Quorum Sensing

3. Add 1 ml of the culture grown instep 2in 2-l conical flasks
   containing 1 l of LB broth supplemented with 100μg/ml
   ampicillin. Let the culture grow for 6–8 h with shaking at
   37 C.


4. When the OD at 600 nm reaches 0.4–0.8, add 0.1 mM IPTG.
   Continue shaking the culture at 37C for 16 h.


5. Harvest the cells by centrifugation at 4000
   gfor 10 min at
   4 C.


6. Resuspend the cells in 40 ml of binding buffer at 4C.


7. Break the cells using a sonicator. Sonicate for 8 min in ice at
   20 Hz, with 5 s intervals “on” and 10 s intervals “off.”


8. Separate the cell debris from the cell supernatant by centrifuga-
   tion at 40,000
   gfor 20 min at 4C.


9. Load the cell supernatant into a Ni2+chelating sepharose col-
   umn (10 ml column volume). Wash the column with the
   following buffers in sequence: 40 ml binding buffer, 40 ml
   wash buffer, and 40 ml histidine-elute buffer. Collect 2 ml
   fractions of the histidine-elute buffer flow through.


10. Check the histidine-elute fractions for the presence of GKL by
    sodium dodecyl sulfate–polyacrylamide gel electrophoresis
    (SDS-PAGE). The expected molecular weight of the purified
    protein is 36.4 kDa.


11. Pool the fractions containing the protein together and concen-
    trate the solution by using a 10 kDa centrifugal filter.


12. Dialyze the concentrated protein fraction 1:200 at 4C against
    the following buffers in sequence: bipyridyl dialysis buffer,
    storage buffer, ZnCl 2 dialysis buffer, and storage buffer.


13. Aliquot 100μl of the dialyzed protein in separate tubes and
    store at 80 C.

3.3 Kinetic Assays of
Lactonase Activity

1. Equilibrate a UV-Vis spectrophotometer to 37C. Set the
   detector to measure the absorbance at 577 nm.


2. In a 1 ml quartz cuvette, add 10μl of bicine buffer, 8μlof
   cresol purple (577 nm,ε¼12,500 M^1 cm^1 ), 1μl of ZnCl 2
   dialysis buffer, 25–50μM homoserine lactone (seeNote 3), and
   1–10μM GKL (seeNote 4). Add enough DMSO to make the
   final concentration of DMSO in the cuvette 1% (vol/vol) (see
   Note 5). Add enough H 2 O to bring the solution to 1 ml.


3. Monitor the change of absorbance at 577 nm for 5–10 min (see
   Note 6).


4. Determine the kinetic parameters by generating a Michaelis-
   Menten curve (seeNote 7).

Directed Evolution of Quorum Quenching Enzymes 317