IGEM:IMPERIAL/2006/Protocols/Biosensortest

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.

Protocol

• Before starting, set up a 25[[:Category:{{{1}}}|{{{1}}}]] waterbath (Dr. O'Hare's Lab?)
• Prepare the following solutions by adding Coloumns 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