microbiology- microorganism's biosensors

1. IN THE NAME OF GOD
Subject:
structure and mechanism of biosensor performance in toxic metal detection
Seyed Reza khedmati
P.H.D student of Microbiology

2. WAT IS BIOSENSOR?
Self-contained integrated device that capable of providing specific
qualitative or semi-qualitative analytical information using a biological
recognition element which is in direct-spatial contact with a transduction
element.(IUPAC,1998)
Biosensor: bio analytical System

3. COMPONENT OF BIOSENSOR

4. STRUCTURE &
MECHANISEM

7. WHAT KIND OF MOLECULES CAN BE DETECT?
• Protein
• Toxin
• Peptide
• Vitamin
• Sugar
• Metal ion
Cholera toxin Glucose Heavy metals

8. HEAVY METAL DETECTION
Heavy
metal
Detectio
n
by
Microorganism
s
Prokaryote
s
Eukaryotes
In Water
(Articles)
In Soil
(Articles)
 A novel approach for rapidly and cost-effectively assessing
toxicity of toxic metals in acidic water using an acidophilic iron-
oxidizing biosensor
 Love-wave bacteria-based sensor for the detection of heavy metal
toxicity in liquid medium
 DETECTING BIOAVAILABLE TOXIC METALS AND METALLOIDS FROM NATURAL
WATER SAMPLES USING LUMINESCENT SENSOR BACTERIA
 Immobilized bacterial biosensor for rapid and effective
monitoring of acute toxicity in water
 Bioluminescent Bacterial Biosensors for the Assessment of Metal
Toxicity and Bioavailability in Soils
 Assessment of heavy metal bioavailability in contaminated
sediments and soils using green fluorescent protein-based
bacterial biosensors
General
Articles
 Heavy metal whole-cell biosensors using eukaryotic
microorganisms: an updated critical review
 Whole-Cell Bacterial Biosensors and the Detection of
Bioavailable Arsenic

9. GENERAL DETECTION OF HEAVY METALS
 Saccharomyces cerevisiae model among yeasts
 Tetrahymena thermophila model for ciliates
 Chlamydomonas model for microalgae
By Selecting The specific Reporter Gene
Heavy metal whole-cell biosensors using eukaryotic microorganisms: an updated critical
review
Reporter gene Advantages Disadvantages
β-Galactosidase(lacZ) Good stability Substrate dependent
Sensitivity depending on
substrate.
Low permeability
No ATP requirement Cellular lysis requirement
Eukaryotic luciferase
(luc)
Rapid response Substrate dependent
Very high sensitivity O2 and ATP
requirement
Low permeability and
stability Cellular lysis
requirement1
Green Fluorescent
Protein (gfp)
Good stability Moderate sensitivity
Substrate independent Lag-time for stable
fluorescence.
No ATP requirement Fluorescence after cell
death
No cellular lysis Autofluorescence
Comparison of the advantages and disadvantages of different reporter genes used in WCBs.
Part
1

10.  Three main chelating molecules are involved in the
cellular
response to metals: glutathione (a tripeptide) , phytochelatins
(oligopeptides), and metallothioneins (proteins;
The two main reporter systems that are the best options for eukaryotic WCBs(whole-cell
biosensor) are:
the eukaryotic luciferase - the GFP(green fluorescent protein )
• CONCLUDING
• (i) eukaryotic microorganisms used as WCBs have certain advantages over prokaryotic cells. Among them, extrapolation of
the results to higher eukaryotic organisms is more reliable than using bacteria;
• (ii) inducible systems are more appropriate for designing heavy metal WCBs; (iii) the decision as to whether to use substrate-
dependent or independent reporters will be determined by the greater or lesser capacity for permeability of the substrate
through the wall or membrane of the cellular system used as WCB;
• (iv) in general, few WCBs are validated using bioassays with real environmental samples;
• (v) the biotechnology for using microalgae as WCBs is still underdeveloped, although these photosynthetic microorganisms
have a great potential as biosensors based on genetic constructs involving photosynthesis genes;
• (vi) ciliates are eukaryotic microorganisms that have a series of advantages over yeasts or microalgae for designing heavy
metal WCBs; and (vii) with regard to heavy metal WCBs for use in real environmental polluted samples, the capacity for
sensitivity of the biosensor is more important than its level of specificity to a metal.

