Present day analytical method such as gas chromatography- mass spectrophotometry (GC-MS), liquid chromatography (LC-MS) and atomic absorption chromatography (AAS) are straight forward approach with high sensitivity, selectivity, accuracy and reproducibility. These are succeeded in selective detection and identification of harmful contaminants from environmental, tissues or food samples. Mean while, suffers from a number of drawbacks such as, they are limited to a pre-determined set of substances, restricted to pre-programmed scope of analytes, fails to indicate bioavailable concentration, time consuming, expensive and requires lot of expertise. Bacteria have long been served as model for explaining the dose response dependent toxicity for specific chemicals in monitoring of environmental contamination. Ever since the conception of bacterial bioreporter in environmental microbiology has been an increases interest in the construction of well challenged report system based on genetic engineering concept. Bacterial bioreporter are living microorganisms that responds to changes in the environment by displaying specific and easily measurable signal. Based on gene expression in presence of toxic/ stress, resistance to heavy metal/ antibiotics, metabolism of organic compounds and other chemicals are explored for construction of reporter system in bacteria by fusion of specific reporter gene with promoter for detection of harmful contaminants. Assaying by using bioreporter for more complex real sample is more challenging because of presence of inhibitory compounds, unknown compounding effects on behavior and sorptive effects of matrix. The bacterial reporters are also explored for foodstuffs for monitoring of arsenic and tetracycline in rice and milk respectively. There are clear, assay miniaturization may provide the basis for the future incorporation of reporter cells into small devices, synthetic biology efforts will further streamline the construction and engineering of the new reporter strains. There are regulatory issues limiting the application of bioreporter assays, owing to the fact that the bacterial in question are genetically modified.

1. Design of Bacterial Bioreporters for Their
Application in Assays of Harmful Chemicals in
Different Environment
Raghu H V & Kumar N.

2. Introduction
Physico-chemical analysis
 High selectivity, sensitivity, accuracy and reproducibility
 Drawbacks–
 Limited to predetermined set of substances,
 Fails to indicate bioavailability
 Time consuming, expensive & requires lot of expertise

3. Bioreporters
• Measures Bioavailable concentration
• Predict the fate & availability
• Cost effective
• Multianalyte approach
• Detects group of compound rather single analyte

4. What are Bioreporter?
A microorganisms, cell culture or cell line, often genetically engineered,
with an activity that reflects changes in environmental conditions in dose
response dependent manner
Signal
Transcription Translation
Promoter
Reporter m RNA
Reporter
gene
protein
Analyte

5. Principle of Bacterial Bioreporters
Analyte Bacterial Signal
Bioreporters
Molecular recognition or
Physico-chemical condition
Class I Class II Class III
Target Stress Compound
compound- Increase in the or Stress
Increase in the out put decrease in
out put the out put
(van der Meer et al., 2004)

6. Cont…
Analyte RNA
degraded to polymerase
effector
Periplasmic Regulator
Analyte A binding A
protein binds
analyte
A
operator promoter Reporter
Transport/ Regulator recruits/ activates
Analyte
Diffusion Diffusion RAN polymerase
binds to
regulator
A
operator promoter Reporter
Signal
Reporter protein
synthesized
A mRNA Reporter
protein,
quantum
operator promoter Reporter yield,
CM stability,
specific
Cytosol
activity
out
(van der Meer et al., 2004)

7. Fundamentals of Bioreporter
Promoter
Transcription Translation
Lights On
Reporter m RNA
gene Reporter
protein
Analyte or
stress
Transcription Lights
Transcription off
Toxic
Translation
Promoter
Reporter
gene Reporter
Transcription protein
No Translation
Toxic
Analyte Lights On
(Xu et al., 2012)

8. Reporter gene
Firefly luciferase (luc)
Bacterial luciferase (lux)
Green fluorescent protein (GFP)
Chloramphenicol acetyltransferase (CAT)
Aequorin
Uroporphyrinogen III methyltransferases
β-galactosidase
β-lactamase
Alkaline phosphatase (SPAP)
(New et al., 2003)

