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Sensing Superfund Chemicals with Recombinant Systems Sensing Superfund Chemicals with Recombinant Systems Sapna K. Deo, S. Xu, D. Ghosh, X. Guan, A. Rothert, J. Feliciano, E. D’Angelo, Leonidas G. Bachas, and Sylvia Daunert Department of Chemistry and Department of Agricultural Sciences University of Kentucky Lexington, KY Molecular Recognition in Analytical Chemistry • Proteins • Cells • High Throughput Screening • Whole Cell-Based Sensing Systems Analyte No Analyte Signal Reporter protein No Expression of Reporter Protein Arsenic Poisoning • Applications • Agriculture • Treatment for diseases • Industrial uses • Long exposure to low doses of arsenic • Skin hyperpigmentation and cancer • Other cancers • Inhibition of cellular enzymes New Bangladesh Disaster: Wells that Pump Poison... New York Times November 10, 1998 Arsenic contamination in the USA U. S. Geological Survey, Fact Sheet FS 063-00, May 2000 Arsenite Resistance in E. coli O/P arsR arsD arsA arsB arsC Schematic Representation of the Antimonite/Arsenite Pump - - AsO2 SbO2 Cytoplasm ADP ATP AsO - ATP 3- 2 AsO4 ADP ArsA ArsA ArsC Periplasm Membrane ArsB - - AsO2 SbO2 Fluorescent Reporter Proteins in Array Detection Protein Excitation Emission λ max λ max GFP 395 (470) 509 EGFP 488 509 BFP 380 440 GFPuv 395 509 YFP 513 527 CFP 433 475 CobA 357 605 RFP 558 583 P H C 3 A Production of fluorescent A P porphyrinoid compounds H3C N HN oxidation NH N A A P A P H C 3 A A A P P P P NH HN H3C N HN UMT sirohydrochlorin NH HN SAM P A NH HN H3C A A A A A UMT P HN P P P P H3C N SAM urogen III Dihydrosirohydrochlorin (Precorrin-2) NH N A CH3 A-CH2COOH A P-CH2CH2COOH SAM - S-adenosyl-L-methionine P P UMT- uroporphyrinogen methyltransferase III trimethylpyrrocorphin Construction of pSD601 plasmid O/P arsR bla arsD PCR pRLUX arsR O/P luxAB arsR + pSD601 ampR cobA cobA cobA pISA417 PCR Time Study for Arsenite 10000 -5 1 x 10 M NaAsO2 8000 6000 4000 2000 Fluorescence (cps) 0 -2000 024681012 Time (h) ALA in Urogen Pathway P A P A COOH COOH A P A P COOH NH HN NH HN ALA PBG Urogen III HO O dehydratase deaminase synthase N NH HN NH HN H P A A A H N H N 2 2 ALA PBG A P P P HMB Urogen III ALA - δ-aminolevulinic acid PBG - Porphobilinogen HMB- Hydroxymethylbilane Urogen- Uroporphyrinogen Time Study of Arsenite with ALA 10000 NO ALA NO ALA 8000 1mM ALA 6000 4000 2000 Fluorescence (cps) 0 -2000 024681012 Time (h) Calibration Plot with ALA 45000 40000 35000 30000 25000 20000 15000 Fluorescence (cps) 10000 5000 0 -8 -7 -6 -5 -4 -3 log[sodium-m-arsenite] Selectivity Study 40 Induction time = 1 h 35 Analyte concentration = 5 x 10-6 M (cps) 30 25 Intensity 20 15 10 Fluorescence 5 0 - - - 3- 2- - - Br Cl NO3 PO4 SO4 AsO2 SbO2 Field Challenges • Conventional analytical techniques • Field-kits • Background signal • Viability of the cells • Freeze drying • Strips (β-galactosidase) Addressing Environmental Analysis with Self-Contained Kits Lyophilized Reagents “The bacteria are dim in tap water (top) but glow brightly when arsenic is present 0 0.1 0.2 0.5 1 (bottom).” Arsenite (µM) Strip Sensors http://www.nature.com/nsu/030929/030929-7.html Centrifugal Microfluidic Platform for Micro-Total Analysis Systems Low power and space requirements Less reagent and sample consumption Easy disposal Short analysis time Integrate washing, sample preparation, and calibration Prototype Compact Disc 7 6 5 4 3 1 2 1. Waste Reservoir 2. Optode 3-7. Solution Reservoirs Prototype CD for four simultaneous analyses Incorporation of Whole-cell Sensing System for Arsenite/Antimonite on the CD Platform 140 130 120 (Counts) Fluorescence110 Intensity 100 90 0 15 Time (min) 30 45 60 0.4 0.5 0. 