IGEM:IMPERIAL/2006/Protocols/Biosensortest
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Biosensor: Calibration & Characterization of the Sensitivity of the Sensor
Motivation
In order to use the biosensor for later experiments, a calibration curve has to be established first. With this testing protocol, the relation between pH and AHL concentration should be extracted. Thus, the sensitivity of the sensor can be determined and characterized.
- JS: is it possible that we can get a continuous readout of the pH, so we can assess the variability in the pH meter over time? Depending upon if it hits a spot of a higher concentration of AHL, it might increase the pH level (or decrease) if it is not stirred properly.
- CS: Yes, the pH will be read in real-time to get an idea of the activity of the enzyme (that is why everything is prepared on ice first). During digest in the 25[[:Category:{{{1}}}|{{{1}}}]]waterbath, the pH should be observed for any changes in time until it reaches a steady value. The whole solution should have been well-mixed before.
Equipment and Materials
Protocol
- Prepare the following solutions ON ICE by adding Coloumns 1, 3 and 6 respectively (add the enzyme at last):
| (1) LB medium (ul) |
(2) Available Stock Concentr of AHL |
(3) Volume of AHL to add (ul) |
(4) Final AHL Concentration |
(5) Amount of AHL present in solution |
(6) Volume of Enzyme to add |
|---|---|---|---|---|---|
| 1800 | 1000uM | 200 | 100uM | 200nm | |
| 1980 | 1000uM | 20 | 10uM | 20nm | |
| 1980 | 100uM | 20 | 1uM | 2nm | |
| 1980 | 10uM | 20 | 100nM | 0.2nm | |
| 1980 | 5uM | 20 | 50nM | 0.1nm | |
| 1980 | 1uM | 20 | 10nM | 0.02nm | |
| 1980 | 500nM | 20 | 5nM | 0.01nm | |
| 1980 | 100nM | 20 | 1nM | 0.002nm | |
| 1980 | 50nM | 20 | 0.5nM | 0.001nm | |
| 1980 | 10nM | 20 | 0.1nM | 0.0002nm | |
| 1980 | 1nM | 20 | 0.01nM | 0.00002nm | |
| 2000 | N/A | 0 | 0nM | 0nm |
- This results in having 11 different AHL concentrations in 2mL LB each
- Vortex solutions shortly to mix the content
- Go over to Dr. O'Hare's Lab (for measuring the pH) - leave everything on ice
- In Dr. O'Hare's Lab:
- Make a note of the time and take the solutions off the ice into a 25 C waterbath (make sure the contents is well-mixed before)
- Immediately after putting the solutions into the waterbath - stick in the pH electrode to get a real-time reading
- Note down the initial pH of all solutions
- Observe and write down all changes in pH over the next 1/2 h
- After 1/2 h the pH should have reached a steady value, if not, leave until it does not change any more
- Write down the final pH
