Design) However, it is possible that the GFP itself is not working or the repressor KRAB-HucR is not strong enough. More experiments to be done to figure out the answer.
Calibration
We expect that there will be a proportional trend when putting K2197300 with uric acid. When we have a positive result in assays, we can use it to determine the effectiveness of K2197400 and K2197500. When uric acid presents, K2197400 can turn uric acid into allantoin by smUOX and K2197500 can absorb uric acid by YgfU. With the result of K2197300, we can estimate the amount of reduced uric acid by comparing the light intensity measured.
Although we do not have a positive result for K2197300, we planned to do the following experiments to interpret the effectiveness of K2197400. For K2197400, we will first add 50ul of uric acid, competent cell and K2197400 to column 1 and 50ul of uric acid, K2197400 and K2197300 to column 2. By comparing the result, we will know the efficiency of K2197400 in turning uric acid into allantoin.
