|Year : 2018 | Volume
| Issue : 1 | Page : 26-30
Construction and genetic improvement of copper bioreporter Escherichia Coli
Kimia Taghavi1, Puria Motamed Fath2, Saman Hosseinkhani3, Mohammad Mirzaei4, Hadi Behrooj4, Arda Kiani5, Atefeh Abedini1, Fatemeh Razavi1
1 Chronic Respiratory Diseases Research Center, National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran
2 School of Engineering, University of Boras, SE-501 90, Boras, Sweden
3 Departments of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
4 Departments of Chemistry, Faculty of Basic Sciences, Shahid Bahonar University, Kerman, Iran
5 Tracheal Diseases Research Center, National Research Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran
|Date of Web Publication||5-Mar-2018|
Dr. Puria Motamed Fath
School of Engineering, University of Boras, Boras 507 44
Source of Support: None, Conflict of Interest: None
Background: Copper is a pollutant compound which can cause earnest toxicity in human and some organisms. Bioreporters are frugal and non-toxic detectors for pollution compounds. Precedent designed recombinant Escherichia coli copper bioreporters with the lux gene of Vibrio fischeri or Aequorin luciferase of Jellyfish does not provide a high sensitivity. The aim of current study was to design an incipient Copper bioreporter with applying firefly luciferase and Copper resistance promoter of P. syringae pv.Tomato in Escherichia coli XL1-Blue. Methods: Recombinant pGL3 was obtained by applying the pGL3-Control vector to Escherichia coli XL1-Blue, by Polymerase Chain Reaction method and double digestion. Recombinant Escherichia coli cells were cultured with applying different concentrations of copper sulphate to study the activity of luciferase by Luminometer. Copper bioreporter specificity was resolute by different concentrations of Zinc sulfate and Ferric sulfate. Results: Recombinant Escherichia coli BL21, with copper promoter gene in pGL3 Vector showed the highest Luciferase activity in 0.1 millimolar of Copper sulfate. The highest Luciferase activity was in 0.09 millimolar and 1.0 millimolar of Zinc sulfate and Ferric sulfate respectively. Conclusion: Current study provided a categorical bioreporter for detecting copper, utilizing firefly luciferase with a high specificity (96.1%). By optimizing inhibitor factors, application of current copper bioreporter can be developed in human life.
Keywords: Copper (D003300), biosensors (D015374), Luciferase (D008156), Escherichia coli (D004926), Vibrio fischeri (D048908), Bioreporter
|How to cite this article:|
Taghavi K, Fath PM, Hosseinkhani S, Mirzaei M, Behrooj H, Kiani A, Abedini A, Razavi F. Construction and genetic improvement of copper bioreporter Escherichia Coli. Biomed Biotechnol Res J 2018;2:26-30
|How to cite this URL:|
Taghavi K, Fath PM, Hosseinkhani S, Mirzaei M, Behrooj H, Kiani A, Abedini A, Razavi F. Construction and genetic improvement of copper bioreporter Escherichia Coli. Biomed Biotechnol Res J [serial online] 2018 [cited 2019 Sep 22];2:26-30. Available from: http://www.bmbtrj.org/text.asp?2018/2/1/26/226579
| Introduction|| |
Over the last decades, increasingly immense amount of organic and inorganic pollutions have threatened the environment. Monitoring of pollutions such as benzene, toluene, phenol, and metal ions is the essential step for decontaminations.
Conventional physical detectors develop low-categorical monitoring in bio pollutions, and chemical detectors such as spectrometry and high-performance liquid chromatography result in hazardous vicissitudes in ecosystems. While, bacterial sensor-reporter (Bioreporter) provids fast, cheap, sensitive, and selectable analysis of pollution samples without any hazardous vicissitudes in ecosystems.,
Copper is a pollutant compound which can cause solemn illnesses in human such as instance Menkes syndrome, yellow nail syndrome, endogenous lipoid pneumonia, and Wilson's disease and also be toxic in some organisms due to its radical-composing characteristics. Recently, some resistance to copper has been reported in some bacteria such as Escherichia More Details coli (E. coli), Mycobacterium tuberculosis, Mycobacterium scrofulaceum, and the plant pathogens Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato (P. syringae pv. tomato).,,,,,
Bacterial sensor-heralds (Bioreporters) are kinds of bacteria with gene-regulatory system which is joined to a herald gene and engenders a categorical protein. The secreted protein can detect categorical chemical and physical targets. Luciferase is the most studied bioreporter protein., The bioreporters are customarily sensitive to 0.01 microgram (μg) to 0.1 milligram (mg) of toxic compounds.
Copper bioreporter has been less studied compared to other metals bioreporters such as Cadmium, Arsenic, Zinc, Iron, and heavy metals. In lots of studies, designed copper bioreporters, Aequorin luciferase from Jellyfish was applied which could not develop a sensitive copper bioreporter.