0. 6 7 Optical Density of Cells Calibration Curves Antimonite Arsenite 120 120 Detection Limit: 1 x 10-6 M Detection Limit: 1 x 10-6 M 100 100 80 80 60 60 40 40 20 Normalized Fluorescence 20 Normalized Fluorescence 0 0 -6.5 -5.5 -4.5 -3.5 -6.5 -5.5 -4.5 -3.5 log (Antimonite, M) log (Arsenite, M) Clc Operon and 3-chlorocatechol Pathway clcR o/p clcA clcB clcD O HOOC OH ClcA COOH ClcB ClcD COOH O COOH O COOH Cl Cl OH clcR clcA clcB′ pSMM50R-B′ r amp lacZ Calibration Curves 5 (5 min induction) ) 5 3-clc 10 4 × 3 2 4-clc 1 catechol Light Intensity (counts 0 -10 -9 -8 -7 -6 -5 -4 -3 -2 log (concentration, M) Selectivity Study 1.5 1) 3-chlorocatechol; 2) 4-chlorocatechol; 3) 4-chlorobiphenyl ) 6 4-7) catechol, biphenyl, 2-chlorophenol, and 4-chlorophenol 1 10 × 1.0 0.5 2 Light Intensity (counts 3 0 4-7 -10 -9 -8 -7 -5 -4 -3 -6 log (concentration, M) Challenges in Environmental Sample Analysis of Chlorocatechol 1. Extraction Method • Free Chlorocatechol Can be extracted by organic solvent O Fe • Bound Chlorocatechol O Cl OH Difficult to be extracted 2. Matrix Effect Selected Chemical Characteristics of Soils Organi Electric cal Oxal atextractablee Soils car cbon onductivity pH P Al Fe -1 -1 (%) (µmhos•cm ) (mg kg ) Acid washed sand 0.02 12 6.1 8 32 54 Maury silt loam 3.3 53 5.0 704 1894 4126 Woolper 7.5 38 5.9 3203 3086 3203 Organic humus 15.3 1099 6.4 415 3575 1106 Optimized Protocol for Soil Analysis Pellet (Intact, induced bacteria ) Supernatant 2 (Water + Bacteria) centrifuge Mixture 1 (10,000 ×g) (Soil + Water +Bacteria) Supernatant Induction (2h), Precipitate (Discarded) centrifuge (400×g) (Soil) Results of Soils HPLC Bacterial sensing system Sample Theoretical Experimental ± Theoretical Experimental ± matrix Value (mg•kg-1) SD (mg•kg-1) Value (mg•kg-1) SD (mg•kg-1) 0.5 0.49 ± 0.02 0.5 0.51 ± 0.02 Sand 2.0 2.03 ± 0.04 2.0 1.95 ± 0.08 10 9.75 ± 0.55 50 52.0 ± 2.6 Woolper 0.5 0.0 ± 0.0 0.5 0.52 ± 0.04 2.0 0.0 ± 0.0 2.0 1.90 ± 0.18 10 9.55 ± 0.75 50 54.0 ± 4.5 Maury 0.5 0.0 ± 0.0 0.5 0.43 ± 0.08 2.0 0.0 ± 0.0 2.0 2.24 ± 0.30 Organic 0.5 0.0 ± 0.0 0.5 0.43 ± 0.09 potting soil 2.0 0.0 ± 0.0 2.0 1.73 ± 0.28 Detection of PCBs Based on Dechlorination Followed by whole cell Sensing Clx Mg/K2Pd6 +H2O Cly - +(X+Y)Cl+H2 PCB Dechlorination Reaction HPLC Chromatogram of Dechlorinated Product: Biphenyl Whole cell Sensing of Biphenyl Based on bph Operon from P. pseudoalcaligenes KF707 pE R1 A1A2(orf3)A3A4 B C No biphenyl No Transcription HOPD O OH Transcription Biphenyl COOH pE Bphoperonof P. pseudoalcaligenes KF707 Biphenyl mol/L Increase in Light Intensity (%) lacZ pSD7001 1x 10-5 81.9 ± 9.5 ampR 1x 10-6 37 ± 6.2 1x 10-7 21 ± 4.8 Degradation Pathway of Hydroxylated-Biphenyl in the strain Pseudomonas azelaica HBP1 hbpR hbpC hbpA hbpD OH OH COOH COOH + + NADH+H NAD O2 O H2O OH OH OH COOH HbpC HbpD O2 H2O HbpA Whole Cell-Based Sensing System for Hydroxylated PCBS 10000 7500 5000 2500 Relative Light Units 0 -9 -8 -7 -6 -5 -4 -3 -2 Log (2-hydroxy-3',4'-dichlorobiphenyl, M) Collaborators Leonidas Bachas Marc Madou Barry Rosen Jan Roelof van der Meer Acknowledgments NIEHS-Superfund Basic Research Program the past and present members of the Daunert Group .
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