Quorum Sensing

of the His-tagged LuxR solo inE. coliin the presence and absence

of AHLs or plant extracts and for testing their solubility and stabil-

ity under native non-denaturing conditions.

1. Clone the gene coding for a LuxR solo in any one of the pQE
   vectors for expression as anN-terminal His-tagged protein.
   The gene has to be cloned in frame with the His-encoding
   tag; plasmid vectors pQE-30, pQE-31, and pQE-32 provide
   the multiple cloning site (MCS) in all three reading frames; use
   one of these depending on the desired reading frame for gen-
   erating the His-LuxR fusion protein. For more detailed infor-
   mation see the QIAexpressionist Handbook (Qiagen, Hilden,
   Germany).


2. Transform the pQE-based His-tagged-luxRsolo (pQELUXR-
   SOLO) plasmid expression construct in heterologousE. coli
   M15(pREP4) and select on LB agar plates supplemented with
   100 μg/ml ampicillin and 50μg/ml kanamycin. The presence
   of pREP4 allows control via the LacI repressor of the promoter
   controlling the expression of theluxRsolo and allowing induc-
   tion of expression upon the addition of inducer (seestep 4
   below).


3. UseE. coliM15 (pREP-4) (pQELUXRSOLO) to inoculate
   10 ml of LB broth supplemented with 100μg/ml ampicillin
   and 50μg/ml kanamycin in a 100 ml Erlenmeyer flask and
   grow overnight at 37C with shaking.


4. For determination of whether it binds an AHL, add 1 ml of the
   overnight culture to two independent 250 ml Erlenmeyer
   flasks containing 50 ml of prewarmed LB broth; of these, one
   is supplemented with 20μM of the AHL(s) of interest (see
   Note 10). Induce expression of the LuxR solo protein by
   adding 1 mM IPTG at an OD 600 of 0.6 and allow growth to
   continue for 3 h at 37C. The culture then needs to be rapidly
   chilled on ice and the cells are harvested by centrifugation
   (8000gfor 10 min at 4C) and frozen at 80 C.


5. For determination whether it binds a plant-derived molecule,
   instead of adding AHLs to the media, plant macerated material
   is provided to LB as follows: 10 g of plant material (leaves, stems,
   or roots depending on the location of the plant-associated bacte-
   ria which is being studied,seeNote 11) is frozen with liquid
   nitrogen and macerated. The resultant plant powder is added to
   100 ml LB broth and autoclaved (alternatively it can be mixed/
   vortexed with LB at room temperature for 30 min and then filter
   sterilized). This media containing plant extract can then be used
   for LuxR solo protein solubilization studies as described instep 4.


6. Resuspend the cell pellet in 5 ml of binding/lysis buffer and
   lyse the cells by sonication using 510 s pulses (200–300 W)
   pausing for 20 s in between each pulse. During sonication the
   sample should be kept on ice at all times.

152 Vittorio Venturi et al.