Post on 11-Oct-2020
Bacterial Bioluminescence from Microbe to Man:Lux Sensing Platforms in Environmental Toxicology g gy
and Biomedical Applications
Gary Sayler,
Center for Environmental Biotechnology,Department of Microbiology, The University of
T d J i t I tit t f Bi l i l S iTennessee and Joint Institute for Biological Sciences, Oak Ridge National Laboratory, Oak Ridge TN USA
China Ecology Forum, May 24, 2013, Beijing
Exploiting Gene Expression for Real time En ironmental Biol minescent SensingEnvironmental Bioluminescent Sensing
• Bacterial Chemical Sensing and Process Control
• BioMicroelectronicSensors
• Eukaryotic Luxu a yo c uexpression in yeast and Mammalian Cells
ObjectivesObjectives
D l i f i i l l d l CDABE• Develop a series of positively regulated lux CDABE transcriptional fusions for Whole Cell Biosensors of Organic PollutantsOrganic Pollutants.
• Apply the Resulting Bioreporter Strains forApply the Resulting Bioreporter Strains for Fundamental Investigations into the Occurrence, Bioavailability and Biodegradation of Pollutantsy g
• Mechanistic Tool for Inter Species Extrapolation in Environmental and Biomedical Sciences
Bio/Environmental RationaleBio/Environmental Rationale
• Real‐Time Analytical Approach for the Detection y ppand Measurement of Bioavailable Contaminants in the Environment and Waste Treatment
• On‐line and In Situ Process Monitoring and Control Strategies for Bioremediation and WasteControl Strategies for Bioremediation and Waste Treatment
• Alternative Endpoints for Clean Up Technology• Alternative Endpoints for Clean Up Technology and Toxicology Assessment
Bi di l I Vi S i d I i i• Biomedical In Vivo Sensing and Imaging in Diagnostics and Therapy
Bioreporter Mechanisticsp490 nm bioluminescence
Signal
P tTranscription Translation
Promoter
Reporter
p
RNA
Flashlight fish
ReporterGene
mRNA Reporter Protein
Flashlight fish
luxCDABEAnalyte
luxCDABEfrom Vibrio
fischeri
Application: Bioreporters in Soils
Removable Cover
O2 Temp Moisture CO2
Fiber 0pticCables PVC Pipes
Containing Biosensors
Air
Pipe for Portable Light Sensing Probe
Inlet LiquidInlet
Clean SoilLysimeter Facility - ORNL
Treatment Zone(GEMs and/or Hydrocarbon)
Irrigation
Fiber Optic BasedLight Detection
System
Clean Soil
Gravel
SystemAir DistributionManifold
d Water
Coarse Sand
Central CoreLeachate
Ground Wa
SupplyRipp et al., Ripp et al., Environ Science TechEnviron Science Tech 34:84634:846Cox et al., Cox et al., Bioremediation JournalBioremediation Journal 4:69 4:69
In Situ Photomultiplier Detection of Bioluminescence
7
8
9
10
esce
nce]
A. With air
3
4
5
6
7
Bio
lum
ine
a
B. Without air
C Control
1
2
0 10 20 30 40
Days
Log
[
PMTPMT--basedbased
C. Control
PMTPMT--based based Days
Monitoring bioluminescence of Monitoring bioluminescence of Pseudomonas Pseudomonas fluorescens fluorescens HK44 in naphthalene contaminated HK44 in naphthalene contaminated soil after addition of a chemically definedsoil after addition of a chemically defined
probeprobe Probe in LysimeterProbe in Lysimeter
soil after addition of a chemically defined soil after addition of a chemically defined medium. Results from A) contaminated soilmedium. Results from A) contaminated soilin contact with air, B) without air, in contact with air, B) without air, and C) soil without naphthalene. and C) soil without naphthalene.
Photograph of Bioreporters in soilPhotograph of Bioreporters in soilPhotograph of Bioreporters in soilPhotograph of Bioreporters in soil
luxCDABE BioreporterspAnalyte Time for
induction Concentration
2,3 Dichlorophenol 2 4 6 T i hl h l
2 h 2 h
50 mg/ L 10 / L2,4,6 Trichlorophenol 2 h 10 mg/ L
2,4-D 20 – 60 min 2 μM – 5 mM 3-Xylene Hours 3 μM 4-Chlorobenzoate 1 h 380 μM – 6.5 mM 4 Nitrophenol 2 h 0 25 mg/ L4-Nitrophenol 2 h 0.25 mg/ LAflatoxin B1 45 min 1.2 ppm Alginate production 1 h 50 – 150 mM NaCl Ammonia 30 min 20 μM Antibiotic effectiveness against Staphylococcus aureus 4 h 100 CFUAntibiotic effectiveness against Staphylococcus aureus infections in mice
4 h 100 CFU
BTEX (benzene, toluene, ethylbenzene, xylene) 1 – 4 h 0.03 – 50 mg/L Cadmium 4 h 19 mg/kg Chlorodibromomethane 2 h 20 mg/ L Chloroform 2 h 300 mg/ LChromate 1 h 10 μM Cobalt Not specified 2.0 mM Copper 1 h 1 μM – 1 mM DNA d ( h d id ) 50 i 6 25 / lDNA damage (cumene hydroperoxide) 50 min 6.25 mg/mlDNA damage (mitomycin) 1 h 0.032 μg/ml Gamma-irradiation 1.5 h 1.5 – 200 Gy Heat shock 20 min Various Hemolysin production Not specified 5 mM cAMP
Hemolysin production Not specified 5 mM cAMPHydrogen peroxide 20 min 0.1 mg/L
luxCDABE Bioreporters (cont.)p ( )Analyte Time for
induction Concentration
in vivo monitoring of Salmonella typhimurium infections 4 h 100 CFUin vivo monitoring of Salmonella typhimurium infections in living mice
4 h 100 CFU
Iron Hours 10 nM – 1 μM Isopropyl benzene 1 – 4 h 1 – 100 μM Lead 4 h 4036 mg/kgLead 4 h 4036 mg/kgMercury 70 min 0.025 nM N-acyl homoserine lactones 4 h Not specified Naphthalene 8 – 24 min 12 – 120 μM Nickel Not specified 0.3 mM Nitrate 4 h 0.05 – 50 μMOrganic peroxides 20 min Not specified PCBs 1 – 3 h 0.8 μM p-chlorobenzoic acid 40 min 0.06 g/l
< 30 i 60 bp-cymene < 30 min 60 ppbPentachlorophenol 2 h 0.008 mg/L Phenol 2 h 16 mg/L Salicylate 15 min 36 μM Tetracycline 40 min 5 ng/mlTetracycline 40 min 5 ng/mlTrichloroethylene 1 – 1.5 h 5 – 80 μM Trinitrotoluene Not specified Not specified Ultrasound 1 h 500 W/cm2 Ultraviolet light 1 h 2.5 – 20 J/m2
gZinc 4 h 0.5 – 4 μM
A li i S ifi I d Ci iApplication Specific Integrated Circuits
•• Fabrication in CMOS Fabrication in CMOS ((Complementary MetalComplementary Metal--Oxide Oxide S i d tS i d t ) ll th) ll thSemiconductorSemiconductor) allows the ) allows the development ofdevelopment of inexpensive, inexpensive, rugged, and functionally rugged, and functionally gg , ygg , ydiverse chips that can be diverse chips that can be mass produced mass produced
•• RF telemetry RF telemetry
•• LowLow--noise signal processingnoise signal processingMichael Simpson, ORNLMichael Simpson, ORNL
•• Global positioningGlobal positioning
JPL P k d Mi hiJPL Packaged Microchip
Low-mass, low-,power, low-volume
Long term storage
No crew intervention
Real-time remoteReal time, remote, on-line sensing
Reproducibility Experiment for the Filter Enclosure
0 05 Ch l 0 h d h lCh l 0 h d h l
0 03
0.04
0.05
nce
Ch 0Ch 1Ch 2
Channel 0 had the lowest Channel 0 had the lowest dark pulse rate and best dark pulse rate and best signal to noise ratiosignal to noise ratio
0 01
0.02
0.03
umin
esce
Ch 2 Comparison with results Comparison with results
obtained with glass tube obtained with glass tube enclosure:enclosure:
0 01
0
0.01
Bio
lu
1.1. ~ 10 to 15 min delay in ~ 10 to 15 min delay in response timeresponse time
-0.010 0.5 1 1.5 2
Hours
2.2. Similar sensitivitySimilar sensitivity
3.3. Similar detection limitsSimilar detection limits
Bioreporters were contained behind filters in the enclosures TheBioreporters were contained behind filters in the enclosures. The liquid phase was circulated on the opposite side of the filter. At hour 0, the BBIC was exposed to 50 ppb salicylate.
