illuminating environmental monitoring with living bioreporters steven ripp the university of...
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Illuminating environmental monitoring with living bioreporters
Steven RippThe University of Tennessee
Center for Environmental Biotechnology
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~27,000 students from over 100 different countries
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Analyte
Promoter
ReporterGene
Transcription
mRNA
Translation
Signal
The anatomy of a bioreporter
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Reporter systems
Reporter gene Disadvantage
Chloramphenicol acetyltransferase (CAT)
Often employs radioisotopes, requires the addition of substrate, requires separation of substrate and product
-galactosidase Endogenous activity, requires the addition of substrate
Aequorin Requires the addition of substrate and high Ca2+
Firefly luciferase (luc) Requires the addition of substrate
Green Fluorescent Protein (GFP)
Requires activation by external source
Bacterial luciferase (lux) Requires oxygen, discrete temperature range
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The attributes of bacterial luciferase (lux)
• Autonomous response• No user interaction required
• Repeatable, re-usable, nondestructive• Near real-time response• Easily measured output (light) with no
requirement for excitation source• But it is a living system
• ATP, O2, NADPH
• Temperature, pH, salinity extremes• Target toxicity• But living is good bioavailability
Bioreporter Sample
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lux-based bioreporter assays
• ‘Lights-off’ assays• Bioreporter continuously produces bioluminescence
(constitutive lux reporter gene system)• A decrease in bioluminescence upon exposure to a
chemical indicates toxicity• i.e., the Microtox® assay
• ‘Lights-on’ assays• Bioreporter generates bioluminescence only when
induced by a specific compound or family of compounds (inducible lux reporter gene system)
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Bioluminescent bioreporter chemical targets• Metals
• Cadmium (4 h, 19 mg/kg)• Chromate (1 h, 10 µM)• Cobalt (not specified, 2 mM)• Copper (1 h, 1 µM)
• Heavy metals• Iron (Hours, 10 nM)• Lead (4 h, 4 g/kg)• Mercury (70 min, 0.025 nM)• Nickel (not specified, 0.3 mM)• Zinc (4 h, 0.5 µM)
• Food/water/air quality• Aflatoxin (45 min, 1.2 ppm)• Ammonia (30 min, 20 µM)• Estrogens/Androgens (1-4 h, 10-11 M)• Histamine (30 min, 20 ppm)• Nitrate (4 h, 0.05 µM)• Tetracycline (40 min, 5 ng/L)
• Organics• 2,4-Dichlorophenol (2 h, 50 mg/L)• 3-Xylene (Hours, 3 µM)• 4-Chlorobenzoate (1 h, 380 µM)• 4-Nitrophenol (2 h, 0.25 mg/L)• BTEX (1-4 h, 0.03 mg/L)• Chloroform (2 h, 300 mg/L)• Dichloromethane (3 h, saturated)• Hydrogen peroxide (20 min, 0.1 mg/L)• Isopropyl benzene (1-4 h, 1 µM)• Naphthalene (8-24 min, 12 µM)• Organic peroxides (20 min, not specified)• PCBs (1-3 h, 0.8 µM)• p-chlorobenzoic acid (40 min, 0.06 g/L)• p-cymene (<30 min, 60 ppb)• Phenol (2 h, 16 mg/L)• Salicylate (15 min, 36 µM)• Trichloroethylene (1-1.5 h, 5 µM)
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The bioreporter Pseudomonas fluorescens HK44
Upper Pathway Lower Pathway
Naphthalene Salicylate Salicylate 2-oxo-4-hydroxypentanoate
A B F C E D R G H I N L J K
A B F C E D R G lux cassette
Naphthalene Salicylate Salicylate 2-oxo-4-hydroxypentanoateX
Wild-type
Bioreporter
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Bioreporter HK44 as a bioremediation process monitoring and control tool
Gravel
Coarse Sand
Contamination Zone(GEMs and Hydrocarbon)
Clean Soil
Clean Soil IrrigationSystemAir Distribution
Manifold
Central Core
Light Sensing Instrument
Leachate
O2 Temp Moisture CO2
Fiber 0pticCables
PVC Pipesfor Biosensors
Removable Cover
AirInlet Liquid
Inlet
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Bioremediation monitoring with P. fluorescens HK44
110+100 to 11090 to 10080 to 9070 to 8060 to 7050 to 6040 to 5030 to 4020 to 3010 to 200 to 10
Time 0 2 years laterNaphthalene
(ppm)
0
1,000
2,000
3,000
0 10 20 30 40
Bio
lum
ines
cenc
e (P
hoto
ns/s
econ
d)
