to the AHL-conjugate in the immunotube and washing as
described in Subheading3.6,step 13, 4 mL of free AHL at a
desired concentration (10 nM–10μM;seeFig. 2) is added to
the immunotube and incubated at room temperature for 2 h
on rolling tumbler.- The eluted phage is mixed with 10 mL of log-phase TG1 cells
and incubated at 37C for 30 min to allow infection. Any
remaining phage bound to the immunotube is recovered by
adding 4 mL of log-phase TG1 cells into the tube and incubat-
ing at 37C for 30 min. - 100μL of each infected culture are serially diluted tenfold in
2 TY medium and plated onto TYE agar plates containing
100 μg/mL ampicillin and 1% (wt/vol) glucose and incubated
at 30C overnight. - The library size after pan 1 or the number of cells infected by
phage recovered from selection is estimated by counting the
number of bacterial colonies on the plate. The remaining
infected culture is centrifuged at 2000gfor 10 min, the
cell pellet suspended in 1 mL of 2TY medium and plated
onto 140 mm Petri dish containing TYE supplemented with
100 μg/mL ampicillin and 1% (wt/vol) glucose and incubated
overnight at 37C. - For helper phage rescue and enrichment of AHL binders,
bacterial colonies grown on 140 mm Petri plates fromstep
18 are recovered by adding 2–3 mL of 2TY medium contain-
ing 30% (vol/vol) glycerol and 10% (wt/vol) glucose. - The bacterial lawn is loosened using a disposable scraper and
the resultant suspension is mixed thoroughly to remove any
bacterial clump. The suspension is stored at 80 Casa
bacterial stock containing and representative of selected
phage from the first round of selection. This is then used as
the starting material for the next round of panning. - From the pan 1 glycerol stock, a small amount is inoculated
into 100 mL of 2TY containing 100μg/mL ampicillin and
1% (w/v) glucose, in order to reach an A 600 between 0.08 and
0.1. The culture is then incubated at 37C with shaking at
250 rpm until an A 600 of 0.4–0.5 is reached. - 50 mL of culture fromstep 21are infected with the M13KO7
helper phage and incubated at 37C for 30 min. The culture is
centrifuged at 2000gfor 10 min and the pellet resuspended
in 100 mL 2TY supplemented with 100μg/mL ampicillin
and 50μg/mL kanamycin, and grown overnight at 30C with
250 rpm shaking. - Phage rescue and precipitation is carried out as described in
steps 6– 10. Second round of selection is carried out on
346 Soumya Palliyil