Sequencing
In order to ensure the integrity of the sequencing, the promoter region was sequenced in the modified plasmid (Fig.1C and 1D). 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 measureable 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 pbrpromoter and expression of the reporter gene (Fig.3). In Fig.3, we aimed to show that the pbr promoter is specific to lead, and other heavy metals such as Zinc (Zn) (Fig.3A), Tin (Sn) (Fig.3B) and Cadmium (Cd) (Fig.3C) do not activate the promoter and significant expression of a reporter gene. 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)
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 200μmol/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 at 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. coliDH5α without plasmid. This resistance may be related to the pbrR regulatory gene (Fig.5B). The resistance genes to heavy metals have heavy metal binding motifs, they can limit the 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 the 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 0.2OD (1 μmol/L concentration) for 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 μM lead can induce luciferase expression. The degree of expression increased with time, with measureable change in Luciferase levels by 2 h measure, and in biological sensors pollution is usually measured at low rates, we chose 2 h for culture of the pGL3-luc/cad-biosensor.