Days
soil with HK44
soil without HK44
BioluminescenceFiber OpticCable
Encapsulated HK44 Cells
Porous MetalHousing
in situ soil bioluminescence
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Long-term (14 year) bioreporter survival
Lysimeter ConditionsNumber of
samples
Quantitative PCR copies/g (% of positive samples)
nahA tetA luxA
1PAH contaminated +
HK4436
Not detected
4455(17%)
Not detecte
d
2PAH contaminated +
HK4436
Not detected
872(17%)
2052 (17%)
Log Time (Days)
1 10 100 1000 10000
Lo
g cf
u/g
so
il
0
2
4
6
8
Chemically contaminated lysimeters (total heterControl lysimeters (total heterotrophs) Chemically contaminated lysimeters (tetracyclinControl lysimeters (tetracycline resistant)
Chemically contaminated lysimeters (total heterotrophs)Control lysimeters (total heterotrophs)Chemically contaminated lysimeters (tetracycline resistant)Control lysimeters (tetracycline resistant)
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Monitoring groundwater contamination at a U.S. Air Force Base
Pseudomonas fluorescens TVA8 bioreporter specific for BTEX (benzene, toluene, ethylbenzene, xylene) jet fuel components
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-5 5 15 40
Upgradient Downgradient
Groundwater Flow
Distance (meters)
Dep
th (
met
ers
abo
ve s
ea le
vel)
65.5
56.5 1 m
0
3 m
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BTEX profiles (ppm)
-15 -10 -5 0 5 10 15-5
0
5
10
15
20
25
30
10.0+8.6 to 10.07.1 to 8.65.7 to 7.1
4.3 to 5.72.9 to 4.31.4 to 2.90.0 to 1.4
-15 -10 -5 0 5 10 15-5
0
5
10
15
20
25
30
10.0+8.6 to 10.07.1 to 8.65.7 to 7.1
4.3 to 5.72.9 to 4.31.4 to 2.90.0 to 1.4
Met
ers
Met
ers
Bioluminescence GC/MS
Source trench
Source trench
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Evanescent optical fiber sensorsOptical claddings consisting of
uniquely ‘colored’ optical bioreporters Fiber core
Fiber length (m)
Lig
ht
atte
nu
atio
n (
spec
tru
m)
Bac
ksca
tter
ligh
t
Cladding
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Other environmental bioreporter sensing applications
On-line detection of wastewater treatment upsets
Remote detection of microbial ‘sick building syndrome’ contaminants
On-board UAV penetration through aerosol clouds
Water toxicity monitoring
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Using reporter bacteriophage (bacterial viruses) to target bacterial pathogens
Rinsate
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CCD imaging of bioluminescence output
105 104 103 102 10 1 0
E. coli cfu/mL
A bioluminescent phage assay for Escherichia coli
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0 2 4 6 8 10 12 14 160
10
20
30
40
50
60
70
Hours
Bio
lum
ine
sc
en
ce
(c
ou
nts
/se
c)
105 104 103 10102
10
3 hour preincubation
A bioluminescent phage assay for Escherichia coli
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Optical Application Specific Integrated Circuits (BBICs)
Encapsulated bioluminescent
bioreportersPhotodetectors
Signal processing
circuitry
Opaque porous barrier
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Whole cell LuxArray Analyte
flow
Removable, reusable circuit board (underneath)
96 distinct cellular reporter fluid
reservoirs
Flow-through output
9 cm
4 cm
Delrin® housing
Reservoir layer
Nanoplotted cellular reporter
membranePrinted circuit board
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Obstacles to overcome
• We are not bacteria!• The effect of a chemical on a bacterial cell does not
adequately profile human/animal toxicity
• ‘Real-world’ applications are rare• Regulatory agencies reluctant to adopt• Public perception of risk difficult to overcome
• Short selling the technology• Bioreporters complement but do not replace conventional
analytical chemical detection methods
• Engineered microorganisms cannot survive in the environment (or can they?)
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Acknowledgements
• The National Science Foundation, grants # CBET-0853780 and DBI-096385
• 490 BioTech Inc.• The Center for Environmental Biotechnology
• Dr. Gary Sayler• Dr. Tingting Xu• Dr. Dan Close• Pat Jegier• Dean Webb• Alexandra Rogers• Clara Beasley