9. Firefly Luciferase (luc)
 luc gene derived from Photinus pyralis
 High light out and rapid response kinetics
Mg2+
luciferase + luciferin + ATP Luciferase. luciferyl-AMP + PPi
Luciferase. luciferyl-AMP + O2 luciferase + oxyluciferin + AMP + C02 + hv
Exogenous addition of luciferin substrate
Not able to react autonomously or monitor
Maximal light output translates into very sensitive assay

10. Luciferase (lux gene)
(Close et al., 2009)

11. lux Gene
• lux AB genes
– Encodes only luciferase
– Exogenous addition of aldehyde (n-dacanol)
– Brighter & easier signal
• lux CDABE genes
– Continuous substrate independent signaling
– Accommodate complete gene cassettes
– Contains full complement of luciferase-luciferrin complex
– Real time to near real time capabilities
• lux CDABE operon synthetically optimized away from its
native AT rich state to towards GC rich MO’s

12. Green Fluorescent Protein (GFP)
Photoprotein clone from jelly fish
Aequorea Victoria
Doesn't require any substrate and
dependent on external light source
Functioning semi-continuously and near
real time
Dual color formats at different spectra

13. Fluorescent Reporter Protein in Array System
Protein Excitation Emission
GFP 395 509
EGFP 488 509
BFP 380 440
GFPuv 395 509
YFP 513 527
CFP 433 475
CobA 357 605
RFP 558 583

14. Aequorin
Ca2+ sensitive luminescent protein – Aequorea aequorea
- Inhibited by Mg2+ and also triggered by Eu2+, Sr2+ and Ba2+ Multifaceted
reporter protein with Affinities (KD) 1-10µL

15. Chloramphenicol Acetyltransferase
(CAT)
CAT gene
Acetyl coenzyme A + Chloramphenicol CoA + CAP-3 Acetate
• Radiolabelled (14 C or 3H) CAP by autoradiography & liquid scintillation
counting
• Fluorescent measurement
• Ex: CAT TOX (L) assay

16. β-Galactosidase
• LacZ gene from E.coli encodes a β-Galactosidase enzyme
• Hydrolysis of β-galactoside disaccharide into monosaccharide yield
colorimetric signal
• SOS chromotest – LacZ fusions to DNA to monitor mutagenic/
Carcinogenic genotoxic compound
• Luminescent, chemiluminescent or fluorescent endpoint also possible
• Contribute to elevated background signal
• Delayed data accumulation

17. Uroporphyrinogen (Urogen) III
Methyltransferases (UMT)
 Important for the biosynthetic pathways of vitamin B12 and siroheme
 Vitamin B12 - cobA genes in Bacillus megatarium, Methanobacterium ivanovii,
Propionibacterium freudenreichii, and Pseudomonas denitrificans.
 Second form of UMT is encoded by the cysG gene in E. coli and S.
typhimurium.
 Bioreporter for the selection of recombinant plasmids, as a marker for gene
transcription, and for the detection of toxic salts such as arsenite and
antimonite.
300 nm
Red to
red-
orange

18. β-lactamases
• Cleaves penicillin and cephalosporin
• TEM-1 β-lactamses (E.coli) engineered into cytosolic membrane
associated forms
• Membrane permeable flourogenic substrate CCF2/AM also enable
the determination of 50 β-lactamses in a cell

19. Construction of Reporter Gene
(Boulin et al., 2006)
Transcriptional Reporters
Translational Reporters
Smg-1 Based Transcriptional reporters

20. Ideal Bioreporter Protein and their Detection
Reporter protein Reporter origin Substrate Detection
genes method
Bacterial luciferase Lux AB Bioluminescent O2, FMNH2 Bioluminescence
or bacteria and long chain
luxCDAB aldehyde
E
β-galactosidase lacZ E.coli Galactopyranos Chemiluminesce
ide nc, colorimetry,
electrochemistry
and fluorescence
Fluorescent protein gfp Aequorea victoria NA Fluorescence
Infrared fluorescent various Bacteriophytochrome NA Fluorescence
proteins family
FMN based various Engineered from None Fluorescence
fluorescent proteins Bacillus subtilis and
P. putida
β-lactamases bla E.coli Lactamides Colorimetric
Spheriodenone crtA Rhodovulum Dimethylsphero colorimetric
sulfidophilum idene

