Quorum Sensing

3.4 Construction of a
Quorum-Quenching
Directed Evolution
Platform (Fig.1)

3.4.1 Amplification and
Cloning of the luxR-
luxCDABE Cassette

1. Amplify theluxR-luxCDABEcassette from the pAL103 vector
   [8] by PCR using the oligonucleotides Lux forward primer and
   Lux reverse primer.


2. Amplify the gene using the following parameters: 98C for
   2 min followed by 30 cycles of 98C for 10 s, 55C for 15 s,
   and 72C for 15 s, and a final extension of 72C for 10 min.


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


4. Purify the band from agarose gel by using a commercial kit.


5. Digest the eluted PCR product and the pUC18R6K-mini-
   Tn7T-GmRvector [1] by using the XhoI and KpnI restriction
   enzymes. Follow the same procedure for DNA digestion, puri-
   fication, ligation, andE. colitransformation as in Subheading
   3.1,steps 7– 11.


6. In this case, verify the presence of theluxR-luxCDABEcassette
   inside the pUC18R6K-mini-Tn7T-GmRvector by restriction
   analysis performed with the XhoI and KpnI restriction enzymes
   on plasmids extracted by the transformedE. colicells by means
   of a commercial kit.

3.4.2 Integration of the
luxR-luxCDABE Cassette
into E. coli JLD271 Cells

Co-transform the plasmid prepared in Subheading3.4.1and the

helper plasmid pTNS2 [1]inE. coli JLD271 cells by

electroporation.

Fig. 1Schematic representation of the quorum-quenching molecular circuit

318 Maybelle Kho Go et al.