11. WHOLE-CELL BACTERIAL BIOSENSORS AND THE
DETECTION OF BIOAVAILABLE ARSENIC
• Bioavailable Arsenic is able to penetrate the membrane of the bacterial biosensor
and trigger the detectable response, luminescence, which can be measured.
bioavailable arsenic can be estimated by this.
• Compounds found are arsenic trioxide, sodium arsenate, and arsenic trichloride.
• In gram-negative bacteria, the arsenic resistance gene remains inactive with the
absence of As(III) in the cell due to the binding of the ars operon repressor protein to
the promoter region of the gene. As(III) activates the system by binding to the
repressor protein and freeing the promoter region for transcription .The freed
promoter region is transcribed to produce various components of the mechanism such
as arsB, an arsenite-translocating protein that serves as a transmembrane efflux
channel. This protein functions either chemiosmotically, without an energy source, or
by ATP hydrolysis when coupled with arsA, an arsenite-specific ATPase. ArsC, the
enzyme arsenate reductase, is also transcribed to reduce As(V) to As(III), since As(V)
cannot pass through the arsB/arsA pump. ArsD is a regulatory protein for additional
control over the expression of the system and arsR is a transcriptional repressor (Figure
1) The mechanism varies slightly in gram-positive bacteria, which lack arsA and arsD.

12. Oraganisms by luxAB in plasmid
p1258:
Staphylococcus aureus
Escherichia coli
Attachment of As ion: Activation of
GFP & luminescence
Detect by :Fluorimeter & Luminometer

14. BIOLUMINESCENT BACTERIAL BIOSENSORS FOR THE ASSESSMENT OF
METAL TOXICITY AND BIOAVAILABILITY IN SOILS
• Important factors
Bioavailability of heavy metals
PH of soil
 organic matter and the clay content of soil
nature of microorganisms
texture and iron oxide concentration & binding of metals by cell walls or by
proteins and extracellular polymers, • formation of insoluble metal sulfides, •
volatilization, and • enhancement of export from cells
attachment factors
either adsorption to the polysaccharide coating or adsorption to binding sites such as
carboxyl, phosphate, sulfhydryl, or hydroxyl groups on the cell surface.
Other factors:
 releasing specific compounds that form complexes with metals and by modifying soil pH
 Gene resistance on plasmids and transposons
DETECTION OF HEAVY METALS IN SOIL
Part
2

15. • lux genes from Vibrio fischeri
• plasmid pI258 (cad A & cad C genes) from Staphylococcus aureus
• genes, zntA and zntR from Escherichia coli
• Genes of Arsenic ion’s resistance in Gram-negative bacteria
• chrA and chrB genes from Ralstonia metalliduran
In orther to using measurable signal :
inaZ coding for ice nucleation protein, lacZ coding for ß-galactosidase, gfp coding for
for the green fluorescent protein, lux genes coding for the bacterial luciferase system,
lue genes coding for firefly luciferase, and genes encoding for the enzyme catechol 2,3-
2,3-dioxygenase

16. • A green fluorescent protein (GFP)-based bacterial biosensor Escherichia coli DH5a
(pVLCD1) was developed based on the expression of gfp under the control of the cad
promoter and the cadC gene of Staphylococcus aureus plasmid pI258
• This mechanism increase detection of heavy metals in soil
To detect the bioavailable heavy metals in soil:
one approach is based on the use of bacteria that are genetically engineered so that a
measurable signal is produced when the bacteria are in contact with bioavailable metal ions:
reporter genes such as lacZ, lux, and luc to detect Cd(II)/Pb(II)
colorimetric enzyme assay and bioluminescence have been very successful as a reporter for
Cd(II)/Pb(II) detection
The gene for green fluorescent protein (GFP) from the jellyfish Aequoria Victoria for detection
Assessment of heavy metal bioavailability in contaminated sediments and soils
using green fluorescent protein-based bacterial biosensors

17. • In this research , researcher describe the construction of a
nonpathogenic Escherichia coli whole-cell biosensor for the detection of Cd(II), Pb(II),
and Sb(III) by employing red-shifted GFP (rs-GFP) as a reporter protein
. The sensor plasmid is based on the expression of rs-GFP under the control of the cad
promoter and the cadC gene of the cadA resistance determinant of Staphylococcus aureus
plasmid pI258
To make new detector plasmid for heavy metal’s:
Colonization and extraction of Plasmid pI258 isolated from S. aureus
PCR primers designed with either EcoRI (forward primer) or BamHI (reverse primer)
recognition sequence extensions and addeded
The resulting recombinant plasmid, pVLCD1 ,was transformed into E. coli DH5a by the
CaCl2 competent cell method
 Schematic organization of the biosensor plasmid pVLCD1.

