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Construction of a sensitive and specific lead biosensor using a genetically engineered bacterial system with a luciferase gene reporter controlled by pbr and cadA promoters
Reza Nedaeinia
Isfahan University of Medical Sciences
Corresponding Author
ORCiD: https://orcid.org/0000-0001-9922-7181
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Background: A bacterial biosensor refers to genetically engineered bacteria that produce an assessable signal in the presence of a physical or chemical agent in the environment.
Methods: We have designed and evaluated a bacterial biosensor expressing a luciferase-reporter gene controlled by pbr and cadA promoters in Cupriavidus metallidurans (previously termed Ralstonia metallidurans) containing the CH34 and pI258 plasmids of Staphylococcus aureus, respectively, and that can be used for the detection of heavy metals. In the present study, we have produced and evaluated biosensor plasmids designated pGL3-luc/pbr-biosensor and pGL3-luc/cad-biosensor, that were based on the expression of luc+ under the control of the cad promoter and the cadC gene of S. aureus plasmid pI258 and pbr promoter and pbrR gene from plasmid pMOL30 of Cupriavidus metallidurans.
Results: We found that the pGL3-luc/pbr-biosensor may be used to measure lead concentrations between 1-100 μM in the presence of other metals, including: zinc, cadmium, tin and nickel. The latter metals did not result in any significant signal. The pGL3-luc/cad-biosensor could detect lead concentrations between 10 nM to 10 μM.
Conclusions: This biosensor was found to be a specific for measuring lead ions in both environmental and biological samples.
Keywords
Lead detection, Bacterial biosensor, Pollution control system
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Ecological heavy metal pollution is a common problem that can exert damage to human health as well as the environment [1]. These heavy metal pollutants may lead to harmful ecological outcomes [2], and hence the development of sensitive, efficient, rapid and cost-effective methods is necessary to screen for the presence of harmful metals in the environment. Lead (Pb) is a toxic heavy metal that is extensively utilized around the world [3, 4]. It has been estimated that the world production of lead is more than 3 million tons per year. It causes widespread environmental contamination in the air, water, soil, and food [5]. This element can find its way through human bodies as well as animals, entering the food chain; in fish it can accumulate in the bone, liver, gills, kidney, ovary, and muscle [6].
Environmental lead may result in an increase in vascular endothelial growth factor (VEGF) and blood concentrations [7, 8], and can lead to neurological and cardiovascular complications [9]. The reproductive system may also be affected by developmental disorders that are highly likely to occur in children [10-13]. Lead can cross the placenta and cause damage to the developing fetal nervous system [14].
The assessment and monitoring of environment heavy metal contamination is very important to prevent harm to human health. Currently, classical analytical methods, such as spectrometry, FIAAS (flow injection atomic absorption spectrometry), ion chromatography, and electrochemical techniques, are the main methods used for measuring environmental heavy metals pollution. The main disadvantage of these methods is the necessity for sample digestion under high temperature and pressure, or acidic conditions in which metal ions in solution are released [15] and subsequent analysis using expensive equipment. Therefore, simpler methods for evaluating heavy metals are required. More importantly, heavy metals are found to be present in biological systems either in bioavailable/toxic or non-available/ non-toxic forms, and current measuring methods are unable to distinguish between toxic and non-toxic fractions of these elements [16]. Furthermore, these methods are both time-consuming and costly [17]. Biosensors have been developed that are an effective alternative to conventional detecting systems. These may be highly sensitivity and simple to use [18]. Cell-based biosensors are biological sensors that contain a reporter gene under the control of a promoter that is sensitive to the presence of an agent, such as environmental contaminants that include heavy metals. Biosensors are used in various designs with different reporters and promoters. At low concentration of heavy bioavailable metals, bioluminescence signals are likely to be suitable[19, 20]. They can also be applied to monitoring bioavailable concentrations of heavy metals [21-27] and piezoelectric biosensors [28-30] as enzyme-based electrochemical biosensors. One of the most obvious advantages of this method is the ability to measure the bioavailable heavy metal at very low concentrations. It is also a cost-effective and time saving method [18]. In these biosensors, the expression of a reporter gene is controlled by a promoter, such as the pbrR promoter in the pMOL30plasmid of Cupriavidus metallidurans CH34 and cadC promoter in pI258 plasmid of Staphylococcus aureus that is sensitive to heavy metals. Most of these promoters originate from bacteria that have resistance systems against heavy metals [31, 32]. In this study, we have designed and evaluated the luciferase reporter gene expression of bacterial biosensor under the control of pbr and cadC promoters in Cupriavidus metalliduransCH34 and pI258 plasmids of Staphylococcus aureus, respectively, for the measurement of lead.
