Quorum Sensing

13. Single colonies fromstep 12are picked from serial dilution
    plates of VH-Vλand VH-Vкlibraries and used as source of
    template DNA in colony PCR to check the presence of the scFv
    inserts. Set up a PCR reaction mix containing 25 pmol of
    AH18REV primer and 25 pmol of GIIIFOR primer, 1μL
    100 mM dNTPmix (final 25 mM each of dATP, dTTP,
    dCTP, dGTP), 2.5μL 50 mM MgCl 2 ,5μL10
    NH 4 Buffer,
    1 μL (1 U) Taq Polymerase, and 38.5μL water to make up the
    volume to 50μL. The reactions are heated up to 95C for
    3 min and then subjected to a 30 cycle program of 95C for
    30 s, 57C for 30 s, and 72C for 1 min, followed by a final
    incubation at 72C for 5 min. The PCR products (5μL) are
    run on a 1.5% (wt/vol) agarose gel alongside with a DNA
    molecular weight marker to analyze the presence of the scFv
    inserts (~750 bp).

3.5 Propagation of
Helper Phage in Large
Quantity

1. M13KO7 helper phage stock is serially diluted as tenfold dilu-
   tions in 2
   TY medium. Ten microliter aliquots of diluted
   helper phage from 10^3 to 10^10 are added into 200μLofE.
   coliER2738 cells grown in 2
   TY medium supplemented with
   12.5μg/mL tetracycline to an A 600 of 0.4–0.5, and incubated
   at 37C for 30 min to facilitate infection with phage particles.


2. After infection, ER2738 cells are added to 5 mL warm H-top
   agar, poured onto TYE agar plates supplemented with
   12.5μg/mL tetracycline and incubated at 37C overnight.


3. Fresh ER2738 cells are grown in 2
   TY medium containing
   12.5μg/mL tetracycline up to A 600 of 0.5, and 3 mL cells are
   infected with a small plaque picked up from overnight grown
   H-top agar plates, by incubating for 1 h at 37C with shaking.


4. Culture fromstep 3is mixed with 500 mL 2
   TY medium in
   a 2 L conical flask and grown at 37C with 250 rpm shaking
   for 1 h.


5. Add 50μg/mL of kanamycin to the culture fromstep 4and
   incubate for additional 16 h at 30C with shaking at 250 rpm.


6. The culture fromstep 5is centrifuged at 10,800
   gfor 15 min
   in sterile 50 mL polypropylene tubes. The supernatant contain-
   ing M13KO7 helper phage is carefully poured out, filter ster-
   ilized using 0.45μm filter, and stored at 4оC.


7. The helper phage titer is determined by performing tenfold
   dilutions of the phage-containing supernatant in 2
   TY
   medium and infecting log phase ER2738 cells as described
   above.


8. After infection, plate 100μL of infected cells onto TYE plates
   supplemented with 50μg/mL kanamycin and 12.5μg/mL
   tetracycline.

342 Soumya Palliyil