IGEM:IMPERIAL/2009/Assays Protocols

Assays

Module 1

Cellulase

Cellulase Assay from Sigma Aldrich
Cellulase Assay (Sigma)

Alternative Cellulase Assay
| Cellulase Assay

Phenylalanine Hydroxylase (PAH)

Phenylalanine Hydroxylase Assay from Sigma Aldrich :
PAH Assay

Module 2

Colonic acid

Quantification of total EPS

1. collect 60 mg of mucoid or non-mucoid bacterial culture from the surface of LB agar plates incubating for 12-h at 37 °C

2. resuspended in 1 ml sterile distilled water by vortex

3. determine cell concentration by cell turbidity at 600 nm

4. To inactivate EPS-degrading enzymes and release EPS

a. boil resuspended culture for 10 min b. cool to room temperature c. centrifuge at16000g for 10min d. save supernatant fraction at - 20 °C for quantification

5. use the anthrone-H2SO4 assay to quantify total EPS Mendrygal and González, 2000 a. prepare 0.4 ml suitably-diluted sample b. mixed it with 0.1 ml of fresh 2% anthrone in ethyl acetate in a 10 ml glass test tube. c. Add 1ml of 95–97% H2SO4 into each sample d. cooled for 10 min to room temperature e. determine the value of the absorbance at 620 nm f. Glucose equivalents in the EPS samples were quantified using a calibration curve with glucose from 10 to 80 g ml- 1 g. Normalise these values by cell turbidity at 600 nm

Note: Perform all experiments with two independent cultures.

Colonic acid assay

This method is based on the specific difference in absorbance at 396 and 427 nm after reacting fucose with sulfuric acid and cysteine hydrochloride

1. Measure the fucose concentration to determine colonic acid concentration

2. Use a L-fucose (Acros Organics) calibration curve from 10–60 g ml- 1

3. normalise these values by cell turbidity at 600 nm

4. For negative control, glucose was assayed. It should not produce a significant signal.

Note: Perform all experiments with two independent cultures.