Sequencing
In order to ensure the integrity of the sequencing by Miligen Company, the promoter region was sequenced in the received plasmid (Fig. 1C and 1D).
Colony confirmation with PCR reaction
PCR was performed using primers designed for the pbr and cadA promoters, and the promoter sequence and regulatory gene were amplified with 634 bp for pbr and 601 bp for cadA (Fig. 2).
Biosensor activity of pGL3-luc/pbr
The expression of the luciferase gene, in the presence of different concentrations of lead, showed that 1 μM of lead was the lowest concentration that could stimulate the promoter and could be distinguished from the basal expression of luciferase, and the highest expression was seen at 100 μmol/L. A good biosensor should have two characteristics: specificity and sensitivity. According to the data obtained from our experiments, this biosensor had a high specificity, and luciferase gene was only expressed in the presence of lead. -
Biosensor specificity for lead in the presence of different concentrations of zinc (ZnCl2), tin (SnCl2) and cadmium (CdCl2)
The biosensor was cultured in the presence of different concentrations of zinc, tin and cadmium, and did not stimulate the pbr promoter and expression of the reporter gene (Fig.3). Data obtained from the expression of the luciferase gene in the presence of various concentrations of tin, zinc and cadmium, indicated that these heavy metals did not stimulate the pbr promoter.
Biosensor activity in the presence of different concentrations of Lead (PbCl3)
The results revealed that lead was the only metal that stimulated the pbr promoter. In the absence of lead, the regulator gene prevents the promoter from activation. Lead ions bind to the regulator gene and inhibits its binding to the operator. As a result, the promoter is activated and the luciferase is expressed. The minimum detectable concentration of this biological sensor was approximately 1 µM and a maximum is 100 μmol/L. The expression of luciferase was no longer linear for value of lead from 100 to 200 μmol/L (Fig. 4A).
The expression of pGL3-luc/pbr-biosensor reporter gene at different times
In order to identify the appropriate time for biosensor growth, a biosensor was cultured at different concentrations of lead for different durations (Fig. 4B). The maximum expression of the luciferase gene was 12 h (Fig. 5A).
The difference in the growth rate of pGL3-luc/pbr-biosensor compared to E. coli strain DH5α
The sensor bacteria had a recombinant plasmid containing the pbr promoter region and the pbrR regulatory gene. These bacteria have a greater resistance to lead than E. coli DH5α without plasmid. This resistance may be related to the pbrR regulatory gene (Fig. 5B). The resistance genes of metals have heavy metal binding motifs, they can result in the non-toxicity of these metals inside the cell, because of these proteins, the relative resistance of the cell to heavy metals.
The activity of pGL3-luc/cad-biosensor at the different concentrations of lead
The lowest and highest concentrations of lead that could stimulate expression of the reporter gene were 10 nmol/L and 10 μmol/L respectively (Fig. 6 and 7A).
Expression of the Luciferase gene in the presence of 1 micro Molar concentration of Lead at different times
The sensor bacteria were incubated at 0.2 OD (1 μmol/L concentration) at different times in the incubator. The expression of luciferase was measured at different times (Fig. 7B). As shown in Fig. 7B, the concentration of 1 υM lead can cause luciferase expression. The amount of expression raised by progressing time, just due to during 2 h the amount of expression is high enough to measure the change in Luciferase, and in biological sensors the pollution is measured at low rates, we chose 2 h for culture the pGL3-luc/cad-biosensor.