Quorum Sensing

3 Methods

3.1 Dichloro-pheno-
lindophenol (DCPIP)
Colorimetric Assay

3.1.1 Preparation
of Stock Solutions

1. Prepare the following stock solutions: 10DCPIP stock solu-
   tion (300–500 μM) in nanopure water, 10 SAM stock
   solution (5–10 mM) in nanopure water, 10HEPES buffer
   (1 M, pH 7.3), and several acyl-CoA stocks (5–20) in nano-
   pure water to cover a range of 1–200 μM final acyl-CoA
   concentrations in the assay (seeNote 4).


2. Dilute acyl-ACP and enzyme freezer stocks using 10 mM MES
   and 20% glycerol storage buffer to make 5–20stock for the
   assay (seeNote 5).


3. Use the following extinction coefficients to determine stock
   concentrations:
   (a) DCPIPε 600 ¼21,000 M^1 cm^1
   (b) SAMε 260 ¼15,400 M^1 cm^1
   (c) Acyl-CoAε 260 ¼13,500 M^1 cm^1
   (d) Acyl-ACPε 280 ¼1490 M^1 cm^1


4. Store both enzyme and substrate solutions in ice throughout
   the experiment.

3.1.2 Determination
of Background Rates

1. Set up the cuvette as mentioned in Table1. Add all compo-
   nents except AHL synthase enzyme. The total cuvette volume
   in the assay is 100μl, which can be scaled up or down depend-
   ing on the type of cuvette/microplate accessory.


2. Instead of enzyme, add 10μl nanopure water to the cuvette
   and record progress curve at 600 nm for 15–20 min.


3. Keep SAM concentration between 200 and 500μM and vary
   the acyl-ACP/acyl-CoA concentrations over a wide range, e.g.,
   10 μM (low), 50 μM (medium), and 100–200μM (high).

Table 1

DCPIP assay setup

Component Volume (μl)

10 HEPES buffer 10

Water 60 x

10 DCPIP 10

10 SAM 10

Acyl-CoA/acyl-ACP x

AHL synthase 10

Total 100

Acyl-Homoserine Lactone Synthase Assays 167