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 cadmium, tin, and zinc 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.
There are several advantages to using bacterial biosensors, including speed, simplicity and cost. Biological sensors containing cadA and pbr promoter regions have been designed by other researchers, the optimization of this cell biological sensor with ability to measure lead comparing the cadA and pbr promoters in a bioassay system was evaluated in this study. The use of biosensors or biological cell sensors containing a reporter gene controlled by promoters susceptible to the heavy metal ions can provide an efficient method to trace particular pollutants in the environment and in a biological solution[33]. The present study assessed a biosensor system for detecting lead ions through construction of a luminescent bacterial sensor containing the luc+ regulated by the cad promoter and cadC gene in plasmid pI258 of S. aureus and the pbr promoter and pbrR gene in pMOL30 plasmid of Cupriavidus metallidurans. Pb specific bacterial biosensors were formerly defined using reporter genes including lacZ, lux,and luc in the transcription fusion constructs [34-36]. In our study, the luciferase reporter gene was used. Luciferases, as a set of heterogeneous enzymes, are able to produce light as a byproduct of catalyzing reactions. They are reporter genes extensively used by prokaryotic and eukaryotic organisms due to their high sensitivity and ease of detection.The quantification of the emitted light, i.e. bioluminescence,is of great importance; it can also be measured using a liquid scintillation counter ,a luminometer, or even a X-ray film [35]. It was concluded that a pGL3-luc/ pbr-biosensor can detect Pb+2 in the range of 1–100 μM using the expression of firefly luciferase as a detector system, and is highly specific, with no expression of reporter in the presence of other metals such as Sn+2, Ni+2, Cd+2 are present. Moreover, this biosensor was 50 times more sensitive when compared with the previous biosensors reported by Chakraborty et al [32]. The R. mettalidurans CH34 strain has several resistance systems that can reduce the concentration of toxic substances to their non-toxic levels. A highly specific system for resistance to lead is known in plasmid pMOL30[37]. It effectively reduced the concentration of lead ions and is equipped with specific mechanisms for the transfer and separation of lead. The pbr operon includes pbrA, pbrB, pbrC and pbrD genes in which pbrD has a role as a chaperone to accumulate lead in the cell and pbrA eliminates lead ions[37]. Our results show that the pGL3-luc/pbr-biosensor is not expressed in the presence of cadmium, zinc, ortin, indicating high sensitivity and specificity of the designed system for lead detection. One of the most important heavy metal transfer systems in Staphylococcus aureus is located in the plasmid pI258. The plasmid has an operon cadA that encodes an ATPas of type P, which causes resistance to metals such as cadmium, lead, zinc, copper, and tin. The expression of the cadA operon is controlled by the cadC homodimeric protein. This protein is able, in a binary manner, to bind to the promoter and metal ions, such as cadmium, lead, zinc, and tin. The cad belongs toArsR / SmtB, a regulating protein family[38]. In our study, the luciferase gene was used as a reporter and E. coli strain of DH5α as a host. Our results showed that the pGL3-luc/cad-biosensor can detect at least 10 nM of lead and the lead toxicity was not observed until a concentration of 300 μM. However, the maximal expression of the reporter gene was performed at 10 μM. Our results are supported by the report of Liao et al that showed the regulating role of cad promoter and the cadC gene in plasmid pI258of S. aureus, the fluorescence emission was intensified with increasing Cd(II), Pb(II), and Sb(III) ions concentrations[39]. For Pb (II), just like our result in pGL3-luc/cad-Biosensor, to induce GFP expression significantly, 10 nM was the low,and 10 μMwas the maximum concentration of lead that induced significantly GFP expression[39]. The metallo regulatory α3N thiolate-rich site in cadC displays a practical selectivity for larger, softer heavy metal like Pb(II), Cd(II), although smaller boundary metal ions such as Zn(II) accommodated[40]. One of the limitations of this method is that bacterial biosensors require the necessary conditions for bacterial growth to operate, and the graphs are based on solving different concentrations of heavy metals in a bacterial culture medium. Therefore, to measure the amount of heavy metals in an unknown environment, it is necessary to optimize the biosensor in the new environment, which would itself require evaluation.
