IGEM:IMPERIAL/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.

Equipment and Materials

  • Enzyme Acylase from Porcine Kidney (more info: sigmaaldrich), stored as powder in freezer
  • Potassium Phosphate (KPO4) buffer
  • MiliQ

Protocol

  • Dissolving the enzyme: (only need to do this the very first time, or if you run out of enzyme mix)
  1. Dissolve 1mg enzyme in 1mL KPO4 buffer (-> concentration of 2000units/mL)
  2. Take 0.1mL of solution made in step 1 and dilute in 0.9mL MiliQ (-> concentration of 200units/mL)
  3. Take 0.1mL of solution made in step 2 and dilute in 0.9mL MiliQ (-> concentration of 20units/mL)
  4. Take 0.1mL of solution made in step 3 and dilute in 0.9mL MiliQ (-> concentration of 2units/mL)


  • Prepare the following solutions by adding Coloumns 1, 3 and 6 respectively (add the enzyme at last & make a note of the time you added):
Label(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
A 0 1000uM 220 1000uM 1um 4uL of 1.) (=8units)
B 110 1000uM 110 500uM 500nm 2uL of 1.) (=4units)
C 0 100uM 220 100uM 100nm 4uL of 2.) (=0.8units)
D 450 1000uM 50 100uM 100nm 4uL of 2.) (=0.8units)
E 110 100uM 110 50uM 50nm 2uL of 2.) (=0.4units)
F 990 1000uM 10 10uM 10nm 4uL of 3.) (=0.08units)
G 990 100uM 10 1uM 1nm 4uL of 4.) (=0.008units)
H 990 10uM 10 100nM 0.1nm 4uL of 5.) (=8E-4units)
I 990 5uM 10 100nM 0.1nm 4uL of 6.) (=8E-5units)
J 990 1uM 10 10nM 0.01nm 4uL of 7.) (=8E-6units)
K 990 500nM 10 5nM 0.005nm 4uL of 8.) (=8E-7units)
L 990 100nM 10 1nM 0.001nm 4uL of 9.) (=8E-8units)
M 990 50nM 10 0.5nM 0.0005nm 4uL of 10.) (=8E-9units)
N 990 10nM 10 0.1nM 0.00005nm 0.5uL of 10.) (=8E-10units)
O 990 1nM 10 0.01nM 0.00001nm 0.5uL of 10.) (=8E-11units)
  • Vortex solutions shortly to mix the content
  • Leave to digest at 25 C for 10min.


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

  • To start "AHL incubation":
    • Label 15 eppendorf tubes with the appropriate letter of the row in the table above
    • Add 400μL of the appropriate AHL sample
    • Add 600 μL of T9002 of OD600 0.1 (dilute the T9002 if necessary)
    • NOTE: for row A-C only use 200μL and 300 μL respectively
    • 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 from each tube to a 96 well plate as outlined in the picture
    • Add 4 x 200uL of growth medium to a well to act as a control
  • Take the plate and to BioChem

<showhide>

  • Add 190ul of ultra pure water to the ependorf, together with 10ul of undiluted GFP standard solution and mix __HIDER__

<hide>

The undiluted GFP is in the BioChem Level 6 Cold Room on our shelf. It is in a small grey plastic box. Pippettes, tips and ultrapure water are located on a shelf above the electroporation machine in the plate reader room.

</hide> </showhide>

  • Add 2 x 200uL of the 200x diluted GFP standard solution to the wells

<showhide>

  • Take 3 readings using the Perkin Elmer Victor III (measure flourescence and absorbance, deselect any wells that are empty__HIDER__

<hide>

Use the preprogrammed Assay under the 'Students' folder called GFP + Abs490

</hide> </showhide> <showhide>

  • Save data files from computer on a memory stick
  • Copy and paste the data into the Results Spreasheet

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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