Quorum Sensing

3.5 Directed
Evolution of GKL

The generation ofgklmutated genes is obtained by PCR amplifica-

tion with a blend of two error-prone DNA polymerases, Mutazyme

II (Agilent).

1. In a 200μl PCR tube, add the following: 41.5μlH 2 O, 5μlof
   10
   Mutazyme II reaction buffer, 1μl of 40 mM dNTP mix,
   0.25μl of 250 ng/μl GKL forward primer, 0.25μl of 250 ng/μ
   l GKL reverse primer, 1μl of 2.5 U/μl Mutazyme II DNA
   polymerase, 1μl of 450 ng/μl PCR product encompassing
   wild-typegklfrom Subheading3.1,step 2(seeNote 9).


2. Spin-down the samples in a benchtop microcentrifuge.


3. Amplify the samples in a thermocycler with the following para-
   meters: 95C for 2 min followed by 30 cycles of 95C for 30 s,
   64 C for 30 s, and 72C for 1 min, and a final extension of
   72 C for 10 min.


4. Quantitate the PCR yield by spectrophotometric analysis at
   260 nm with quartz cuvettes.


5. Clone the PCR products into pBAD33 via NdeI-BamBI
   restriction as previously indicated for wild-typegkl.


6. Transform the mutant plasmid library into electrocompetentE.
   coliXL1 Blue cells (Stratagene). Add the cell suspension into a
   0.2 cm gap-width electroporation cuvette and electroporate at
   1.8 kV.


7. Immediately add 1 ml of LB medium and incubate with shak-
   ing for 1 h at 37C.


8. Plate 100μl of the cell suspension onto an LB agar plate
   supplemented with 30μg/ml chloramphenicol. Incubate at
   37 C overnight.


9. Resuspend the resulting colonies in 5 ml LB broth supplemen-
   ted with 30μg/ml chloramphenicol. Grow overnight at 37C
   with shaking, harvest the cells by centrifugation, and purify the
   plasmids using a commercial kit for plasmid DNA extraction
   from bacterial cells.


10. Transform the library of mutant plasmids into 50μl of chemi-
    cally competentE. coliJLD271 reporter cells, as described in
    Subheading3.3,step 1. In this case, use LB agar plates sup-
    plemented with 30μg/ml chloramphenicol as selective plates.
    Incubate the plates at 37C for 16–24 h.


11. Streak the resulting colonies onto LB agar plates supplemented
    with 30μg/ml chloramphenicol, 0.02% (wt/vol) arabinose,
    and an AHL substrate (seeNote 4). Incubate the plates over-
    night at 37C.


12. Detect for enhanced quorum-quenching activity by biolumi-
    nescence (seeSubheading3.4.4,step 3).

320 Maybelle Kho Go et al.