Illuminating environmental monitoring with living bioreporters

Steven RippThe University of Tennessee

Center for Environmental Biotechnology

~27,000 students from over 100 different countries

Analyte

Promoter

ReporterGene

Transcription

mRNA

Translation

Signal

The anatomy of a bioreporter

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

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

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)

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)

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

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

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

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)

Monitoring groundwater contamination at a U.S. Air Force Base

Pseudomonas fluorescens TVA8 bioreporter specific for BTEX (benzene, toluene, ethylbenzene, xylene) jet fuel components

-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

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

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

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

Using reporter bacteriophage (bacterial viruses) to target bacterial pathogens

Rinsate

CCD imaging of bioluminescence output

105 104 103 102 10 1 0

E. coli cfu/mL

A bioluminescent phage assay for Escherichia coli

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

Optical Application Specific Integrated Circuits (BBICs)

Encapsulated bioluminescent

bioreportersPhotodetectors

Signal processing

circuitry

Opaque porous barrier

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

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?)

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