2 mL PBS, spin for 10 min at 17,000gon a tabletop
centrifuge to remove any bacterial debris. The phage obtained
after double PEG-NaCl precipitation can be stored at 4C for
immediate use or as a glycerol stock (15% (wt/vol) glycerol) at
80 C for long-term storage.- To determine the titer of PEG-NaCl precipitated rescued
phage, precipitated phage is titrated by performing tenfold
serial dilutions of phage stock in PBS and infecting 900μLof
log-phase TG1 cells with 100μL of each dilution. - After incubation at 37C for 30 min, 100μL of infected cells
are plated onto TYE plates supplemented with 100μg/mL
ampicillin and 1% (wt/vol) glucose, and incubated at 30C
overnight. - Determine the phage titer by counting the number of plaques
present on the agar plates. - Panning rescued phage against AHL-conjugates is aimed at
selecting phages that bound to the conjugate of interest (i.e.,
AHL-BSA or AHL-TG conjugates). The panning strategy is
designed to drive the selection towards enrichment of cross-
reactive clones as shown schematically in Fig.2. Coat Nunc
Maxisorp Immuno tubes (Thermo Fisher Scientific) by adding
200 μg of HSL-conjugate dissolved in 4 mL of PBS and
incubating overnight at 4C. The immunotube is then washed
three times with PBS, blocked with MPBS for 2 h at 37оC, and
further washed three times with PBS. - To select for specific binding, 1 mL of rescued phage mixed
with 3 mL of MPBS is added to the immunotube and incu-
bated for 30 min at room temperature on a rolling tumbler. - The tube is then allowed to stand for 90 min at room tempera-
ture. Following incubation, the unbound phage is discarded by
washing the tube ten times with PBS containing 0.1% (vol/vol)
Tween 20, and then ten times with PBS. - For triethylamine elution of AHL-binding phages, following
an initial PBS wash, the bound phage is eluted from the immu-
notube by adding 1 mL of 100 mM triethylamine and rotating
for exactly 10 min at room temperature. Triethylamine is alka-
line and increasing the pH of the immunotube causes phage
release. The solution is then transferred to another tube and
neutralized by adding 500μL of 1 M Tris–HCl (pH 7.4).
Eluted phage can be stored at 4C or as glycerol stock at
80 оC by adding 15% (vol/vol) glycerol. - Free antigen elution of AHL-binding phages. This additional
elution strategy is included from the second and subsequent
rounds of selection to increase the chance of recovering “free
AHL” (non-conjugated) binding phage. After phage binding
344 Soumya Palliyil