Biofilm Electrode ChipBiofilm Electrode Chip
InterdigitatedInterdigitated 100100μμm Au and m Au and carbon carbon nanofibernanofiber electrodes electrodes on 100on 100μμm pitch.m pitch.
Fabricated at ORNL’s CenterFabricated at ORNL’s Center Fabricated at ORNL s Center Fabricated at ORNL s Center for for NanophaseNanophase Materials Materials Science user facility.Science user facility.
New Directions in Eukaryotic Cells
Whole animal imaging and implantable chip targets for environmental sensing
A Broad Range ofA Broad Range of Natural and Synthetic y
Hormonally Active Compounds
Under TSCA EPA has a Minimum of 70 000Minimum of 70,000 Chemicals to Screen(Not Including Drugs
d d dd )and Food Additives)
CWA & SDWA are theCWA & SDWA are the Broader EPA Interface to the Environment
Effects of Endocrine Disrupters on Wildlife• News reports beginning in the 1990’s• Feminized Fish
– United Kingdom– Europe– United States
• Chinook Salmon in WashingtonWhit P h i th G t L k• White Perch in the Great Lakes
• Small Mouth Bass in the Potomac River• White Suckers in Boulder Creek
• Links to increases in reproductive disorders and cancers– Breast cancer, prostate cancer, testicular cancerp
Even 21 ng/L of 17β-estradiol (E2) or 3 ng/L of 17α ethin l estradiol (EE2) ca ses preg lation17α-ethinyl estradiol (EE2) causes upregulation
of vitellogenin in male fish.
Trace Organic gContaminants
Why does the public care?
What are they?
Where do we find them?them?
How much isHow much is present?
Screening gApproachesReceptor Binding assaysReceptor Binding assays
Receptor Transactivation
Uterothrophic Assay
Hershberger Assays
SAR‐QSAR
Receptor‐ Reporter Assays
Hormones and Synthetic Chemicals
Human Estrogen ReceptorEstrogenic
Human Estrogen Receptor Compound
Nucleus
luxCERE
luxA luxBluxE
frp
luxD
frp
Light
Bioluminescent Bioreporters
Transcription
luxC luxD luxB luxEluxApromoter
Transcription
NADPHATP
AMP+PPiMembrane Lipids
SynthetaseReductase
Myristic Acid
NADPAMP+PPi
Transferase
Myristol‐ACP
( luxCE )
( luxD )
Myristyl Aldehyde
LuciferaseFMNH2
H2O
Acetyl‐CoAO2
Myristyl Aldehyde
LightFMN
O2
H2O( luxAB )
( frp )
NADP
FMN
NADPH2
Construction ofConstruction of S. cerevisiae bioreporters
human androgen receptor + androgen + plasmid = LIGHT
1st to Express the Full Cassette in E k tEukaryotes
A 30
• Saccharomycestransformed with
A
20
transformed with luxCDABE generated light
b b k d
10
2500X above background• Gupta, R.K., et al., Expression of the
Photorhabdus luminescens lux genes (luxA B
B020
15Photorhabdus luminescens lux genes (luxA, B, C, D, and E) in Saccharomyces cerevisiae. Fems Yeast Research, 2003. 4(3): p. 305‐313.
10
55
0
EDC high‐throughput 96‐well microtiter plate format imaged in real time with a Xenogen IVIS Lumina imaging system.
A
B BLYRa (A)
C
BLYES (B) BLYRa (C)BLYAS (D)
C
DD
Yeast Bioassays Response to E2Yeast Bioassays – Response to E2
Calculate EC50Measure light production over time
ce (C
PS)
10000
12000
14000
(540
nm
)
2.0
2.5S. cerevisiae BLYES (r2=0.97)S. cerevisiae YES (r2=0.96)
Bio
lum
ines
cenc
4000
6000
8000
Opt
ical
Den
sity
1 0
1.5
EC50
17-estradiol Concentration (M)
10-12 10-11 10-10 10-9 10-8 10-7
B
2000
000 O
0.5
1.0
17 estradiol Concentration (M)
D60
0)180
20017-estradiol (R2 = 0.98)17-ethynyl estradiol (R2 = 0.99)
e (c
ps/O
D
140
160
y y ( )estrone (R2 = 0.99)17-estradiol (R2 = 0.98)
nesc
ence
100
120
Bio
lum
in
60
80
S] (M
)
-8.0
-7.8
-7.6
y = 0.78x - 1.03R2 = 0.99
rmal
ized
0
20
40
log
[BLY
ES
-8.6
-8.4
-8.2
10-11 10-10 10-9 10-8 10-7 10-6 10-5
No 0
log [YES] (M)-10.0 -9.8 -9.6 -9.4 -9.2 -9.0 -8.8 -8.6 -8.4
-8.8
Concentration (M)
S. cerevisiae BLYES1e-7
17-estradiol spiked water samplesWater samples17-estradiol 1x10-7 M
1e-8
1e 7 va
lent
s
1e-9
nic
Equ
iv
1e-10
Est
roge
1e-11
1e-12
1e-13
Raw Wastewater
WastewaterTreatmentEffluent
Raw DrinkingWater
FinishedDrinkingWater
DownstreamUpstream17β‐estradiolstandard
1 2 3 4 5 6 7 8 9 10 11 12Plate map for high throughput testing of environmental samples…
A
B
Standard: 17β‐estradiol
S d d 17β di l S l Bl k T Bl kB
C
Standard: 17β‐estradiol Solvent Blanks True Blanks
Sample 1 Sample 7
D Sample 2
E Sample 3
F
G
Sample 4
S l 5
Sample 8
G
H
Sample 5
Sample 6
Methanol&Media ‐ ctrl Media ‐ ctrl
BLYES expose to 17b‐estradiol (1E‐7M to 2.5E‐12M)
BLYAS expose to DHT (1E‐6 to 2.5E‐11)
BLYR
Surface water samples – Brazil(used for source drinking water)
Jardim et al. 2012
Analyte Sampling PointsSampling PointsTanque Campinas Atibaia Sorocaba Cotiaq p
Chemical Chemical analysisanalysisE1 ND <LOQ ND <LOQ <LOQE1 ND <LOQ ND <LOQ <LOQE2 ND ND ND ND NDE3 ND 1 48 /L ND ND 7 7 /LE3 ND 1.48 ng/L ND ND 7.7 ng/L
EE2 ND ND ND ND NDBPA ND ND 2.76 ng/L 3.53 ng/L 11.4 ng/LOP ND 1.96 ng/L ND ND <LOQg QNP ND 1.24 ng/L ND ND ND
Predicted bioassay responsePredicted bioassay responsePredicted bioassay responsePredicted bioassay responseE2equiv. BDL BDL BDL BDL BDL
Bioassay analysisBioassay analysisE2equiv. ND 0.13 ng/L ND 0.7 ng/L 3.1 ng/L
Jardim et al. 2012
Sample Date of collection
Chemical detected by
chromatographic l i a
Calculated estrogenic
equivalents ( E2 i L 1)
BLYES(ng E2 equiv. L-1)
BMAEREluc/ERα (ng E2 equiv. L-1)
analysisa (ng E2 equiv. L-1)
Barueri-treated water
June-09 BDL BDL BDL BDLSept-09 BDL BDL BDL BDL
M h 10 BDL BDL BDL BDLMarch-10 BDL BDL BDL BDL
Barueri raw
June-09E3 7.7 ng L-1
BPA 11.4 ng L-1 BDL 3.1 1.9
E3 3 80 ng L-1Barueri- raw surface water
Sept-09E3 3.80 ng L-1
BPA 33.6 ng L-1 BDL 7.1 BDL
