Difference between revisions of "Team:Hong Kong UCCKE/Experiments"
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<a href="https://2017.igem.org/File:T--Hong_Kong_UCCKE--assay_results.png"> | <a href="https://2017.igem.org/File:T--Hong_Kong_UCCKE--assay_results.png"> | ||
<img src="https://2017.igem.org/File:T--Hong_Kong_UCCKE--assay_results.png" alt="" style="width:100%;"> | <img src="https://2017.igem.org/File:T--Hong_Kong_UCCKE--assay_results.png" alt="" style="width:100%;"> | ||
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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 04:02, 28 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 incubate the cells for 24 hours and then pipette them into different concentrations of uric acid. 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, and K2197500 respectively.
General Steps:
- Pipette 100ul of water into A4-A12
- Pipette 100 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 100 ul of cultured Cells (part 400 and 500) into Column 7-12 and let it set for 1 hour
- Add 100 ul of cultured Cells (part 300) into Column 4-12
- 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.
