Abstract and Figures

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
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© 2018 Biomedical and Biotechnology Research Journal (BBRJ) | Published by Wolters Kluwer - Medknow
26
Original Article
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.[1,2]
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 coli (E. coli), Mycobacterium tuberculosis,
Mycobacterium scrofulaceum, and the plant pathogens
Xanthomonas campestris pv. vesicatoria and Pseudomonas
syringae pv. tomato (P. syringae pv. tomato).[3-8]
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.[9,10] The bioreporters
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
1Chronic Respiratory Diseases Research Center, National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical
Sciences, Tehran, Iran, 2School of Engineering, University of Borås, SE‑501 90, Borås, Sweden, 3Departments of Biochemistry, Faculty of Biological Sciences, Tarbiat
Modares University, Tehran, 4Depar tments of Chemistry, Faculty of Basic Sciences, Shahid Bahonar University, Kerman, 5Tracheal Diseases Research Center, National
Research Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran
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 scheri or Aequorin luciferase of Jellysh does not provide a high sensitivity. The aim of current study was to design an incipient
Copper bioreporter with applying rey 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 specicity 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 rey luciferase
with a high specicity (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 scheri (D048908),
Bioreporter
Address for correspondence: Dr. Puria Motamed Fath,
School of Engineering, University of Boras, Boras 507 44, Sweden.
E‑mail: puria.motamed@yahoo.com
Abstract
Access this article online
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DOI:
10.4103/bbrj.bbrj_65_17
How to cite this article: Taghavi K, Fath PM, Hosseinkhani S, Mirzaei M,
Behrooj H, Kiani A, et al. Construction and genetic improvement of copper
bioreporter Escherichia Coli. Biomed Biotechnol Res J 2018;2:26-30.
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Taghavi, et al.: Construction of a copper‑sensitive Bioreporter
Biomedical and Biotechnology Research Journal ¦ Volume 2 ¦ Issue 1 ¦ January‑March 2018 27
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.[11]
In addition, recombinant E. coli by the lux gene of Vibrio
scheri did not distribute a concrete copper bioreporter.[12]
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, specicity of recombinant
E. coli XL1-Blue with Firey luciferase and copper-resistance
promoter of P. syringae 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
purication 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 efciency of Gene Runner software [Figure 1].
PCR was performed at 61.7°C annealing temperature, and
AccuPrep® Gel Purication Kit was utilized for DNA band
extraction and purication. 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 quantied
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 specicity.
Calibration diagram for different concentrations of copper
sulfate was quantied 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 quantications were carried out in
utilizing an air and acetylene ame at ow 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, ve samples of PCR colony were collected to check
the recombinant plasmid. In Figure 2, it is clear that all ve
samples (recombinant pGL3) engendered desirable DNA band
at 186 bp. Two other loaded samples were as control + (pGL3)
and control with no plasmid.
Figure 3 presents recombinant pGL3 plasmid under double
digestion by Nhe I and Sac I enzymes.
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.
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 Figures 5 and 6.
Forward Primer
10 20 29
CGATGAGCTC ATACTAAAAA AACTGAAAG
Reverse Primer
10 20 28
CTTAGCTA GC ACGCGCGGGT CCAGGTAG
Nhe I
Figure 1: Forward and reverse primers of Nhe I enzyme sequences
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Taghavi, et al.: Construction of a copper‑sensitive Bioreporter
Biomedical and Biotechnology Research Journal ¦ Volume 2 ¦ Issue 1 ¦ January‑March 2018
28
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 quantied by atomic absorption instrument.
Figure 7 represents luciferase assay calibration diagram
dIscussIon
In two recent decades, environment cordial materials instead
of chemical methods have been in the center of attentions.
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 −)
Figure 7: Calibration diagram for CuSO4 5H2O which was measured by
atomic absorption instrument
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)
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
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
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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Taghavi, et al.: Construction of a copper‑sensitive Bioreporter
Biomedical and Biotechnology Research Journal ¦ Volume 2 ¦ Issue 1 ¦ January‑March 2018 29
Recently, many studies have been done to design qualied
biosensors and bioreporters (Bacterial sensor-herald) which
are able to detect metal pollutions.[13,14] 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 rst 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.[15]
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.[16]
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 quantiable 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 rey luciferase which in comparison with the
previous studies whit insertion of copper promoter afore
lux gene or Aequorin without applying rey luciferase
gene, showed higher specicity (96.1%, 91.2%, and 82.7%,
respectively).[11,17]
Digestion of pGL3 was performed more than ve 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:
1. Low facility of Xho I enzyme to cut pGL3 due to its high
supercoiled structure
2. Low concentration of intrigued gene after digestion and
purication from the gel.[18,19]
In the current study, these quandaries were solved by designing
two methods:
(1) Synthesis of fascinated gene (Copper-resistance promoter)
and insertion it to the pGL3
(2) 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 rst 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 magnication 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 magnication
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 specically 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 quantication
may affect the sensitivities and induction coefcients of the
biosensor. By optimizing inhibitor factors, application of the
current copper bioreporter can be developed in human life.
Acknowledgment
We would like to express our deepest thanks to all specialists
and professors for their valuable contribution.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conicts of interest.
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