Quorum Sensing

1. Add 50 ng of the LuxR-LuxCDABE/pUC18R6K-mini-
   Tn7T-GmR plasmid and 50 ng of the pTNS2 plasmid into
   100 μlofE. coliJLD271 electrocompetent cells (seeNote 8)
   and transfer the cell suspension into a 0.2 cm gap-width elec-
   troporation cuvette.


2. Electroporate at 2.5 kVand immediately add 1 ml of LB broth.
   Incubate with shaking for 1 h at 37C.


3. Plate 100μl of the cell suspension onto a selective LB agar plate
   supplemented with 10μg/ml gentamycin. Incubate at 37C
   overnight.


4. Pick five to ten colonies that grew on the selective plate and
   screen for the insertion of theluxR-luxCDABEcassette at the
   attTn7 by PCR using 300 nM Gm-up primer and 300 nM
   Gm-down primer. Use the following thermocycler parameters:
   95 C for 5 min followed by 30 cycles of 95C for 45 s, 59C
   for 30 s, and 72C for 20 s, and a final extension of 72C for
   10 min.


5. Check gene amplification by agarose gel analysis as indicated in
   Subheading3.1,steps 3– 6. In this case, the expected amplicon
   is of 6800 bp.


6. For positive clones, extract the genomic DNA by using a
   commercial kit and verify chromosomal insertion of theluxR-
   luxCDABEcassette by sequencing.

3.4.3 Subcloning of the
gkl Gene into a Modified
pBAD33 Plasmid

1. Digest the GKL/pET15b plasmid prepared in Subheading3.1,
   step 16, and modified pBAD33 plasmid with the restriction
   enzymes NdeI and BamHI. Follow the same procedure for
   DNA digestion, purification, ligation, andE. colitransforma-
   tion as in Subheading3.1,steps 7– 11.


2. In this case, verify the presence of thegklgene inside the
   modified pBAD33 vector by restriction analysis performed
   with the NdeI and BamHI restriction enzymes on plasmids
   extracted by the transformedE. colicells by means of a com-
   mercial kit.

3.4.4 Generation of the
GKL Quorum-Quenching
Molecular Circuit

1. Transform the GKL/pBAD33 plasmid into chemically compe-
   tentE. coliJLD271 reporter cells (generated in Subheading
   3.4.2), as described in Subheading3.3,step 1. In this case, use
   LB agar plates supplemented with 30μg/ml chloramphenicol
   as selective plates. Incubate the plates at 37C for 16–24 h.


2. Pick five to ten colonies grown on the selective plates and streak
   them each onto LB agar plates supplemented with 30μg/ml
   chloramphenicol, 0.02% (wt/vol) arabinose, and an AHL sub-
   strate (seeNote 4). Incubate the plates overnight at 37C.


3. Detect bioluminescence using a luminescent image analysis
   system. Set exposure time ranging from 5 s to 1 min.

Directed Evolution of Quorum Quenching Enzymes 319