September 23, 2010
( JV)
Objective: Characterized catechol degradation by xylE enzyme
Method: Measured absorbance of catechol (275nm) and 2-hydroxymuconate semialdehyde (380nm).
- Protocol:
- 1) Grow cells in M9 minimal medium
- 2) Take 1/10 dilution of cells
- 3) Introduce 1µL of 0.05M catechol solution into the cell dilution. (Final concentration of 50µM;).
- 4) Quench the reaction with 5A% w/v trichloroacetate at certain time points. (0,15sec, 30sec, 45sec, 60sec, 2min, 3min, 4min, 5min, 10min).
- 5) Spin down cells.
- 6) Measure absorbance of supernatant.
Results: