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