IGEM:IMPERIAL/2006/Protocols/BiosensorEnzymeAct

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Contents

Biosensor: Checking & assessing the activity of the enzyme (acylase)

Motivation

We would like to check whether the enzyme is actually degrading the AHL present in solution (regardless of whether we get a pH change or not). In order to assess the activity of the enzyme, the protocol below should be carried out. Since the enzyme is being used in the J37015 Testing Protocol, this protocol is to assess the activiy of the enzyme for these purposes as well.

Equipment and Materials

Equipment:

  • pH meter
  • Eppendorf Tubes
  • Small White Cap Tubes
  • Gilson Pippettes
  • 37[[:Category:{{{1}}}|{{{1}}}]] shaker
  • Wallac Victor 3 Multi-Well Fluorimeter

Materials:

- has been diluted into different stock concentrations (the solutions are stored in the -20[[:Category:{{{1}}}|{{{1}}}]] freezer)
  • (Potassium Phosphate (KPO4) buffer)
  • MiliQ
  • (LB Medium)
  • T9002 assay being cultured up following the T9002 testing protocol

Protocol

  • This protocol is dependent on the T9002 testing protocol:
    • Follow the T9002 testing protocol up to the AHL incubation, then follow the protocol as outlined below
    • NOTE: Since the T9002 testing is carried out in parallel, any T9002 cultures needed can be diluted from that experiment (about 4mL of T9002 cells of OD600 0.1 are needed)


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) 


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 50nM 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)
P 1000 N/A N/A N/A N/A N/A

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


After having carried out the pH measurement, as described in the Biosensor testing protocol:

Use the T9002 Protocol as AHL assay as outlined below:
<bbpart>T9002</bbpart> Protocol Section (slightly revised)

  • To start "AHL incubation":

<showhide>

  • Label 9 eppendorf tubes for rows A,B,C,E,H,K,N,O,P with the appropriate letter of the row in the table above__HIDER__

<hide>

We are only testing some samples of the above table since if the highest and lowest concentrations of AHL are degraded, all the other concentrations in betweeen should be degraded as well.

</hide>

  • Add 400μL of the appropriate AHL-enzyme mixture (NOTE: Add 200μL for row A,B,C,E)
  • Add 600 μL of T9002 of OD600 0.1 (dilute the T9002 if necessary) (NOTE: Add 300μL for row A,B,C,E)
  • At this stage, a pH buffer might be added (pH 7 or 9)
  • Vortex each tube
  • Incubate the 15 new eppendorf tubes in a 37°C shaker for 4 hours so GFP expression can reach steady state
  • After 4 hours:
    • Add a 200uL sample (with repeats) from each tube to a 96 well plate as outlined in the picture
    • Add 4 x 200uL of LB medium to a well to act as a control
  • Take the plate to BioChem
  • Add 190ul of MiliQ to the eppendorf, together with 10ul of undiluted GFP standard solution and mix
  • Add 4 x 200uL of the 200x diluted GFP standard solution to the wells
  • Take 3 readings using the Perkin Elmer Victor III: Measure flourescence and absorbance, deselect any wells that are empty
  • Save data files from computer on a memory stick
  • Copy and paste the data into the Results Spreadsheet

Expected Results

  • Assuming the enzyme degrades all the AHL present in the solution, all the solutions with previously different concentrations of AHL being added should show no fluorescence induced by AHL. The fluorescence values should all equal since all should have the same background fluorescence.


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