Our results show that the maximum expression of reporter gene was found in the presence of 100 μM of Lead in pGL3-luc/pbr-biosensor and 1 μM of lead in pGL3-luc/cad-biosensor. In this study, the specificity and sensitivity of the two heavy metal susceptive probes, pbr and cadA, was investigated. Sensors containing these two promoter regions were able to detect the concentration of lead between 1-100 μM and 10 nM to 10 μM of lead, respectively. For other heavy metals such as mercury, copper, nickel, manganese, zinc and cadmium, different biological sensors can be made and their presence in the environment can be measured with very high accuracy. To determine the accuracy of biosensors, a standard curve of Luciferase gene expression was plotted at different lead concentrations. The standard curve was constructed from triplicates values, we evaluated the accuracy of the biosensor with the specific concentrations that we had obtained from lead metals. By developing these sensors, the time required to identify environmental pollution can be minimized.
Chemicals
Analytical reagents, media and buffer solutions like TBE-EDTA buffer (Tris-borate-Ethylenediaminetetraacetic acid), NaOH (Sodium hydroxide), CaCl2 (calcium chloride), boric acid, Tris base, and agarose were all purchased from Merck (Germany). Fermentas (Lithuania) supplied the restriction endonucleases Nco1 and Hind3, T4 DNA ligase, and molecular ladder 10000-300bp. We also supplied the DNA polymerase (TaKaRa LA Taq®. DNA Polymerase), dNTP and MgCl2 from Takara (Beijing, China). In addition, the plasmid extraction kit and primers were brought from Bioneer (Seoul, South Korea).
Construction of biosensor plasmid
pMOL30 (X71400 AJ278984) and PI258 (GQ900378.1) containing the pbrR gene (634 bp) and CadC gene (601bp) (Accession number: pbrR: WP_003103716.1and CadC: WP_000726009, respectively, were synthesized and supplied by Millegen company. To ensure the accuracy of synthesized plasmid, the promoter region was sequenced. PGl3-control as a vector containing the Luciferase gene and E.coli strain DH5α as the host were used in our study. To obtain a large amount of pMA-T plasmid (a synthetic plasmid) which contains p-promoter sequences and the regulatory gene was sent to MilliGen, after evaluation at the NCBI site, for the synthesis of sequences. Synthesized sequences consisted of both pbr_pMA-T plasmids containing the promoter sequence of the pRR operon and the pbrR regulator gene including; cadA pMS-RQ-Bs plasmid containing the promoter region of the cadAp and the cadA gene regulating gene), it was cloned to E. coli host. Afterwards, pMA-T was extracted using plasmid extraction kit, and its quantity and quality were both examined by spectrophotometry and agarose gel, respectively, before they got digested by HindIII and NcoI. The promoter regions with the regulator genes were also purified from the gel electrophoresis. The received sequence and pGL3-control vector were cut using the same restriction enzyme (Nco1 and Hind3) and ligation reaction at 37°C for 3–4 h with ligase enzyme. The firefly luciferase gene was placed under the control of the received promoter sequences and recombinant plasmids of cad and pbr promoters were named pGL3-luc/pbr-biosensor and pGL3-luc/Cad-biosensor, respectively. Recombinant plasmids pGL3-luc/pbr-biosensor (Fig. 1A) and pGL3-luc/Cad- biosensor (Fig. 1B) were transferred to the DH5α bacteria using the chemical method of CaCl2 and then were screened using selective plates containing antibiotic Ampicillin. After plasmid extraction, PCR was performed to detect colonies containing the promoter region of pbr and cadA using primers designed for the cloned fragments. After these processes, recombinant plasmids were used to evaluating and measuring different concentrations of heavy metals.
