1. The Development of Bioluminescent Biosensors for Air Environment Monitoring in Closed Ecosystems Li Yang, 2005 SLSTP Trainee Carnegie Mellon University June 23, 2005 Dr. Valentina Kratasyuk, Principal Investigator Biophysical Department, Krasnoyarsk State University (Russia) Li Yang, 2005 SLSTP Trainee Carnegie Mellon University June 23, 2005 Dr. Valentina Kratasyuk, Principal Investigator Biophysical Department, Krasnoyarsk State University (Russia)

2. 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 Introduction Future Space Travel

3. Glow to Grow Experiments Bioluminescent 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

4. Hypothesis 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

5. Bioluminescent Biosensors Convert 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 stem

6. Materials and Methods 1. Initial Air Sampling from Mars Green House and Environmental Chambers 2. Bioluminescent Bioassay in vivo Luminous Bacteria Assay: Photobacterium phosphoreum 3. Bioluminescent Bioassay in vitro Coupled Enzyme System: NADH- FMN:Oxidoreductase-luciferase 4. Test NanoCeram Filters with Bioluminescent Biosensors in H 2 0 and Air

7. Materials and Methods Experimental Reagents Materials 1.E.coli recombinant 2.(LUX-gene) 3.Tryptic Soy Broth 4.NaCl – 3% solution 5.Luciferase 6.Oxidoreductase 7.Tetradecanal 8.NADH 9.FMN 10.Ethanol 11.Phosphate buffer pH 7.0 Enzymes NADH-FMN- oxidoreductase- luciferase E.coli recombinant LUX-gene from luminous bacteria Bioluminometer

8. FMN + NADH + H + FMNH 2 + NAD + FMNH 2 + R 1 COH + O 2 FMN + R 1 COOH + H 2 O + R Membrane Inhibitors Ag chlorine Cytoplasmic membrane 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 phosphorylation uncouplers (phentachlorophenol, SDS) Respiratory blockers eg cyanide Membrane ATPase Cytoplasmic constituents L h 490 nm Luminous Bacteria

9. Luminous Bacteria Assay Scheme of Analysis luminescence I с Luminous bacteria, Enzymes control test luminescence I е sample

10. Luciferase Enzymatic Reactions Coupled Enzyme System: oxidoreductase-luciferase Bioluminescence is the emission of light, produced from a chemical reaction, which originates within a living organism FMN NAD(P)* NAD(P)H Oxidoreductase FMNH 2 Luciferase RCHO RCOOH O2O2 H2OH2O H+H+ NADH:FMN-oxidoreductase NADH (NADPH) + H+ + FMN  NAD(NADP)+ + FMNH2 (1) luciferase FMNH2 + RCHO + O2  FMN + RCOOH + H2О + h (2)

11. Analysis of Luminescent Intensity Maximum Emission (I 0 ) Sampling Emission (I s ) LIBI I s / I 0 = Bacterial Indexes (BI) calculated from the Luminous Bacteria Assay Luciferase Indexes (LI) calculated from the Coupled Enzyme System =

12. NanoCeram 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.

13. Expected 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 with my Biosensors

14. Pitfalls 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.

15. Timetable of Scheduled Activities Week 1: Introduction to Experiment and General ResearchJune 13-19 Week 2: Experimental Design and Operation of BioluminometerJune 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 SLSTPJuly 18-24

16. Acknowledgements Dr. Valentina Kratasyuk, Prof. Biophysical Department, Krasnoyarsk State University (Russia) Dr. Sergey Gusev, Biophysical Department, Krasnoyarsk State University (Russia) Diane Shoeman SIFT 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 Directors SLSTP (Spaceflight and Life Sciences Training Program) Project Counselors SLSTP (Spaceflight and Life Sciences Training Program) Elizabeth Raffi SLSTP (Spaceflight and Life Sciences Training Program) All SLSTP Trainees SLSTP (Spaceflight and Life Sciences Training Program)

17. References 1.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.

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