21. Selection of Promoter
• Sensitivity & specificity for the chemicals considered
Specificity
Degree to which the expression cassette is responsive towards one
specific compound not to other
Affinity of the regulatory system, driving the reporter gene through
interaction with the promoter
Stresses, induced lesions, side products by toxicological reaction
Group specific
Compound specific
Metabolite specific reporter

22. Sensitivity of Promoter
• Level of compound generates a significant signal which can be
detected or measures comparable to LOD/LOQ
• System determines the sensitivity by cellular up take & affinity of the
compound for the regulatory system
• E.coli possess different system for uptake of compound
– Hydrophobic- diffusion
– Hydrophilic - porins
• Affinity of the compound to the regulatory protein determines the level
of protein/ compound complex induces the cellular promoter
– Higher affinity, lower the compound and higher sensitivity

23. Bacterial Bioreporter Design
Existing signaling pathway monitored by artificial output
Reporter protein is artificially controlled by sensory
regulatory system
To detect chemical compound or sample toxicity
Other possibilities for Bioreporters is oscillators or
riboregulated transcriptional cascade counter

24. Toxicity Bioreporter Design
 Promoter-reporter fusion
 Recombination & repair protein A (Rec A) – Lex A
regulated SOS response
 SOS response network in E.coli & S. typhimurium induces
toxicity response inducible gene expression–umu C, recN,
sfiA, rec A and colicin D gene

25. Heat shock response to detect compound leading to protein damage –
dnaK, grpE, and lon reporter construction
Antoxoidative defense regulons – oxy R and soxRS
New toxicity inducible
promoters
E.coli
Shotgun chromosomal library of
random 1.8kb fragment fused
Reporter Protein
Lux CDABE
reporter gene

26. Design Of Compound Specific
Bioreporters
• Isolated from bacteria displaying resistance mechanisms to specific
compounds or metabolize that toxic compounds Gene
Regulatory gene
Promoter Reporter gene
Reporter gene
Regulatory
protein
Regulatory Reporter
protein protein
Reporter
protein
(Van der Meer et al., 2010)

27. Bioreporters for mercury & arsenic
(Merulla et al., 2012)

28. Bioreporter for Mercury
Hg2+ Mercuric Secondary
Activator transport reductase regulator
repressor
mer R mer T mer P mer C mer A mer D
O/P
Repressor
Activation
Reporter gene
Mer R
Hg2+
Reporter Signa
Mer R/Hg protein l

29. Bioreporters for Heavy Metals
Analyte promoter Reporter Bacteria Time for Detection limit
detection
Aluminiu, fliC (E. coli) luxAB (V. harveyi) E. coli 20 min 40–400 mM
Antimonit, arsRD’ lacZ E.coli 17 h 100 mM
Arsenite
Arsenate, arsRDABC, luxAB (V. harveyi) S. aureus 1h ca. 0.01–10 mM
arsenite arsRBC
E. coli, S.
aureus) luc (firefly)
arsR
Cadmium cadA (S. luxAB (V. harveyi) S. aureus, 1–2 h 1–100 mM
aureus), cadA, blaZ E. coli
cadC S. Aureus 1.5 h 0.5–100 mM
(S. aureus) luc (firefly)
cadCo/p B. cereus 3h 10nM
Inorganic mer (Tn21) Luc (firefly) E.coli <0.1FM
mercury Mer (Tn21) luxAB(V. haevey) E.coli 2-3min 10-8M
Lux CDABE
Mer (Tn21) E.coli 40 min 0.5-5 µM

30. Bioreporter For Organic Chemicals
• Direct or indirect intracellular reaction of catabolic regulatory proteins
• Genetic dissection of pathways helped to disclose the different
compound recognition specificities of the proteins can be exploited
P. Fluorescence 5 R (nah+, sal+)

