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Raghu H V & kumar N.

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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.

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Raghu H V & kumar N.

  1. 1. Design of Bacterial Bioreporters for TheirApplication in Assays of Harmful Chemicals in Different Environment Raghu H V & Kumar N.
  2. 2. IntroductionPhysico-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. 3. Bioreporters• Measures Bioavailable concentration• Predict the fate & availability• Cost effective• Multianalyte approach• Detects group of compound rather single analyte
  4. 4. What are Bioreporter?A microorganisms, cell culture or cell line, often genetically engineered,with an activity that reflects changes in environmental conditions in doseresponse dependent manner Signal Transcription Translation Promoter Reporter m RNA Reporter gene protein Analyte
  5. 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 StressIncrease in the out put decrease in out put the out put (van der Meer et al., 2004)
  6. 6. Cont… Analyte RNA degraded to polymerase effector Periplasmic RegulatorAnalyte 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. 7. Fundamentals of Bioreporter Promoter Transcription Translation Lights On Reporter m RNA gene Reporter proteinAnalyte 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. 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. 9. Firefly Luciferase (luc) luc gene derived from Photinus pyralis High light out and rapid response kinetics Mg2+ luciferase + luciferin + ATP Luciferase. luciferyl-AMP + PPiLuciferase. 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. 10. Luciferase (lux gene) (Close et al., 2009)
  11. 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. 12. Green Fluorescent Protein (GFP)Photoprotein clone from jelly fishAequorea VictoriaDoesnt require any substrate anddependent on external light sourceFunctioning semi-continuously and nearreal timeDual color formats at different spectra
  13. 13. Fluorescent Reporter Protein in Array SystemProtein Excitation Emission GFP 395 509 EGFP 488 509 BFP 380 440GFPuv 395 509 YFP 513 527 CFP 433 475 CobA 357 605 RFP 558 583
  14. 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. 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. 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. 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. 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. 19. Construction of Reporter Gene (Boulin et al., 2006) Transcriptional Reporters Translational Reporters Smg-1 Based Transcriptional reporters
  20. 20. Ideal Bioreporter Protein and their DetectionReporter 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 Fluorescencefluorescent proteins Bacillus subtilis and P. putida β-lactamases bla E.coli Lactamides Colorimetric Spheriodenone crtA Rhodovulum Dimethylsphero colorimetric sulfidophilum idene
  21. 21. Selection of Promoter • Sensitivity & specificity for the chemicals consideredSpecificityDegree to which the expression cassette is responsive towards onespecific compound not to otherAffinity of the regulatory system, driving the reporter gene throughinteraction with the promoterStresses, induced lesions, side products by toxicological reaction Group specific Compound specific Metabolite specific reporter
  22. 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. 23. Bacterial Bioreporter DesignExisting signaling pathway monitored by artificial outputReporter protein is artificially controlled by sensoryregulatory systemTo detect chemical compound or sample toxicityOther possibilities for Bioreporters is oscillators orriboregulated transcriptional cascade counter
  24. 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. 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. 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. 27. Bioreporters for mercury & arsenic (Merulla et al., 2012)
  28. 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 RepressorActivation Reporter gene Mer R Hg2+ Reporter Signa Mer R/Hg protein l
  29. 29. Bioreporters for Heavy Metals Analyte promoter Reporter Bacteria Time for Detection limit detectionAluminiu, fliC (E. coli) luxAB (V. harveyi) E. coli 20 min 40–400 mMAntimonit, arsRD’ lacZ E.coli 17 h 100 mMArseniteArsenate, arsRDABC, luxAB (V. harveyi) S. aureus 1h ca. 0.01–10 mM arsenite arsRBC E. coli, S. aureus) luc (firefly) arsRCadmium 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 10nMInorganic 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. 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. 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. 32. Bacterial Reporter Construction Sensors protein Host Promoter- Chemical Detection chassis reporter targets sensitivity fusionXylR of P. putida E. coli Pu-lucFF Benzene, toluene 40 µM & XyleneDmpR of P. putida P. putida Po-luxAB Phenol 3 µMFruR of E. E. herbicola fruBp-gfp Fructose & 2 µMherbicola (AAV) sucroseAraC of E.coli E.coli pBAD-gfpuv L-arabinose 0.5 µMArsR of E.coli E.coli arsRp-luxAb Arsenite & 5 nM antimoniteMerR of E.coli E.coli merTp- Hg2+ 1 nM luxCDABECadC of S. aureus Bacillus cadCp-lucFF Cd2+, Pb, Sn and 3 nM subtilis Zn
  33. 33. Bioreporters for different environment Sensors Host chassis Promoter- Chemical Detection protein reporter targets sensitivity fusionZntR of E.coli E.coli zntAp- Zn, Pb and Cd 5, 0.7µM luxCDABE and 10nM respectivelyTetR of E. coli E.coli TetAp- Tetracycline 45nM LuxCDABEMphR of E.coli E.coli mphAp-lacZ Macrolides 10µMSOS response B. subtilis yorBp-lucFF Various 60 nMproteins of B. antibiotics (ex.subtilis Ciprofloxacin)Ada of E.coli E.coli alkAp- DNA alkylating 70 nM luxCDABE agents
  34. 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 fusionHbpR of E.coli E.coli hbpR-luxAB Hydroxylated -- Human serum, polychlorinate urine d biphenylArsR 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 luxCDABENahR of P. P. nahR-luxAB naphthalein 10nM Soil, Gas,putida putida aqueous phase
  35. 35. Next generation BioreportersBioreporter cells immobilized on Silicon based CMOS surface to detect thebioluminescenceCells deposited on to the photodiodes, light emitting diodes or field effecttransistorsHydrogels and other polymers used for long term maintenance of viabilityMicroarrays of living E.coli GFP reporter cells in PEG diacryliate Hydrogels withoptical trap Bioluminescent bioreporter integrated circuit sensors
  36. 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. 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. 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

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