In addition, recombinant E. coli by the lux gene of Vibrio fischeri did not distribute a concrete copper bioreporter.
The aim of current study was to design a copper bioreporter E. coli whit competency of detecting the copper in the culture. In this incipient-designed study, specificity of recombinant E. coli XL1-Blue with Firefly luciferase and copper-resistance promoter of P. syringa e pv. tomato in detecting copper and not iron or Zinc was studied.
| Methods|| |
In the case of encoding proteins which give the bacteria resistance to copper, plasmid pPT23D of P. syringae pv. tomato was collected. COP operon, as the promoter sequence of the gene, was utilized afore luciferase gene and initial 172 nucleotides of COP operon were inserted into expression vector. According to its multiple cloning sites, the pGL3-control vector was applied to enable COP operon to insert into expression vector. Sac I and Xho I restriction sites were selected for cloning. Xho I enzyme (Fermentas, Germany) was not felicitous for double digestion of pGL3 due to high supercoiled structure of plasmid. In addition gel purification of double-digested promoter was not acceptable. Thus, polymerase chain reaction (PCR) method was applied. Forward and Reverse primers of Nhe I enzyme were designed by the efficiency of Gene Runner software [Figure 1].
PCR was performed at 61.7°C annealing temperature, and AccuPrep ® Gel Purification Kit was utilized for DNA band extraction and purification. PCR product of pGL3 plasmid was double digested by Nhe I and Sac I enzymes (Fermentas, Germany) and phosphate cessations of pGL3 plasmid which inhibit self-ligation in the host cells, were abstracted by SAP and Shrimp Alkaline Phosphatase. E. coli XL1-Blue host cells were treated with CaCl2 which enabled ligation of pGL3 vector and gene COP promoter. PCR colony with 186 bp size band designated correct transformation and insertion. Recombinant plasmids were extracted by AccuPrep Nano-Plus Plasmid Mini Extraction Kit and designed. Conclusively, some recombinant vectors were sent for sequencing after approving the quality of digestion with gel electrophoresis. Recombinant pGL3 was extracted from E. coli XL1-Blue by Mini-prep method and was transferred to BL-21. Recombinant pGL3 was extracted from E. coli XL1-Blue by Mini-prep method and was transferred to BL-21. Recombinant E. coli cells were cultured in Lysogeny broth medium (LB broth) and then induced with different concentrations of copper sulfate (Copper Industry Co., Iran) and after 4-h incubation at 37°C, activity of luciferase was quantified by luminometer after integration of luciferin 5 mM as substrate. This process was reiterated for different concentrations of Zinc sulfate (ZnSO4.H2O) (M. W.: 179.45 g/mole) and Fe2(SO4)3.5H2O (M. W.: 399.86 g/mole) to find copper bioreporter specificity.
Calibration diagram for different concentrations of copper sulfate was quantified by Varian SpectraAA 220 (Murglave, Australia) atomic spectrometer to demystify precision and precision of dissimilar densities of copper which were yare and integrated to the bacteria culture during luciferase assay. The hollow cathode lamp of copper was utilized as sources of radiation. The sensitive wavelength was 222.6 nm, lamp current was 4 mA, and slit width was 0.2 nm for the tenacity of all of the absorbance quantifications were carried out in utilizing an air and acetylene flame at flow rates of 3.5 and 1.0 L/min.
| Results|| |
The best annealing temperature was 61.7°C for copper Bioreporter Promoter amplification. After prosperous transformation which conceived superior colonies in the plates, five samples of PCR colony were collected to check the recombinant plasmid. In [Figure 2], it is clear that all five samples (recombinant pGL3) engendered desirable DNA band at 186 bp. Two other loaded samples were as control + (pGL3) and control with no plasmid.
|Figure 2: Gel electrophoresis of polymerase chain reaction colony products (Line 1: DNA Ladder, Line 2–6: polymerase chain reaction products of different colonies, Line 7: Control +, and Line 8: Control -)|
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[Figure 3] presents recombinant pGL3 plasmid under double digestion by Nhe I and Sac I enzymes.
|Figure 3: Double digestion of recombinant pGL3 after polymerase chain reaction colony. However, DNA Ladder is not clear (Line 1: DNA Ladder and Line 2: polymerase chain reaction product)|
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Luciferase assay of recombinant E. coli (Copper bioreporter) is shown in [Figure 4] which represents effects of different concentrations of CuSO4.5H2O on recombinant E. coli. The bioluminescent light was quantified with three different samples, and average numbers were shown by mean + SD diagram. Recombinant E. coli BL-21, with copper promoter gene in pGL3 Vector, showed the highest Luciferase activity in 0.1 mM of CuSo4.
|Figure 4: Different concentrations of CuSO4 which were added to LB medium consisting of recombinant Escherichia coli BL-21, with copper promoter gene in pGL3 Vector|
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Recombinant E. coli BL-21, with copper promoter gene in pGL3 Vector, showed the highest luciferase activity in 0.09 mM and 1.0 mM of ZnSO4 and Fe2(SO4)3.5H2O, respectively. The results of luciferase assays on different concentrations of ZnSO4.H2O and Fe2(SO4)3.5H2O are presented in [Figure 5] and [Figure 6].
