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 paper
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
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.
Another assay for colonic acid from here
Principle : Measure fucose concentration
1. Dilute 10 to 100 μl of the colanic acid preparation to 1 ml with distilled water
2. mixed it with 4.5 ml of H2SO4/H2O (6:1; v/v) at room temperature
3. heat at 100 °C for 20 min
4. cooled it down to room temperature
5. For each sample measure absorbance at 396 nm and 427 nm was measured
a. directly (control sample (A-co))
b. After addition of 100 μl of cysteine hydrochloride (cysteine sample (A-cy)).
6. Heat with H2SO4 to yield brown products absorbing between 396 nm and 427 nm.
7. Subtract the absorption due to this unspecific reaction from the total absorption of the sample: A396-co and A427-co were subtracted from A396-cy and A427-cy, respectively, to obtain ΔA396 and ΔA427.
8. Values of (ΔA396–ΔA427) were directly correlated to methylpentose concentration by using a standard curve obtained with a fucose concentration ranging from 5 μg/ml to 100 μg/ml.