31. Compound Specific Bioreporter
• Report circuit based on LysR-type transcriptional activators
(NahR)
• Environmental compound concern are toluene, xylenes, &
ethyl benezene (XylE or TbuT), phenols (DmpR),
hydroxylated biphenyls (HbpR), Phenathathrene (PhnR)

32. Bacterial Reporter Construction
Sensors protein Host Promoter- Chemical Detection
chassis reporter targets sensitivity
fusion
XylR of P. putida E. coli Pu-lucFF Benzene, toluene 40 µM
& Xylene
DmpR of P. putida P. putida Po-luxAB Phenol 3 µM
FruR of E. E. herbicola fruBp-gfp Fructose & 2 µM
herbicola (AAV) sucrose
AraC of E.coli E.coli pBAD-gfpuv L-arabinose 0.5 µM
ArsR of E.coli E.coli arsRp-luxAb Arsenite & 5 nM
antimonite
MerR of E.coli E.coli merTp- Hg2+ 1 nM
luxCDABE
CadC of S. aureus Bacillus cadCp-lucFF Cd2+, Pb, Sn and 3 nM
subtilis Zn

33. Bioreporters for different environment
Sensors Host chassis Promoter- Chemical Detection
protein reporter targets sensitivity
fusion
ZntR of E.coli E.coli zntAp- Zn, Pb and Cd 5, 0.7µM
luxCDABE and 10nM
respectively
TetR of E. coli E.coli TetAp- Tetracycline 45nM
LuxCDABE
MphR of E.coli E.coli mphAp-lacZ Macrolides 10µM
SOS response B. subtilis yorBp-lucFF Various 60 nM
proteins of B. antibiotics (ex.
subtilis Ciprofloxacin)
Ada of E.coli E.coli alkAp- DNA alkylating 70 nM
luxCDABE agents

34. Bioreporter application in different
Environment
• Simple laboratory principle for the functioning
• Complex real world samples is more challenging
– Presence of inhibitory compound
– unknown compounding effect of chemical mixture
Sensor Host Promoter- Chemical Detection Matrix
protein reporter target limit
fusion
HbpR of E.coli E.coli hbpR-luxAB Hydroxylated -- Human serum,
polychlorinate urine
d biphenyl
ArsR of E.coli E.coli arsR-luxAB Arsenic 40–400 Rice powder,
mM portable water
(10ppb)
TetR of E.coli E.coli tetR- Tetracycline 100ppb milk
luxCDABE
NahR of P. P. nahR-luxAB naphthalein 10nM Soil, Gas,
putida putida aqueous phase

35. Next generation Bioreporters
Bioreporter cells immobilized on Silicon based CMOS surface to detect the
bioluminescence
Cells deposited on to the photodiodes, light emitting diodes or field effect
transistors
Hydrogels and other polymers used for long term maintenance of viability
Microarrays of living E.coli GFP reporter cells in PEG diacryliate Hydrogels with
optical trap
Bioluminescent bioreporter integrated circuit sensors

36. Commercially Available Bioreporters
• umu-Chromo Test kit, for rapid detection of genotoxicity
or DNA damage (ISO/ CD 13829)
• Microtox test (US EPA & ISO 11348) – natural
bioluminescence based method

37. Future Prospects
• Synthetic biology approach for further streamline the
construction and engineering of new reporter strains
• Multistrain bioreporter assay for addressing the effects of
chemical mixtures
• Predictive performance in comparison with standards
techniques
• Preservation of bioreporter cells in active form
• Regulatory issues limiting the bioreporter assay

38. Conclusions
 Based on gene expression in presence of toxic/ stress, heavy metals
antibiotics, organic compounds etc exploited for construction of
Bioreporters by fusion of specific reporter gene with promoter
 Assaying more complex real sample is more challenging, because of
possible presence of inhibitory compounds, unknown compounding
effects on behaviors & sportive effect
 Bioreporters also explored in foodstuffs for the detection of arsenic in rice
and tetracycline residues in milk below 100ppb
 Technical hurdle for limiting the application bioreporter assay is limiting
use of genetic modification of the reporter cell
 Overcome this barrier it is imperative that the bioreporter tests are
accredited as internationally accepted test