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).