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.