Culture of bacteria and measuring biosensor activity of Luciferase enzyme
To study the efficiency of promoters the detection of heavy metals, a luciferase enzyme measurement performedin the presence of lead and other heavy metals such as tin, zinc and cadmium. In this process, E.coli stains carrying pGL3-luc/Cad-Biosensor and pGL3-luc/pbr-Biosensor were cultured in Luriae Bertani (LB) broth that contained 100 µg/mL ampicillin at 37˚C, overnight. Then 50 µl from overnight grown culture of pGL3-luc/pbr-Biosensor for 12 h and pGL3-luc/Cad-Biosensor with optical density (OD600) 0.8 for 2h were cultured in the presence of heavy metals at different concentrations [41]. Next, the culture was centrifuged at 5000 rpm for 10 min at 4°C metals for bacterial sedimentation. Then the medium was removed, and lysis buffer was added to the plate and sonicated at low temperature. Then, the amount of luciferase expression was measured by a luminometer (Berthold Company).
Statistical analysis
All the experiments were repeated at triplicate to minimize error. The Student t- test and one-way analysis of variance (ANOVA) were used to compare the statistically significant between the two groups and each group was compared with the baseline through the same method. . Statistical significance was set at *p<0.05. Data are shown as mean values± standard deviation (SD). Linear regression was used to model the standard curve. Analysis of Data was performed using SPSS version 22 statistical software (IBM, Chicago, IL, USA).
Compliance with ethical standards
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Informed consent
For this type of study, formal consent is not required.
Competing interests
The authors have declared no conflict of interest.
Funding information
This study was supported by Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University.
Consent for publication
All authors have given consent for publication
Availability of data and materials
All data generated or analyzed during this study are included in this published article
Author Contributions
MS and SH designed research; EN and RN performed research; HKS and NE analyzed data; EN and MN wrote the manuscript; AM, MN and ZF performed statistical analysis; MG and MR contributed new reagents or analytical tools. GAF revising the manuscript critically for important intellectual content. All authors read and approved the manuscript.
Acknowledgements:
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Pb: Lead; VEGF: Vascular endothelial growth factor; Flow injection atomic absorption spectrometry: FIAAS; LB: Luriae Bertani; RLU: Relative luminescence units.
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Construction of a sensitive and specific lead biosensor using a genetically engineered bacterial system with a luciferase gene reporter controlled by pbr and cadA promoters
Reza Nedaeinia
Isfahan University of Medical Sciences
Corresponding Author
ORCiD: https://orcid.org/0000-0001-9922-7181
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Background: A bacterial biosensor refers to genetically engineered bacteria that produce an assessable signal in the presence of a physical or chemical agent in the environment.
Methods: We have designed and evaluated a bacterial biosensor expressing a luciferase-reporter gene controlled by pbr and cadA promoters in Cupriavidus metallidurans (previously termed Ralstonia metallidurans) containing the CH34 and pI258 plasmids of Staphylococcus aureus, respectively, and that can be used for the detection of heavy metals. In the present study, we have produced and evaluated biosensor plasmids designated pGL3-luc/pbr-biosensor and pGL3-luc/cad-biosensor, that were based on the expression of luc+ under the control of the cad promoter and the cadC gene of S. aureus plasmid pI258 and pbr promoter and pbrR gene from plasmid pMOL30 of Cupriavidus metallidurans.
Results: We found that the pGL3-luc/pbr-biosensor may be used to measure lead concentrations between 1-100 μM in the presence of other metals, including: zinc, cadmium, tin and nickel. The latter metals did not result in any significant signal. The pGL3-luc/cad-biosensor could detect lead concentrations between 10 nM to 10 μM.
Conclusions: This biosensor was found to be a specific for measuring lead ions in both environmental and biological samples.
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Ecological heavy metal pollution is a common problem that can exert damage to human health as well as the environment [1]. These heavy metal pollutants may lead to harmful ecological outcomes [2], and hence the development of sensitive, efficient, rapid and cost-effective methods is necessary to screen for the presence of harmful metals in the environment. Lead (Pb) is a toxic heavy metal that is extensively utilized around the world [3, 4]. It has been estimated that the world production of lead is more than 3 million tons per year. It causes widespread environmental contamination in the air, water, soil, and food [5]. This element can find its way through human bodies as well as animals, entering the food chain; in fish it can accumulate in the bone, liver, gills, kidney, ovary, and muscle [6].
