Quorum Sensing

A thermostable lactonase fromGeobacillus kaustophilus(GKL)

was used as the template for the in vitro evolution of enhanced

quorum-quenching activity. GKL was chosen as the template for

in vitro evolution because of its high solubility and expression and

high thermostability. However, it was found to only catalyze a

limited range of medium to long-chain AHLs. Thus, this study

created a robust and tunable directed evolution platform to screen

for enhanced quorum-quenching activity against a broad range of

AHLs. This will allow the identification of mutants that will have

better or broader range of lactonase activity compared to the wild-

type GKL.

2 Materials

Prepare all solutions using distilled and deionized water and analyt-

ical grade reagents. Prepare and store all reagents at room tempera-

ture (unless indicated otherwise). Diligently follow all waste

disposal regulations when disposing waste materials.

1. 1
   TAE buffer [tris(hydroxymethyl)aminomethane (Tris)-
   Acetate-Ethylenediaminetetraacetic acid (EDTA)]: dissolve
   4.84 g Tris and 0.37 g EDTA disodium salt in 500 ml H 2 O
   while stirring, add 57.1 ml glacial acetic acid and bring the
   volume to 1 l.


2. 1% (wt/vol) agarose gel: dissolve 0.5 g of agarose in 50 ml 1
   
   TAE buffer. Heat the solution until the agarose is fully dis-
   solved. Add 5μl of a 10 mg/ml ethidium bromide solution.
   Swirl the solution until the ethidium bromide is fully dissolved.
   Pour the hot solution carefully onto a tray of a DNA electro-
   phoresis apparatus and place an appropriate gel comb. Cool the
   molten gel at room temperature until it is solid.


3. Antibiotic stock solutions: 100 mg/ml ampicillin in H 2 O;
   30 mg/ml chloramphenicol in ethanol; 10 mg/ml gentamycin
   in H 2 O. Filter the stock solutions using 0.22μm membrane.
   Store the solutions at 20 C. The stock solutions are stable for
   at least 1 year. Upon addition of the antibiotics in LB media or
   LB agar, the media or plates are stable for 2 months at 4C.


4. Luria-Bertani (LB) broth: 10 g/l tryptone, 5 g/l yeast extract,
   10 g/l NaCl. Shake the solution on a magnetic stirrer to
   prevent clumps. Autoclave the medium at 121C for 20 min.
   Cool the solution to room temperature prior to use. If required
   add antibiotics: 100μg/ml ampicillin; 30μg/ml chloramphen-
   icol; 10μg/ml gentamycin.


5. LB/antibiotic agar plates: 10 g/l tryptone, 5 g/l yeast extract,
   10 g/l NaCl, 15 g/l agar. Shake the solution on a magnetic
   stirrer to prevent clumps. Autoclave the medium at 121C for

312 Maybelle Kho Go et al.