20 min. Cool the molten agar solution to 60C before adding
the appropriate amount of antibiotic (100μg/ml ampicillin;
30 μg/ml chloramphenicol; 10μg/ml gentamycin). Transfer
15 ml of the molten LB/antibiotic agar into sterile Petri dishes.
Let the agar solidify in sterile conditions at room temperature.
- Binding buffer: dissolve 0.34 g imidazole, 29.22 g NaCl, and
2.42 g Tris in 1 l deionized water while stirring. Adjust the pH
to 8.0 with 12 M HCl. Store the buffer at 4C.
- Wash buffer: dissolve 6.99 g imidazole, 29.22 g NaCl, and
2.42 g Tris in 1 l deionized water while stirring. Adjust the
pH to 8.0 with 12 M HCl. Store the buffer at 4C.
- Histidine-elute buffer: dissolve 15.51 gL-histidine, 29.22 g
NaCl, and 2.42 g Tris in 1 l deionized water while stirring.
Filter the solution using 0.22μm membrane. Store the buffer
at 4C.
- Bipyridyl dialysis buffer: dissolve 15.62 mg 2,2-bipyridyl,
1.17 g NaCl, and 2.42 g Tris in 1 l deionized water while
stirring. Store the buffer at 4C.
- Storage buffer: dissolve 1.17 g NaCl and 2.42 g Tris in 1 l
deionized water while stirring. Store the buffer at 4C.
- ZnCl 2 dialysis buffer: dissolve 13.63 mg ZnCl 2 , 1.17 g NaCl,
and 2.42 g Tris in 1 l deionized water while stirring. Store the
buffer at 4C.
- Bicine buffer: dissolve 40.79 g bicine in 1 l deionized water
while stirring. Adjust the pH to 8.0 using 6 M HCl. Store the
buffer at 4C.
- Cresol purple: dissolve 2.0 mgm-cresol sodium salt in 1 ml
deionized water. Store the indicator at 4C.
- 2% (wt/vol) arabinose: dissolve 0.2 gL-arabinose in 10 ml
deionized water. Filter the solution using 0.22μm membrane.
Store the solution at 4C.
- 1 M isopropyl-β-D-thiogalactoside (IPTG): dissolve 0.24 g in
1 ml deionized water. Filter the solution using 0.22μm mem-
brane. Store the solution at 20 C.
- Dimethyl sulfoxide (DMSO).
- Quartz cuvette.
- Spectrophotometer.
- Kit for DNA extraction from agarose gel.
- PCR reagents.
- Oligonucleotides:
(a) GKL forward primer: 5^0 -GAAAGGGGTGAAATTAA-
TATGGCGGAGATGGTAGAAACGG-3^0.
Directed Evolution of Quorum Quenching Enzymes 313
