Sequencing
In order to ensure the integrity of the sequencing by Miligen Company, the promoter region was sequenced in the received plasmid (Fig. 2).
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. 3).
Biosensor activity of pGL3-luc/pbr
The expression of the luciferase gene in the presence of different concentrations of lead showed that 1 μM lead is the lowest concentration that could stimulate the promoter and distinguish the expression of luciferase from the basal state 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 expressed only in the presence of lead. In addition, it has relatively great sensitivity.
Biosensor specificity in the presence of different concentrations of Zinc (ZnCl2), Tin (SnCl2) and Cadmium (CdCl2)
The biosensor cultured in the presence of different concentrations of zinc, tin and cadmium did not stimulate the pbr promoter and expression of the reporter gene (Fig. 4). Data obtained from the expression of the luciferase gene in various tin, zinc and cadmium concentrations indicate that these heavy metals had no affect on the stimulating of the pbr promoter.
Biosensor activity in the presence of different concentrations of Lead (PbCl3)
The results revealed that lead was the only metal that could stimulate the pbr promoter. In the absence of lead, the regulator gene prevents the promoter from activation. Lead is binds 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 by this biological sensor is 1 µM and a maximum is 100 μmol/L. The expression of luciferase was decreased with a slight gradient from 100 to 200 μmol/L (Fig. 5).
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. 6). The maximum expression of the luciferase gene was 12 h (Fig. 7).
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. 8). 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 results showed that 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. 9 and 10).
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. 11). As shown in Fig. 11, the concentration of 1 micro Molar 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 Luciferase, and in biological sensors the pollution is measured at low rates, we chose 2 h for culture the pGL3-luc/cad-biosensor.