The Development of Bioluminescent Biosensors for Air Environment Monitoring in Closed Ecosystems Li Yang, 2005 SLSTP Trainee Carnegie Mellon University.

The Development of Bioluminescent Biosensors for Air Environment Monitoring in

Closed Ecosystems

The Development of Bioluminescent Biosensors for Air Environment Monitoring in

Closed Ecosystems

Li Yang, 2005 SLSTP TraineeCarnegie Mellon University

June 23, 2005

Dr. Valentina Kratasyuk, Principal InvestigatorBiophysical Department, Krasnoyarsk State University (Russia)

Li Yang, 2005 SLSTP TraineeCarnegie Mellon University

June 23, 2005

Dr. Valentina Kratasyuk, Principal InvestigatorBiophysical Department, Krasnoyarsk State University (Russia)

•NASA missions rely on closed

ecological life support systems

•Monitoring toxicity in closed

ecological system is a problem

•Physical/chemical tests can only

tell us chemical composition

•Biological assays of the toxicity

of environment must be

developed

•We suggest Bioluminescence

IntroductionFuture Space Travel

IntroductionFuture Space Travel

Glow to Grow ExperimentsBioluminescent Biosensors for Space Biotechnology

Glow to Grow ExperimentsBioluminescent Biosensors for Space Biotechnology

•Bioluminescence - biological emission of light in enzymatic reactions with luciferase

•Bioluminescence property of living organisms gives rapid response rate to toxicity

•Bioassays will be used to measure biological toxicity

HypothesisHypothesis

•The prediction of this project is that bioluminescent systems will respond to the toxicity of environmental conditions in closed ecological systems.

•Contaminants in the environment that are toxic to live organisms will act as inhibitors and interrupt bioluminescent reactions- causing measurable reduction in light intensity

Bioluminescent BiosensorsBioluminescent BiosensorsConvert biological light emissions to electrical signals

•Bioluminescent sensors: toxicity assay for living organisms, highly sensitive and accurate bioassay, small, portable, simple, and low cost, rapid response rate, quantitative

•Physical/chemical tests (gas-chromatography and mass-spectroscopy etc.) : will not reveal whether substance is harmful to living organism and require large complicated devices

Plant stemPlant stem

Materials and MethodsMaterials and Methods1. Initial Air Sampling from Mars

Green House and Environmental Chambers

2. Bioluminescent Bioassay in vivoLuminous Bacteria Assay: Photobacterium phosphoreum

3. Bioluminescent Bioassay in vitroCoupled Enzyme System: NADH- FMN:Oxidoreductase-luciferase

4. Test NanoCeram Filters with

Bioluminescent Biosensors in H20 and

Air

Materials and MethodsExperimental Reagents

Materials and MethodsExperimental Reagents

Materials1.E.coli

recombinant 2.(LUX-gene) 3.Tryptic Soy Broth4.NaCl – 3%

solution5.Luciferase6.Oxidoreductase7.Tetradecanal8.NADH9.FMN 10.Ethanol11.Phosphate

buffer pH 7.0

EnzymesNADH-FMN-

oxidoreductase-luciferase

E.coli recombinant

LUX-gene from luminous bacteria

Bioluminometer

FMN + NADH + H+ FMNH2 + NAD+

FMNH2 + R1COH + O2 FMN + R1COOH + H2O

+

R

MembraneInhibitorsAg chlorine

Cytoplasmicmembrane

Aldehydes,Cationic agents,Heavy metals

Heavy metals,Phenols

Polar agents,solvents,alcohols

Wall

Heavy metals(cadmium),Phenolics(Dichlorophenol)

Protonmotive force

Electron transport system

Non-polar organics(Toluene)Oxidative phosphorylationuncouplers(phentachlorophenol, SDS)

Respiratory blockerseg cyanide

Membrane ATPase

Cytoplasmic constituents

L h490 nm

Luminous BacteriaLuminous Bacteria

Luminous Bacteria AssayScheme of Analysis

Luminous Bacteria AssayScheme of Analysis

luminescence Iс

Luminous bacteria, Enzymes

control

testluminescence

Iе

sample

Luciferase Enzymatic Reactions Coupled Enzyme System:

oxidoreductase-luciferase

Luciferase Enzymatic Reactions Coupled Enzyme System:

oxidoreductase-luciferaseBioluminescence is the emission of light, produced from a chemical reaction, which originates within a living organism

