Quorum Sensing

4 Notes

1.S-adenosyl-L-methioninep-toluenesulfonate salt may also be

used in place of chloride salt. The tosylate salt usually is more

pure and thus has a lower background rate. However, we

observed that the initial rates for AHL synthase with SAM-

tosylate are lower than with SAM-chloride, possibly due to

nonspecific enzyme inhibition byp-toluenesulfonate. If possi-

ble, SAM should be synthesized in the laboratory using SAM

synthase to achieve the highest purity. The enzymatically

synthesized SAM should be stored at pH<3 to minimize

sample degradation. The pH of this sample must be raised to

neutral pH prior to use in the enzyme assay.

2. Acyl-ACP and AHL synthases were purified according to pro-
   tocols well documented in literature [6, 8–20].


3. It is preferable to use nonbinding plates for colorimetric and
   UV-transparent plates for coupled assay.


4. The volume of acyl-substrate used will depend on the stock
   concentration. For instance, in a 100μl total reaction volume,
   5 μlofa20stock is required to achieve 1substrate concen-
   tration in the assay.


5. The enzyme stocks are typically stored in 10 mM MES and 20%
   glycerol.


6. The path length for the light passing through the cuvette is
   assumed to be 1 cm. Each DCPIP reduces two CoA or holo-
   ACP thiols. After the incubation period, the background rate is
   usually low around 0.1μM/min, which does not fluctuate with
   substrate concentration. Occasionally, depending on the purity
   of substrate used, the background could be>0.2μM/min that
   could increase with higher substrate concentrations. If the
   variation in background rate between low and high substrate
   concentrations is<20%, then the rates can be averaged to
   estimate the background. However, if this rate varied more
   than 20%, then the true background rate at each substrate
   concentration must be determined.


7. Michaelis-Menten (top) and substrate inhibition (bottom)
   equation:

v 0 ¼

kcat½ŠEt½ŠS

Kmþ½ŠS

v 0 ¼

kcat½ŠEt½ŠS

Kmþ½ŠS 1 þ½ŠKSi



vois initial rate,kcatis turnover number,Kmis Michaelis con-

stant, andKiis inhibition constant.

174 Daniel Shin and Rajesh Nagarajan