2.4 Conjugation assay
Prior to the conjugation assay, the E. coli strains were
incubated overnight at 30°C on solid lysogeny broth (Lennox) (LB) agar
(VWR) supplemented with antibiotics, 30 ug kanamycin/ml for donor/R27
and 10 ug tetracycline/ml for donor/pB10. A single colony per replicate
used, was grown in 5 ml LB for 3 hours and enumerated by flow cytometry.
Donors and recipients were resuspended in 0.9 % (9 g/l) NaCl solution
and mixed in a 1:1 ratio. The 0.2 μm mixed cellulose ester (MCE) filter
(Advantec®) was placed on solidified LB agar plates and the mixture was
pipetted on to 54 mm2 corresponding to an initial cell
density of 3.6 x 105 cells/mm2. The
plates were then incubated for 20 hours at 30°C, at which state cells
were harvested; the filter was transferred to a 0.9 % NaCl solution
where cells were washed off and the suspension was stored at 4°C.