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Tolerance Assay

A 0.5 M (10x) stock solution of catechol was made in a 5 ml volume of autoclaved, distilled water. This solution was added to 25 ml of sterile MM2 media to make up a volume of 30 ml. This was added to 20 ml of MM2 innoculated with E.coli DH5a (approximate density 1x10e8 cells per ml) for a final volume of 50 ml (catechol concentration 50mM). Control samples were created as above minus catechol. Treated and untreated samples were placed in a 37C shaker incubator and aliquots were removed every two hours. 0.5 ml aliquots were serially diluted in 4.5 ml of MM2 media and four dilutions representing 1 x 10-3, 1 x 10-4, 1 x 10-5 and 1x10-6 were plated by spreading 200 ul of the diluted sample on LB agar plates. After drying, plates were incubated in an inverted position in a 37C incubator overnight. Plates with between 30 and 300 colonies were counted to determine the number of colony forming units using the method described by Imperial College (iGEM 2009 OpenWetWare).