Morphology of wild type vs. hybrid Qβ phage plaques.

<p>Panel A) wild type Qβ phages; Panel B) Qβ-FMDV VP1 G-H loop phages; Panel C) Qβ-GFP rescued phages from <i>E. coli</i> SURE (expression host with F⁺) over-expressing A1-GFP protein infected with wild type Qβ. Panel D) QβΔA1 phages. All at very low multiplicity of infection (MOI), and all plates are exactly 1 day (24 hours) old when photographed.</p

Field light micrograph of modified Qβ.

<p>A) Unlabelled Qβ virion: the original particle projection obtained with conventional transmission electron microscopy of a negatively stained sample was treated by Marham rotation 3 times 120\grad\intervals and printed at reversed contrast; Magnification Bar: 50 nm. B) Negatively stained Qβ, modified at A1 gene products by additional of FMDV G-H loop motif decorating the corners by IgG of mAb SD6 against VP1 G-H loop motif at 120\grad\intervals (arrows); Magnification Bar 25 nm. C) Phage particles projection depicted in B was treated by Makham rotation and printed at reversed contrast; arrows showing antibody against integrin motif attached to the corners of the virus particle; Magnification 25 nm.</p

Ouchterlony double diffusion assay.

<p>A) Wells 1 and 2 represent Qβ-FMDV phages; wells 3 and 4 represent QβΔA1 phages; wells 5 and 6 represent wild-type Qβ; center well contains polyclonal serum from immunized guinea pig (labeled “Ab”). B) Same as panel A but with 1/3 of the serum concentration. C) Wells 1 and 2 Qβ-FMDV are the same as panel A; wells 5 and 6 contain half the phage titer of wells 1 and 2; well 3 represents phages from pBRT7QβΔA1 and well 4 represents phages from pQβ8ΔA1; center well contains IgGs purified from serum panel A and B (labeled “Ab”). The line of precipitation is visible as a white haze forming a half-circle around some of the wells in the experiments.</p

RT-PCR of RNA purified from Qβ-phage plaques.

<p>Panel A) Lane 2: Qβ-HisJ; lane 3: Qβ-tHisF; lane 4: soft agar stab from HisJ plate; lane 4: soft agar stab from tHisF. Panel B) Lanes 2 and 4: wild type Qβ; Lanes 3 and 5: Qβ-GFP. Panel C) Lanes 2 and 4: Qβ-FMDV; Lanes 1 and 5: wild type Qβ (positive control). The 100 bp and 1 kb DNA ladder were used.</p

Agarose gel electrophoresis of the RNA display system vector construction.

<p>Panel A) Lanes 1–3: positive recombinant pUCHisJ plasmid clone (cl) restricted with <i>Afl</i>II and <i>Nsi</i>I; Lanes 5–7: positive pUCtHisF and Lane 8: negative clone. Panel B) Lanes 2–6: positive recombinants pBRT7QβHisJ restricted with <i>Afl</i>II and <i>Nsi</i>I; Lane 7: negative clone. Panel C) Lanes 2–7: positive recombinants pBRT7QβtHisF restricted with <i>Afl</i>II and <i>Nsi</i>I. Panel D): Lane 1: pQβ8 negative control; Lanes 2 and 3: positive recombinants pQβ8ΔA1; Lanes 4 and 5: positive recombinants pBRT7Qβ-FMDV; Lanes 6 and 7: positive recombinants pBRT7QβΔA1 all restricted with <i>Nhe</i>I. Lanes “ladder” were loaded with the 100 bp or 1 kb DNA ladder.</p

Schematic representation of the RNA phage display vector construction.

<p>General cloning procedure from PCR fragments to pBRT7Qβ with transient cloning in the pUC18-cassette working plasmid. Step 1: cloning of PCR fragment into pCR2.1 vector; Step 2: cloning of the foreign gene from PCR into the pUC-cassette (with <i>Nsi</i>I) using <i>Af</i>lII and <i>Esp</i>I sites; Step 3: Cloning of the foreign gene into pBRT7Qβ using <i>Afl</i>II and <i>Nsi</i>I. P: promoter; <i>Amp</i>: ampicillinase gene; <i>Kan</i>: kanamycin resistance gene; ori: origin of replication.</p

Table adapted from Drake [29].

a<p>In the cases where multiple targets were measured, the average is presented.</p>b<p>Taken from Domingo <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113069#pone.0113069-Domingo3" target="_blank">[48]</a>.</p><p>Table adapted from Drake <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113069#pone.0113069-Drake1" target="_blank">[29]</a>.</p