IGEM:IMPERIAL/2009/M2/Assays/3.2

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Functional Trehalose Assay

Functional Assay:
By simulating stressful environmental conditions (notably dessication), and monitoring the change on the activity of catalase, we can see the effects of changes on the activity of catalase present within the cells. This will give us a quantifiable output as to the protein protection offered by the trehalose.


Our testing construct for the trehalose functional assay involves the testing construct shown below.
II09 OTSAOTSB.JPG


Protocol

1) Take a 50ml culture of cells and start trehalose production by inducing with 50uL of 1M HSL(Final Conc = 1mM HSL).

2) After induction (including time=0), take a sample every half hour up to 5 hours. This means the trehalose concentration will increase throughout, to a steady state.

3) With each sample take out a 0.5ml sample to take an OD reading. Take 2 X 1ml samples out and put in eppendorf tubes. According to the OD readings given, dilute to give the same cell number per unit volume. Use OD=0.7 as standard??

Medical Isotonic solutions have a NaCl concentration of 0.9% (w/v)


4) Set up the oxygen electrode and add 1ml of 50mM Na2HPO4-KH2PO4-pH7.0 Buffer. In the case of the dessication add NaCl???

5)Allow to equilibrate to 25'C and establish recorder setting near 100%

6) Bubble buffer system with N2 gas until zero setting is established, and close off reaction chamber to atmosphere.

7) Add 10uL H2O2 (33.5mM conc) solution through injection port and allow to equilibrate.

8) Add 50uL of suitably diluted catalase sample and the initial velocity of reaction may be established from the kinetics of the O2 production rate. (Note that the non-enzymatic production of O2 should be deducted ~ respiration found to be negligible in another paper )

9) Normalise with respect to the cell density, and from that work out the catalase activity per cell.


O2 Electrode Basic Assay
Old Catalase Assay, Main Points Useful