Difference between revisions of "Team:Hong Kong UCCKE/Experiments"
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<p style="text-align:left !important;">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) | <p style="text-align:left !important;">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. </p> | 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. </p> | ||
Revision as of 10:52, 30 October 2017
Assay
We have done assays on the cultured cells.
The aim of doing these assays was to test whether part 300,400 and 500 are valid. We expect there will be a positively proportional trend when putting part 300 into increasing concentrations of uric acid. The higher the concentration is, the more GFP expression. Below I explain the steps and calculations involved in these assays in detail.
We first centrifuge the cell and resuspend it with PSD. We then add uric acid into the cell for growth. We also grow cell with uric acid in different concentrations for the second experiment. We expect that there will be an increase in GFP expression when it was put in higher uric acid concentration. When there is no uric acid, or in very low concentration, the strong repressor KRAB-HucR will stop the expression and thus no GFP expression. For Column 1-3, 4-6, 7-9, and 10-12, they are uric acid with water, K2197300, K2197400 with K2197300, and K2197500 with K2197300 respectively.
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.
