Quorum Sensing

Solution C: 50% Glycerol in water, autoclave.

Solution D: 0.1 M Arginine dissolved in water, filter sterilize.

To 960 ml of Solution A, add 10 ml of Solution B, 20 ml of

Solution C, and 10 ml of Solution D to make 1.0 l of AB

medium.

5. Antibiotics: Stocks of ampicillin (100 mg/ml) and kanamycin
   (100 mg/ml) prepared in water are used with growth media to
   the desired final concentrations by diluting the stocks to
   1000
   fold in growth media (e.g., add 1 ml of antibiotic
   stock to 1.0 l of growth medium). Before use, stocks are filtered
   sterilized with 0.22μm PVDF membrane syringe filter disc.
   Aliquots of 1.0–5.0 ml are stored at 20 C.


6. Solid agar media plates: Prepare using 15 g/l of select agar in
   desired bacterial growth media and autoclave for 20–30 min.


7. Incubator shaker fitted with desired adaptors for bacterial cul-
   ture growth.

2.3 Protein
Overexpression
and Purification

1. 1.0 M Isopropyl thiogalactoside (IPTG) is dissolved in distilled
   water and filter sterilized with 0.22μm PVDF membrane
   syringe filter disc.


2. Lysozyme stock of 50 mg/ml in distilled water, stored as 25μl
   aliquots at 20 C.


3. 0.1 M Phenylmethyl sulfonyl fluoride (PMSF) in isopropanol,
   stored at 20 C(seeNote 1).


4. Following stocks in water are prepared and autoclaved: 1.0 M
   NaH 2 PO 4 , 1.0 M Na 2 HPO 4 , and 3.0 M NaCl.


5. A buffer stock of 1.0 M NaH 2 PO 4 -Na 2 HPO 4 (pH 8.0) is
   prepared by mixing appropriate volumes of 1.0 M NaH 2 PO 4
   and 1.0 M Na 2 HPO 4. For 100 ml of stock add 94.7 ml of
   Na 2 HPO 4 and 5.3 ml of NaH 2 PO 4 and check the final pH
   using pH electrode.


6. 2.5 M Imidazole in water, adjust pH to 7.5 with HCl and filter
   sterilize. Store at 4C.


7. Buffer A (column equilibration/wash buffer): 25 mM
   NaH 2 PO 4 -Na 2 HPO 4 (pH 8.0), 35 mM NaCl, and 10 mM
   imidazole.


8. Buffer B (lysis buffer): To buffer A solution add 15 mM 2-
   mercaptoethanol (2-ME), 1 mM PMSF, and 0.2 mg/ml lyso-
   zyme (seeNote 1).


9. Buffer C (elution buffer): 25 mM NaH 2 PO 4 -Na 2 HPO 4
   (pH 8.0), 35 mM NaCl, and 50 mM imidazole.


10. Ni-NTA affinity gel (seeNote 2) and empty 2.5
    30 cm (or
    similar) chromatography column for affinity gel packing.

76 Sathish Rajamani and Richard Sayre