- Pick colonies that have enhanced quorum-quenching activity
relative to theE. coliJLD271 reporter cells expressing wild-
type GKL. - Grow selected colonies overnight at 37C in 5 ml of LB broth
supplemented with 30μg/ml chloramphenicol. - Harvest the cells by centrifugation and extract the plasmid by
using a commercial kit for plasmid DNA extraction from bac-
terial cells. - Sequence the plasmids to obtain the DNA sequence of thegkl
mutant genes with increased lactonase activity.
4 Notes
- Transform 1μl of the commercial pET15b vector into 50μlof
chemically competentE. coliXL1 Blue cells (Stratagene). Pick a
colony and grow in 5 ml LB broth supplemented with 100μg/
ml ampicillin. Prepare a frozen stock of the cells by adding
850 μl of the culture to 250μl of sterile glycerol. Store the
stock in 80 C. The stock will be stable for at least 3 years.
You may prepare other vectors in the same manner. - You may prepare a batch of chemically competent cells instead
of using the commercially prepared cells. Grow overnight at
37 C a 5 ml LB culture of theE. colicells that you need to use
(for XL1 Blue, grow in LB supplemented with 10μg/ml
tetracycline). Inoculate a 100 ml LB culture the next day with
200 μl of the starter culture. Grow the culture at 37C with
shaking until the OD at 600 nm reaches 0.4–0.8. Harvest the
cells by centrifugation at 6000gat 4C for 10 min. Gently
resuspend the cells in ice-cold water with 10% (vol/vol) glyc-
erol and 0.1 M CaCl 2. Do not vortex. Repeat this step three
times. Resuspend the cells in 500μl of ice-cold water with 10%
(vol/vol) glycerol and aliquot 60μl of the cells in different
tubes. Freeze the tubes at 80 C. The cells will be stable for at
least 3 years. Thaw cells on ice prior to use. - Homoserine lactones have different solubility in DMSO and
H 2 O. Their solubility ranges from 100 to 500 mM in DMSO
and from 0.2 to 5 mM in H 2 O. You need to check test the
solubility of the homoserine lactone that you want to use first
before proceeding with the experiment. The homoserine lac-
tones used in this project are shown in Fig.2. - Different homoserine lactones have different activity with
GKL. You need to test their activities with GKL first before
proceeding with the experiment. - DMSO will inactivate the enzyme at high concentrations. Add
a maximum of 1% (vol/vol) DMSO in the reaction. This will
Directed Evolution of Quorum Quenching Enzymes 321