|Figure 5: Different concentrations of ZnSO4 which were added to LB medium consisting of recombinant Escherichia coli BL-21, with copper promoter gene in pGL3 Vector|
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|Figure 6: Different concentrations of Fe2(SO4) which were added to LB medium consisting of recombinant Escherichia coli BL-21, with copper promoter gene in pGL3 Vector|
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To identify precision of dissimilar densities of copper which was integrated to the bacteria culture during luciferase assay, luciferase calibration diagram for different concentrations of copper sulfate was quantified by atomic absorption instrument. [Figure 7] represents luciferase assay calibration diagram
|Figure 7: Calibration diagram for CuSO45H2O which was measured by atomic absorption instrument|
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| Discussion|| |
In two recent decades, environment cordial materials instead of chemical methods have been in the center of attentions. Recently, many studies have been done to design qualified biosensors and bioreporters (Bacterial sensor-herald) which are able to detect metal pollutions., Copper bioreporter has been less studied compared to other metals bioreporters. The application of copper on luc gene site has not been tested yet in the researches. Therefore, copper in the first phase of experiments was applied on E. coli BL-21 which harbors pET-16b. Compared to the previous study result on SAMDC1 gene, luciferase activity decremented gradually from Blank to 1.7 mM CuSO4 and reduced rapidly until 3 mM CuSO4.
However, when lactose as an inducer was integrated into the culture, two results were observed. On the one hand, the results demonstrated higher luciferase activity compared to the absence of Lactose. On the other hand, the activity of luciferase increased from Blank to 1 mM CuSO4.
In a former antecedent study, copper worked as an inhibitor in the medium which diminished the activity of cells and thereupon expression of luciferase gene reduced. Even at higher concentration of CuSO4, it caused cell death.
These observations inspirited us to design and engender a copper bioreporter to be able to detect different concentrations of copper sulfate in the medium and react to it by engendering luminescent light which was quantifiable by luminometer. In regard to the toxic effect of CuSO4 on luciferase activity or luciferase expression in E. coli that could be a felicitous progenitor for quantifying amount of copper. The current study provided a bioreporter categorical for copper tenacity utilizing firefly luciferase which in comparison with the previous studies whit insertion of copper promoter afore lux gene or Aequorin without applying firefly luciferase gene, showed higher specificity (96.1%, 91.2%, and 82.7%, respectively).,
Digestion of pGL3 was performed more than five times but the results of digestion were not acceptable. Infelicitously Xho I could not cut plasmid as well as Sac I. This trouble was solved by applying gel electrophoresis.
| Conclusion|| |
In anterior studies, two distractions were observed after digestion of pGL3 and PCR2.1 plasmids by Xho I and Sac I enzymes which inhibited progression of the experiment:
- Low facility of Xho I enzyme to cut pGL3 due to its high supercoiled structure
- Low concentration of intrigued gene after digestion and purification from the gel.,
In the current study, these quandaries were solved by designing two methods:
- Synthesis of fascinated gene (Copper-resistance promoter) and insertion it to the pGL3
- Supersession of Nhe I enzyme instead of Xho I, and design congruous primers and running PCR to obtain high concentration of intrigued gene from PCR 2.1
The first method was so simple procedure if it had been applied, but it was not an experimental and logical technique, due to synthesized gene and inserted to pGL3 vector by a company product and only luciferase assay performance by researcher. In the current study, the second method was applied.
In the current study, at 0.05 and 0.2 mM concentrations the luciferase activity was virtually 50%, but by incrementing amount of CuSO4 in the medium, the activity of luciferase was decremented conspicuously. Currently designed bioreporter showed acceptable activity at 0.08 and 0.1 mM densities of CuSO4. The results could be cognate to the lethal effect of high concentration of copper sulfate on the recombinant cell which inhibits magnification of bioreporter. Currently designed bioreporter showed concrete copper detection capability under situations which were designed and considered. Higher concentrations of Zinc (>0.4 mM) inhibited the magnification of the more preponderant amount of bacteria in the culture. This issue can be due to the bactericide feature of Zinc. The currently designed copper bioreporter showed little increase in activity at higher concentration of Fe2(SO4)3 (0.2-1 mM) but did not specifically detect Ferric Sulfate in the medium. The contradictory result can be due to some personal or experimental mistakes.
We endeavored to accomplish the current study under standard states. However, several adscititious factors including host strain, medium composition, magnification phase of the harvested bacteria, and amount of bacteria per quantification may affect the sensitivities and induction coefficients of the biosensor. By optimizing inhibitor factors, application of the current copper bioreporter can be developed in human life.
We would like to express our deepest thanks to all specialists and professors for their valuable contribution.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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