18. • The induction of the sensing system by a variety of metal
ions,
including As(III), Co(II), Cu(II), Fe(II), Hg(II), Mn(II), Ni(II), Sn(II), Cd(II),
Pb(II), Sb(III), and Zn(II) was studied by measuring the green fluorescence produced
For chemical analysis, concentrations of Cd(II), Pb(II) in water extracts of soil samples
were determined with inductively coupled plasma atomic emission spectroscopy analyzer
(PerkinElmer 3000SC, Norwalk, CT, USA).
Result:
Selectivity of the bacterial biosensor to metal ions. DH5a (pVLCD1) was treated with 1 mmol L1 of various individual metal ions or
mixtures of metal ions for 2 h. Induction intensity (in %) is defined as value of culture specific fluorescence (in SFI) with metal
treatment minus culture specific fluorescence (in SFI) of control then divided by culture specific fluorescence (in SFI) of control.
. Fluorescence of biosensor strain carrying pVLCD1
exposed to Sb(III). DH5a (pVLCD1) was treated with 1
mmol L1 of Sb(III) for 2 h at 37 C in LuriaeBertani (LB)
medium

19. Time-dependent induction of green
fluorescence with effectors. The DH5a cells
harboring the pVLCD1 plasmid were exposed
to 4 mmol L1
Cd(II), Pb(II), or Sb(III), and the specific
fluorescence intensity (in SFI) was determined
after different exposure periods

20. • Detection technique using a change of color, colorimetric, is widely used in the detection of
biomolecules, metal ions, and the presence of other compounds because the response can be
seen directly with the naked eye without the need for specialized instruments
• Immobilization is a technique that allows the bacteria can be used for long-term without
reducing its ability as bioreceptor
• Among the immobilization techniques, trapping method is one method which can be used for
cell immobilization.
• . In this method, the bacteria will be in a trap in the form of a matrix polymer
• example of matrix polymers: agarose, acrylamide, chitosan and alginate
Part
3
DETECTION OF HEAVY METALS IN WATER
Immobilized bacterial biosensor for rapid and effective monitoring of acute toxicity in water

21. IN THIS STUDY:
• E. coli was entrapped in the form of beads using calcium alginate
• Other materials as a reagent for sensing toxicant : Sodium alginate - calcium chloride -
Potassium ferricyanide -sodium chloride- ferric chloride - sulfuric acid, hydrochloric acid,
disodium hydrogen phosphate, sodium dihydrogen phosphate
Using :E. coli ATCC 25922
Culture & incubation & centrifuge & mix by calcium alginate
Absorbance value measured using spectrophotometer ( 600 nm)
Prussian blue was a colorimetric indicator of the process of
reduction of ferricyanide to ferrocyanide during bacterial
respiration with the addition of FeCl3 and can be measured
using spectrophotometer

22. • The relative activity of bacterial beads for monitoring
various
toxicants with different concentration levels (below Fig ) proved that
the higher the toxicant concentration, the smaller the response value. The higher the
toxicant concentration, the less the amount of K3[Fe(CN)6] converted to K4[Fe(CN)6]
during bacterial respiration process resulted in the amount of Prussian blue produced
was also decreased because toxicant inhibits bacterial respiration

23. Profile of reuse cycles of bacterial
biosensor beads for monitoring of
The stability of bacterial biosensor beads after
different storage times at 4 for monitoring toxicants
in water

24. • The ®re¯y (Photinus pyralis) luciferase (Luc) gene was used as a reporter in sensor strains
constructed.
• Luciferase catalyzes the oxidation of the heterocyclic substrate D-luciferin in the presence of
ATP producing visible light.
• The quanti cation of light emission . bioluminescence is one of the most sensitive means of
detection and it can be measured from living cells with a liquid scintillation counter, a
luminometer or even with X-ray ®lm
DETECTING BIOAVAILABLE TOXIC METALS AND METALLOIDS FROM
NATURAL WATER SAMPLES USING LUMINESCENT SENSOR BACTERIA
It presents schematically how the sensor bacteria work. In the absence of the inducing metal the expression of the
reporter gene is repressed, once the metal is added the expression of the reporter gene is induced and after substrate
addition (D-luciferin) the cells become luminescent, which can easily be measured

25. • Freeze-dried cells were reconstituted by two diffrent methods. Bacillus subtilis strains,
BR151(pTOO21) and BR151(pTOO24), and toxicity measurement strains Staphylococcus
aureus RN4220(pCSS810) and Escherichia coli MC1061(pCSS810) were reconstituted
• containing 0.5% casein hydrolysate for E. coli and S. aureus strains detecting mercury and
arsenite,
• containing 0.1% casein hydrolysate for the arsenite detecting B. subtilis strain
After addeding heavy metals:
The luminescence was measured with a microtiter plate reading luminometer, Luminoskan
(Labsystems, Helsinki, Finland)

26. RESULT
Spiked water samples. Metal sensing ability presented as induction coefficients of natural
water samples spiked with arsenite (AsOÿ
2 ), cadmium (Cd2+), lead (Pb2+) and mercury
(Hg2+).

27. Thank you