Measurement of E-GFP:
The modified over-expression plasmids (pAPD8, pAPDp, AKVSS1, and
pAKVSS2) along with wild type (pAPD7) were conjugatively mobilized inA. brasilense Sp245 already having pAPD11 plasmid. The effect of
deletions and mutations of two conserved motifs of RpoE10 onabm:gfp were monitored. For this, overnight grown cultures of
recombinants and control were inoculated in the same media to maintain
OD600 to ~ 0.1 for all cultures. When
bacterial growth reached up to ~0.6
OD600 then each culture was divided into two equal
parts. One part of the culture was induced by IPTG (0.5 mM) and allowed
to grow for another 6 h. After sample collection and washing with
phosphate buffer saline, GFP intensity was measured by exciting at 405
nm and emission 567 at 485 nm to 525 nm in Varian spectrofluorimeter.
The second part of the culture was used for plate assay in which cells
with equal OD were kept on a MM agar plate as a drop for 48 h in the
presence and absence of IPTG. GFP expression was observed under UV light
and photographs taken by digital camera.