Environmental lead may result in an increase in vascular endothelial growth factor (VEGF) and blood concentrations [7, 8], and can lead to neurological and cardiovascular complications [9]. The reproductive system may also be affected by developmental disorders that are highly likely to occur in children [10-13]. Lead can cross the placenta and cause damage to the developing fetal nervous system [14].
The assessment and monitoring of environment heavy metal contamination is very important to prevent harm to human health. Currently, classical analytical methods, such as spectrometry, FIAAS (flow injection atomic absorption spectrometry), ion chromatography, and electrochemical techniques, are the main methods used for measuring environmental heavy metals pollution. The main disadvantage of these methods is the necessity for sample digestion under high temperature and pressure, or acidic conditions in which metal ions in solution are released [15] and subsequent analysis using expensive equipment. Therefore, simpler methods for evaluating heavy metals are required. More importantly, heavy metals are found to be present in biological systems either in bioavailable/toxic or non-available/ non-toxic forms, and current measuring methods are unable to distinguish between toxic and non-toxic fractions of these elements [16]. Furthermore, these methods are both time-consuming and costly [17]. Biosensors have been developed that are an effective alternative to conventional detecting systems. These may be highly sensitivity and simple to use [18]. Cell-based biosensors are biological sensors that contain a reporter gene under the control of a promoter that is sensitive to the presence of an agent, such as environmental contaminants that include heavy metals. Biosensors are used in various designs with different reporters and promoters. At low concentration of heavy bioavailable metals, bioluminescence signals are likely to be suitable[19, 20]. They can also be applied to monitoring bioavailable concentrations of heavy metals [21-27] and piezoelectric biosensors [28-30] as enzyme-based electrochemical biosensors. One of the most obvious advantages of this method is the ability to measure the bioavailable heavy metal at very low concentrations. It is also a cost-effective and time saving method [18]. In these biosensors, the expression of a reporter gene is controlled by a promoter, such as the pbrR promoter in the pMOL30plasmid of Cupriavidus metallidurans CH34 and cadC promoter in pI258 plasmid of Staphylococcus aureus that is sensitive to heavy metals. Most of these promoters originate from bacteria that have resistance systems against heavy metals [31, 32]. In this study, we have designed and evaluated the luciferase reporter gene expression of bacterial biosensor under the control of pbr and cadC promoters in Cupriavidus metalliduransCH34 and pI258 plasmids of Staphylococcus aureus, respectively, for the measurement of lead.
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 cadmium, tin, and zinc 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.
There are several advantages to using bacterial biosensors, including speed, simplicity and cost. Biological sensors containing cadA and pbr promoter regions have been designed by other researchers, the optimization of this cell biological sensor with ability to measure lead comparing the cadA and pbr promoters in a bioassay system was evaluated in this study. The use of biosensors or biological cell sensors containing a reporter gene controlled by promoters susceptible to the heavy metal ions can provide an efficient method to trace particular pollutants in the environment and in a biological solution[33]. The present study assessed a biosensor system for detecting lead ions through construction of a luminescent bacterial sensor containing the luc+ regulated by the cad promoter and cadC gene in plasmid pI258 of S. aureus and the pbr promoter and pbrR gene in pMOL30 plasmid of Cupriavidus metallidurans. Pb specific bacterial biosensors were formerly defined using reporter genes including lacZ, lux,and luc in the transcription fusion constructs [34-36]. In our study, the luciferase reporter gene was used. Luciferases, as a set of heterogeneous enzymes, are able to produce light as a byproduct of catalyzing reactions. They are reporter genes extensively used by prokaryotic and eukaryotic organisms due to their high sensitivity and ease of detection.The quantification of the emitted light, i.e. bioluminescence,is of great importance; it can also be measured using a liquid scintillation counter ,a luminometer, or even a X-ray film [35]. It was concluded that a pGL3-luc/ pbr-biosensor can detect Pb+2 in the range of 1–100 μM using the expression of firefly luciferase as a detector system, and is highly specific, with no expression of reporter in the presence of other metals such as Sn+2, Ni+2, Cd+2 are present. Moreover, this biosensor was 50 times more sensitive when compared with the previous biosensors reported by Chakraborty et al [32]. The R. mettalidurans CH34 strain has several resistance systems that can reduce the concentration of toxic substances to their non-toxic levels. A highly specific system for resistance to lead is known in plasmid pMOL30[37]. It effectively reduced the concentration of lead ions and is equipped with specific mechanisms for the transfer and separation of lead. The pbr operon includes pbrA, pbrB, pbrC and pbrD genes in which pbrD has a role as a chaperone to accumulate lead in the cell and pbrA eliminates lead ions[37]. Our results show that the pGL3-luc/pbr-biosensor is not expressed in the presence of cadmium, zinc, ortin, indicating high sensitivity and specificity of the designed system for lead detection. One of the most important heavy metal transfer systems in Staphylococcus aureus is located in the plasmid pI258. The plasmid has an operon cadA that encodes an ATPas of type P, which causes resistance to metals such as cadmium, lead, zinc, copper, and tin. The expression of the cadA operon is controlled by the cadC homodimeric protein. This protein is able, in a binary manner, to bind to the promoter and metal ions, such as cadmium, lead, zinc, and tin. The cad belongs toArsR / SmtB, a regulating protein family[38]. In our study, the luciferase gene was used as a reporter and E. coli strain of DH5α as a host. Our results showed that the pGL3-luc/cad-biosensor can detect at least 10 nM of lead and the lead toxicity was not observed until a concentration of 300 μM. However, the maximal expression of the reporter gene was performed at 10 μM. Our results are supported by the report of Liao et al that showed the regulating role of cad promoter and the cadC gene in plasmid pI258of S. aureus, the fluorescence emission was intensified with increasing Cd(II), Pb(II), and Sb(III) ions concentrations[39]. For Pb (II), just like our result in pGL3-luc/cad-Biosensor, to induce GFP expression significantly, 10 nM was the low,and 10 μMwas the maximum concentration of lead that induced significantly GFP expression[39]. The metallo regulatory α3N thiolate-rich site in cadC displays a practical selectivity for larger, softer heavy metal like Pb(II), Cd(II), although smaller boundary metal ions such as Zn(II) accommodated[40]. One of the limitations of this method is that bacterial biosensors require the necessary conditions for bacterial growth to operate, and the graphs are based on solving different concentrations of heavy metals in a bacterial culture medium. Therefore, to measure the amount of heavy metals in an unknown environment, it is necessary to optimize the biosensor in the new environment, which would itself require evaluation.
Our results show that the maximum expression of reporter gene was found in the presence of 100 μM of Lead in pGL3-luc/pbr-biosensor and 1 μM of lead in pGL3-luc/cad-biosensor. In this study, the specificity and sensitivity of the two heavy metal susceptive probes, pbr and cadA, was investigated. Sensors containing these two promoter regions were able to detect the concentration of lead between 1-100 μM and 10 nM to 10 μM of lead, respectively. For other heavy metals such as mercury, copper, nickel, manganese, zinc and cadmium, different biological sensors can be made and their presence in the environment can be measured with very high accuracy. To determine the accuracy of biosensors, a standard curve of Luciferase gene expression was plotted at different lead concentrations. The standard curve was constructed from triplicates values, we evaluated the accuracy of the biosensor with the specific concentrations that we had obtained from lead metals. By developing these sensors, the time required to identify environmental pollution can be minimized.
Chemicals
Analytical reagents, media and buffer solutions like TBE-EDTA buffer (Tris-borate-Ethylenediaminetetraacetic acid), NaOH (Sodium hydroxide), CaCl2 (calcium chloride), boric acid, Tris base, and agarose were all purchased from Merck (Germany). Fermentas (Lithuania) supplied the restriction endonucleases Nco1 and Hind3, T4 DNA ligase, and molecular ladder 10000-300bp. We also supplied the DNA polymerase (TaKaRa LA Taq®. DNA Polymerase), dNTP and MgCl2 from Takara (Beijing, China). In addition, the plasmid extraction kit and primers were brought from Bioneer (Seoul, South Korea).