March-10E3 18.3 ng L-1
BPA 37 7 ng L-1 BDL 0.35 BDLBPA 37.7 ng L 1
Cerquilho-treated water
June-09 BDL BDL BDL BDLSept-09 BDL BDL BDL BDL
March 10 BDL BDL BDL BDLMarch-10 BDL BDL BDL BDL
Cerquilho-raw surface
June-09 BPA 3.53 ng L-1 BDL 0.7 BDL
Sept-09E1 0.76 ng L-1
BPA 6 26 ng L-1 BDL 2.1 9.0raw surface
water BPA 6.26 ng L
March-10E1 2.60 ng L-1
BPA 27.9 ng L-1 BDL 0.53 BDL
H it lHospital Effluent
June-09 E3 182 ng L-1 0.3 20.5 13.1
Blank BDL BDL BDL BDL
Hallsdale-Powell Utility District
Hallsdale-Powell Wastewater Treatment Facility
Experimental Design
Activated Sludge Fill & Draw Bioreactor Experiment
30mL Filtered
Wastewater
30mL SpikedFiltered
Wastewater
30mL Filtered
Wastewater
Activated Sludge100 mL
Activated Sludge
AS
Filtered Raw W t t
-
Activated Sludge E2 EE2 & DHT
AS+RefillBioreactors.
Incubated in 30 °C, 200rpm.
Field Sampling
Wastewater+ E2, EE2 & DHT
30mL were collected30mL were collectedat at 0, 1, 3, 7 0, 1, 3, 7 days. days.
Raw Wastewater
EXTRACTIONConcentrate liquid phase 40X
in methanol using
Yeast Bioluminescence in methanol using
Solid Phase ExtractionAssay
ff f
Effluent test results - HPUDWastewater Effluents Monitored for 9 months
100
Traditional Activated SludgeM b Bi t
og n
g/L) 10
Membrane Bioreactor
alen
cy (l
o
1
nic
Equ
iva
0.1
Est
roge
n
0.01
1 2 3 4 5 6 7 8 9 0 1 2 3 4 6 8 20 22 25 27 29 33 35 38
0.001
Wee
k 1W
eek 2
Wee
k 3W
eek 4
Wee
k 5W
eek 6
Wee
k 7W
eek 8
Wee
k 9W
eek 1
0W
eek 1
1W
eek 1
2W
eek 1
3W
eek 1
4W
eek 1
6W
eek 1
8W
eek 2
0W
eek 2
2W
eek 2
5W
eek 2
7W
eek 2
9W
eek 3
3W
eek 3
5W
eek 3
8
Effluent test resultsEstrogenicity test result ( 6-14-2012 samples)
70000
60000
MBR effluentsTAS effluentts
e (C
PS
)
50000
nesc
ence
40000
Bio
lum
i
30000
20000
Concentration Factor
0.01 0.1 1 10 100 100010000
Conclusions• Androgenic compounds are attenuated by
wastewater treatment in HPUD to below-detection-limit level.
i f• Estrogenic compounds can be detected from HPUD effluent from both MBR and traditional activated sludge.M b Bi i idi• Membrane Bioreactor is providing on average 10 fold cleaner effluent than gtraditional activated sludge in terms of estrogenic compoundsestrogenic compounds.
Measurement of estrogenic substances in samples of the the Deepwater Horizon oil spillsamples of the the Deepwater Horizon oil spill
Goals: Determine if oil, weathered oil, or dispersants contain estrogenic
b tsubstances.
Hypothesis: ypOil, weathered oil, and dispersants will contain estrogenic substances.
Gulf Oil SamplesGulf Oil Samples
Conclusions• S. cerevisiae BLYES detected estrogenic
substances in oil and weathered oil samplessubstances in oil and weathered oil samples.
