IGEM:IMPERIAL/2006/Protocols/Biosensortest

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 25waterbath, 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
Equipment:
 * pH meter
 * Eppendorf Tubes
 * Small White Cap Tubes
 * Gilson Pippettes

Materials:
 * Enzyme Acylase from Porcine Kidney (more info: sigmaaldrich), stored as powder in freezer
 * AHL (N-(β-Ketocaproyl)-DL-homoserine lactone) (more info & catalogue no: sigmaaldrich), comes as powder to be stored in -20 freezer
 * - has been diluted into different stock concentrations (the solutions are stored in the -20 freezer)


 * (Potassium Phosphate (KPO4) buffer)
 * MiliQ
 * LB Medium
 * T9002 assay being cultured up following the T9002 testing protocol

Protocol
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)
 * Dissolving the enzyme: (only need to do this the very first time, or if you run out of enzyme mix)

1.) Prepare the solutions as outlined in the table by adding Columns 1,3 and 6 on ice. Add the enzyme at last.
 * Before starting, set up a 25 waterbath
 * NOTE: For an initial test, only test rows D, F, G

2.) Again, prepare the solutions as outlined in the table. However, instead of using LB in Coloumn 1, use MiliQ. Add Columns 1 (MiliQ),3 and 6 on ice; add the enzyme at last.


 * REMARK: Because of the size of the pH glass electrode, the volumes used (see table below) had to be at least 1.5mL. If a smaller pH electrode is used, smaller volumes can be used to be tested.

(Note: The seemingly arbitrary numbers in row A-E were chosen because of the limited stock and concentrations of AHL available)

If activity of the enzyme has to be checked:
 * Add the enzyme just before putting the mixtures into 25 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 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
 * Follow the Biosensor Testing: Enzyme Activity protocol afterwards

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