Development of plasmid constructs
A polycistronic cassette, expressing KP, VD and LANP genes in tandem under the control of synthetic nirB promoter reported by Nasr and Eidgahi was designed based on the ANP preprohormone sequence (Genbank accession number: NP_006163). For visualization of bacteria at tumor sites, another polycistronic system expressing green fluorescent protein (GFP) was linked to the previous cassette through a Lambda transcription termination sequence. A ribosomal binding site (RBS) sequence was included upstream of GFP and each of the three gene sequences. For facilitating excretion of expressed cardiac peptides, an immunoglobulin κ chain signal peptide (IgK) leader sequence was embedded between RBS and each of the gene’s sequence. A terminating codon was also located at the end of each gene sequence. Lambda transcription termination sequence (a rho independent transcription termination sequence) was allocated between the two polycistronic expressing systems to separate expression of corresponding mRNAs from each other. The DNA sequence of GFP was obtained from the GFP commercial plasmid pLOX-EWgfp and fused to the rest of the construct synthesized by Cinnagen Inc. (Iran) performing SOEing-PCR. At the end, the flanking sequences of pET-32 Ek/LIC vector was added to the endings of the construct performing another round of PCR and the construct was cloned into the vector based on previously described method (10).
Transformation of thecardiac peptide/GFP expressing plasmid DNA (pET-CP/GFP)
The rpoS(Am) rph-1 λ  rrnB3 ΔlacZ4787 hsdR514 Δ(araBAD)567 Δ(rhaBAD)568 rph-1 Escherichia coli strand, BW25133, was grown in Luria-Bertani (LB) media at 37 °C until mid-log phase, and then harvested at 4 °C. After confirmation of correct cloning, pET-CP/GFP constructs were transformed in to E. coli BW25133 strain using calcium chloride method. Bacteria were then maintained and selected in LB media applying 50 mg/ml Ampicillin and 10 mg/ml tetracycline. Confirming function of synthetic nirB promoter, bacteria transformed with pET-CP/GFP were grown overnight on LB medium containing 50 mg/ml Ampicillin and 10 mg/ml tetracycline at 37 °C. The anaerobic condition was induced by bubbling filtered helium gas through the medium for 10 min. plates were then completely sealed in a way that no air could pass to the medium and incubated for 24h at 37 °C. fluorescent microscope imaging was used to detect GFP proteins expressed by bacteria.