Flat bottomed plates

Plasma anti-OVA IgG titers were measured using ELISA as follows: 96-well flat-bottomed plates (Nunc) were coated overnight with OVA (Sigma-Aldrich) in phosphate-buffered saline (PBS). After washes with PBS/0.05% Tween 20 (PBS-Tween), non specific binding sites were blocked with PBS/5% foetal calf serum (FCS). Plates were then incubated with plasma. Horse raddish peroxidase conjugated polyclonal rabbit anti-rat IgG (Abcam) was added. Finally, TMB substrate (Thermoscientific) added to each well. The reaction was stopped with 2N H₂SO₄ and plates read at 450 nm using an automatic Infinite M200 microplate reader (Tecan). Titers were expressed as ×10³ of the highest plasma dilution, giving an optical density at least twice the blank value.

Escherichia coli DH10B bacteria bearing or lacking (as a reference) the viral chimera were grown overnight in LB at 37 °C. Thereafter, the growth culture was diluted and the bacteria were grown until their O.D.₆₀₀ reached 0.2. Subsequently, 50 μL of bacterial culture was dispensed into 96-well flat-bottomed plates (Nunc, Roskilde, Denmark) containing 50 μL of the different treatments. Unless stated otherwise, IPTG was added to the cells to final concentrations ranging from 0 to 100 μM. d-glucose was added to a concentration of 1%. Moreover, 96-well plates were incubated for 16 h at 37 °C in an Infinite 200 from the Tecan Group (Männedorf, Switzerland) at a constant high shaking rate. O.D.₆₀₀ readings were recorded every 15 min. For every measurement, duplicates or triplicates were conducted.
For the Escherichia coli LB650 bacteria, the same protocol was used, except that growth was done in LBK overnight. Subsequently, the bacteria were diluted and grown until their O.D.₆₀₀ reached 0.2, after which the media was replaced to LB and diluted twofold with the various treatments in each well. Unless stated otherwise, IPTG was added to the LB650 bacteria to a final concentration of 10 μM.