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
- Dissolving the enzyme: (only need to do this the very first time, or if you run out of enzyme mix)
Soln 1: Dissolve 1mg enzyme in 1mL KPO4 buffer (-> concentration of 2000units/mL) Soln 2: Take 0.5mL of Soln 1 and dilute in 0.5mL MiliQ (-> concentration of 1000units/mL) Soln 3: Take 0.1mL of Soln 1 and dilute in 0.9mL MiliQ (-> concentration of 200units/mL) Soln 4: Take 0.1mL of Soln 2 and dilute in 0.9mL MiliQ (-> concentration of 100units/mL) Soln 5: Take 0.1mL of Soln 3 and dilute in 0.9mL MiliQ (-> concentration of 20units/mL) Soln 6: Take 0.1mL of Soln 5 and dilute in 0.9mL MiliQ (-> concentration of 2units/mL) Soln 7: Take 0.1mL of Soln 6 and dilute in 0.9mL MiliQ (-> concentration of 0.2units/mL) Soln 8: Take 0.1mL of Soln 7 and dilute in 0.9mL MiliQ (-> concentration of 0.02units/mL) Soln 9: Take 0.15mL of Soln 8 and dilute in 1.35mL MiliQ (-> concentration of 0.002units/mL) Soln 10: Take 0.5mL of Soln 9 and dilute in 0.5mL MiliQ (-> concentration of 0.001units/mL) Soln 11: Take 0.1mL of Soln 10 and dilute in 0.9mL MiliQ (-> concentration of 0.0002units/mL) Soln 12: Take 0.1mL of Soln 10 and dilute in 0.9mL MiliQ (-> concentration of 0.0001units/mL) Soln 13: Take 0.1mL of Soln 11 and dilute in 0.9mL MiliQ (-> concentration of 0.00002units/mL) Soln 14: Take 0.1mL of Soln 13 and dilute in 0.9mL MiliQ (-> concentration of 0.000002units/mL)
- Before starting, set up a 25[[:Category:{{{1}}}|{{{1}}}]] waterbath (Dr. O'Hare's Lab?)
- Prepare the following solutions by adding Columns 1,3 and 6 ON ICE. Add the enzyme at last:
Label | (1) LB medium (μL) |
(2) Available Stock Concentr of AHL |
(3) Volume of AHL to add (μL) |
(4) Final AHL Concentration |
(5) Amount of AHL present in solution |
(6) Volume of Enzyme to add |
---|---|---|---|---|---|---|
A | 0 | 1000uM | 200 | 1000uM | 1um | 4uL of Soln 1 (=8units) |
B | 100 | 1000uM | 100 | 500uM | 500nm | 4uL of Soln 2 (=4units) |
C | 0 | 100uM | 200 | 100uM | 100nm | 4uL of Soln 3 (=0.8units) |
D | 450 | 1000uM | 50 | 100uM | 100nm | 4uL of Soln 3 (=0.8units) |
E | 100 | 100uM | 100 | 50uM | 50nm | 4uL of Soln 4 (=0.4units) |
F | 990 | 1000uM | 10 | 10uM | 10nm | 4uL of Soln 5 (=0.08units) |
G | 990 | 100uM | 10 | 1uM | 1nm | 4uL of Soln 6 (=0.008units) |
H | 990 | 10uM | 10 | 100nM | 0.1nm | 4uL of Soln 7 (=8E-4units) |
I | 990 | 5uM | 10 | 100nM | 0.1nm | 4uL of Soln 8 (=8E-5units) |
J | 990 | 1uM | 10 | 10nM | 0.01nm | 4uL of Soln 9 (=8E-6units) |
K | 990 | 500nM | 10 | 5nM | 0.005nm | 4uL of Soln 10 (=4E-6units) |
L | 990 | 100nM | 10 | 1nM | 0.001nm | 4uL of Soln 11 (=8E-7units) |
M | 990 | 50nM | 10 | 0.5nM | 0.0005nm | 4uL of Soln 12 (=4E-7units) |
N | 990 | 10nM | 10 | 0.1nM | 0.00005nm | 4uL of Soln 13 (=8E-8units) |
O | 990 | 1nM | 10 | 0.01nM | 0.00001nm | 4uL of Soln 14 (=8E-9units) |
(Note: The seemingly arbitrary numbers in row A-E were chosen because of the limited stock and concentrations of AHL available)
- Add the enzyme just before putting the mixtures into 25[[:Category:{{{1}}}|{{{1}}}]] waterbath
- Vortex solutions shortly to mix the content
- Measure and write down the pH of all solutions before taking them off ice
- Make a note of the time and take the solutions off the ice into a 25[[:Category:{{{1}}}|{{{1}}}]] 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 10min. (take a reading every minute)
- After 10min. the pH should have reached a steady value, if not, leave until it does not change any more
- Write down the final pH
If activity of the enzyme has to be checked:
- Follow the Biosensor Testing: Enzyme Activity protocol afterwards