Construction of biosensor plasmid
pMOL30 (X71400 AJ278984) and PI258 (GQ900378.1) containing the pbrR gene (634 bp) and CadC gene (601bp) (Accession number: pbrR: WP_003103716.1and CadC: WP_000726009, respectively, were synthesized and supplied by Millegen company. To ensure the accuracy of synthesized plasmid, the promoter region was sequenced. PGl3-control as a vector containing the Luciferase gene and E.coli strain DH5α as the host were used in our study. To obtain a large amount of pMA-T plasmid (a synthetic plasmid) which contains p-promoter sequences and the regulatory gene was sent to MilliGen, after evaluation at the NCBI site, for the synthesis of sequences. Synthesized sequences consisted of both pbr_pMA-T plasmids containing the promoter sequence of the pRR operon and the pbrR regulator gene including; cadA pMS-RQ-Bs plasmid containing the promoter region of the cadAp and the cadA gene regulating gene), it was cloned to E. coli host. Afterwards, pMA-T was extracted using plasmid extraction kit, and its quantity and quality were both examined by spectrophotometry and agarose gel, respectively, before they got digested by HindIII and NcoI. The promoter regions with the regulator genes were also purified from the gel electrophoresis. The received sequence and pGL3-control vector were cut using the same restriction enzyme (Nco1 and Hind3) and ligation reaction at 37°C for 3–4 h with ligase enzyme. The firefly luciferase gene was placed under the control of the received promoter sequences and recombinant plasmids of cad and pbr promoters were named pGL3-luc/pbr-biosensor and pGL3-luc/Cad-biosensor, respectively. Recombinant plasmids pGL3-luc/pbr-biosensor (Fig. 1A) and pGL3-luc/Cad- biosensor (Fig. 1B) were transferred to the DH5α bacteria using the chemical method of CaCl2 and then were screened using selective plates containing antibiotic Ampicillin. After plasmid extraction, PCR was performed to detect colonies containing the promoter region of pbr and cadA using primers designed for the cloned fragments. After these processes, recombinant plasmids were used to evaluating and measuring different concentrations of heavy metals.
Culture of bacteria and measuring biosensor activity of Luciferase enzyme
To study the efficiency of promoters the detection of heavy metals, a luciferase enzyme measurement performedin the presence of lead and other heavy metals such as tin, zinc and cadmium. In this process, E.coli stains carrying pGL3-luc/Cad-Biosensor and pGL3-luc/pbr-Biosensor were cultured in Luriae Bertani (LB) broth that contained 100 µg/mL ampicillin at 37˚C, overnight. Then 50 µl from overnight grown culture of pGL3-luc/pbr-Biosensor for 12 h and pGL3-luc/Cad-Biosensor with optical density (OD600) 0.8 for 2h were cultured in the presence of heavy metals at different concentrations [41]. Next, the culture was centrifuged at 5000 rpm for 10 min at 4°C metals for bacterial sedimentation. Then the medium was removed, and lysis buffer was added to the plate and sonicated at low temperature. Then, the amount of luciferase expression was measured by a luminometer (Berthold Company).
Statistical analysis
All the experiments were repeated at triplicate to minimize error. The Student t- test and one-way analysis of variance (ANOVA) were used to compare the statistically significant between the two groups and each group was compared with the baseline through the same method. . Statistical significance was set at *p<0.05. Data are shown as mean values± standard deviation (SD). Linear regression was used to model the standard curve. Analysis of Data was performed using SPSS version 22 statistical software (IBM, Chicago, IL, USA).
Compliance with ethical standards
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Informed consent
For this type of study, formal consent is not required.
Competing interests
The authors have declared no conflict of interest.
Funding information
This study was supported by Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University.
Consent for publication
All authors have given consent for publication
Availability of data and materials
All data generated or analyzed during this study are included in this published article
Author Contributions
MS and SH designed research; EN and RN performed research; HKS and NE analyzed data; EN and MN wrote the manuscript; AM, MN and ZF performed statistical analysis; MG and MR contributed new reagents or analytical tools. GAF revising the manuscript critically for important intellectual content. All authors read and approved the manuscript.
Acknowledgements:
None
Pb: Lead; VEGF: Vascular endothelial growth factor; Flow injection atomic absorption spectrometry: FIAAS; LB: Luriae Bertani; RLU: Relative luminescence units.
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