Design of construct and expression of cardiac peptides and GFP
The overall structure of the construct co-expressing the cardiac peptides and GFP under the control of synthetic nirB promoter has been represented in figure 1A. Presence of a single band at 550 bp in electrophoresis confirmed correct performance of SOEing PCR (figure 1B). results of colony PCR also confirmed the presence of constructs in recombinant bacteria (figure 1C). following induction of anaerobic condition, collected supernatant of bacteria was concentrated and subjected to SDS-PAGE analysis to evaluate expression of cardiac peptides. As demonstrated in figure 2A, presence of a single band at about 5 kDa confirmed expression of cardiac peptides. As all the expressed cardiac peptides possess a molecular weight very close to each other, three single bands could have been overlapped together and it is not surprising to observe only one band in SDS-PAGE analysis. Extracted proteins form cytoplasm of bacterial pellets in lysis buffer were also subjected to SDS-PAGE analysis for evaluation of expression of GFP. The band at 27 kDa relates to the molecular weight of GFP which is absent in the bacterial samples non-transformed with the cassette (figure 2B).