C it 9500 (di t d t th• Corexit 9500 (dispersant used at the Deepwater Horizon spill site) is toxic at high concentrations.
Whole animal imaging and implantable chip targets for environmental sensing
Mammalian Expression StrategyMammalian Expression Strategy
ll bj d d i i i• All Genes Subjected to Codon Optimization– Match human codon preference– Remove restriction and regulatory sites
• Separate Using IRES ElementsSeparate Using IRES Elements– Mimic bacterial organizationIncrease translational efficiency– Increase translational efficiency
• Divide Expression Between Two Plasmids(l f ) d ( b )– pLuxAB (luciferase) and pLuxCDEfrp (substrate processing)
Further Research and New Directions
• Dealing with Mixtures and Interaction
• Molecular Dynamic Simulations‐Protein
• Automation to Decrease Processing Time
Docking Models
• On Line Real Time Monitoringo o g
• Eukaryotic Lux expression in Vertebrate andin Vertebrate and Mammalian Cells
Developing A Conditional Reporterp g pReplace with TET response element and CMVmini promoterCMVmini promoter
Toward Whole Animal Lux BioluminescenceToward Whole Animal Lux Bioluminescence
Comparison of Visual DetectionFLuc lux GFP
S.C. Injection
I.P. Injection
Comparison Between Yeast and T‐47D assay
18
14
16
18
BLYEST-47D
duct
ion
10
12
14
Fold
of I
nd
6
8
2
4
Time (Hour)
0 5 10 15 20 25 300
Whole animal imaging and implantable chip targets for environmental sensing
Zebrafish FunctionalityZebrafish Functionality
• Expression of• Expression of luxAB acts as a biosensor forbiosensor for aldehyde contamination in the Zebrafish model system
Confidential
l d l fComputational Modeling of interactions between humaninteractions between human
estrogen receptor alpha mutants estrogen receptor alpha mutantsand endocrine disruptors
Jun Wang
Human Estrogen Receptor α Ligand Binding Domain
17β‐estradiol in hER binding pocket
Binding affinity vs. transcriptional activity
Dock vs. Experimental
120S
core
100R2 = 0.513
enta
l Act
ivity
S
60
80
Expe
rime
40
60
2B1Z docking score
-18 -16 -14 -12 -10 -820
2B1Z docking score
AcknowledgementsT dd D k & N N
Acknowledgements• Todd Dykes & Nancy Newman
-Hallsdale Powell Utility DistrictAlice Layton, John Sanseverino and Dan
CloseParts of the reported work were Funded
in part by US EPA, NIH, and Eastman Ch i l CChemical Company
C ll b tCollaborators• Mike Simpson Mitch DoktyczTim McKnight• Mike Simpson, Mitch DoktyczTim McKnight,
Anatoli Melechko, Kate Klein; Oak Ridge National Laboratoryy
• Linda Del Castillo, NASA Jet Propulsion Laboratoryy
• Jay Garland, Michele Birmele, William McLamb; Kennedy Space Center Life ScienceMcLamb; Kennedy Space Center Life Science Support Facility
• Ben Blalock; University of Tennessee ElectricalBen Blalock; University of Tennessee Electrical and Computer Engineering Department
Primary Contributors and SponsorsPrimary Contributors and Sponsors
• Mike Simpson, Tim McKnight, & Anatoli Melechko; TED Henry ORNL/CEBMelechko; TED Henry ORNL/CEB
• Alice Layton, John Sanseverino, Dan Close, David Nivens Steve Ripp Rakesh Gupta StaceyNivens, Steve Ripp, Rakesh Gupta, Stacey Patterson, Liz Mitchell, Bin Wan, Bruce Applegate, Scott Moser Ben Blalock Sayed Islam; UT/CEBScott Moser, Ben Blalock, Sayed Islam; UT/CEB
• Mike Flickinger, Univ. of MinnesotaNASA EPA DARPA DOE NIH P ki El• NASA, EPA, DARPA, DOE, NIH, Perkin Elmer, Dynamac