FMN

NAD(P)*

NAD(P)H

Oxidoreductase

FMNH2

Luciferase

RCHO

RCOOH

O2

H2O

H+

NADH:FMN-oxidoreductase

NADH (NADPH) + H+ + FMN NAD(NADP)+ + FMNH2 (1)

luciferase

FMNH2 + RCHO + O2 FMN + RCOOH + H2О + h (2)

Analysis of Luminescent Intensity

Analysis of Luminescent Intensity

time, s

I,mV Sampling

I0 Is

Maximum Emission (I0)

Sampling Emission (Is)

LI BI Is / I0=

• Bacterial Indexes (BI) calculated from the Luminous Bacteria Assay

• Luciferase Indexes (LI) calculated from the Coupled Enzyme System

=

NanoCeram FiltersNanoCeram Filters• Bioluminescent sensors will test whether the NanoCeram

Filters remove the contaminants in liquid solutions.

• We will take a liquid solutions samples.

• Attach the filter and push the liquid solutions through the filter

• Bioluminescence Biosensors will be used to test the samples before and after filtrations.

Expected OutcomesExpected Outcomes•To clarify whether

bioluminescent biotests should be recommended as the alarm test to control acute toxicity from a variety of sources such as air, water or soil samples in Closed Ecological Systems.

•To design bioluminescent biosensors for control of the air and water quality surrounding plants grown in closed environments (the toxicity of the gas and liquid phases ) using the bioluminescent organisms or their enzymatic reactions

•To live and work on Mars withmy Biosensors

Pitfalls and LimitationsPitfalls and Limitations•Challenging new methods

•Unknown effects of gases on bioluminescence

•Using the plates instead of cuvettes in

bioluminometer.

•Air samples must be loaded into micro-tray. We

are not sure if the air samples can be directly

injected into the tray.

Timetable of Scheduled Activities

Timetable of Scheduled Activities

•Week 1: Introduction to Experiment and General Research June 13-19

•Week 2: Experimental Design and Operation of Bioluminometer June 20-26

•Week 3 & Week 4 : Continue Glowing Experiments June 27-10

•Week 5: Data analysis, Poster preparations, and ASGSB abstracts, July 11-17

•Week 6: Finale Poster Presentations, Dinner Banquet SLSTP July 18-24

AcknowledgementsAcknowledgements• Dr. Valentina Kratasyuk, Prof. Biophysical Department,

Krasnoyarsk State University (Russia)

• Dr. Sergey Gusev, Biophysical Department, Krasnoyarsk State University (Russia)

• Diane ShoemanSIFT Employee from Merritt Island High School

• Dr. Ray Allen Bucklin, Prof., Agriculture and Biological Engineering Department, University of Florida

• Dr. Melanie Correll, Asst Prof., Agriculture and Biological Engineering Department, University of Florida

• Program DirectorsSLSTP (Spaceflight and Life Sciences Training Program)

• Project CounselorsSLSTP (Spaceflight and Life Sciences Training Program)

• Elizabeth RaffiSLSTP (Spaceflight and Life Sciences Training Program)

• All SLSTP TraineesSLSTP (Spaceflight and Life Sciences Training Program)

ReferencesReferences1.Kratasyuk V.A. etc. The use of bioluminescent biotests

for study of natural and laboratory aquatic ecosystems. Chemosphere, 42 (2001) 909-915.

2.Kratasyuk V.A. Esimbekova E.N. Polymeric Biomaterials, The PBM Series, V.1:Introduction to Polymeric Biomaterials, Arshady R (Ed), Citus Books, London 2003, pp 301-343

3.Paddle, Brian. Biosensors for Chemical and Biological agents of defense interest. Review Article. Biosensors and Bioelectronics Vol. 11 No. 11 pp. 1079-113, 1996

4.Farre M., Barcelo D. Toxicity Testing of water and sewage sludge by Biosensors, bioassays and chemical analysis. Trends in Analytical Chemistry, Vol. 22, No. 5, 2003.

QuestionsQuestions?

??

?

¿ ¿

¿ ¿?

?