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1. BIOSENSORS In the laboratory of: Professor Kate Ziemer Northeastern University
3. PURPOSE:
To use liquid cultures of genetically altered bacteria as a living sensory detection system for potentially poisonous organic liquids and gases in enclosed environments (space stations, submarines)
4. MATERIALS AND EQUIPMENT: Pseudomonas putida TVA8 organic chemicals Luminometer Spectrophotometer gyratory shaker
5. THE BIOSENSOR:
The gene for Bioluminescence (fire flies) has been isolated and transferred into a bacteria named Pseudomonas putida TVA8
In the presence of organic liquids and gases the gene will make protein enzymes causing the bacteria to undergo bioluminescence.
6. The Bioluminescence is detected by inoculating the bacteria into an instrument:
Luminometer: measures RLU (Relative Luminescence Units)
The growth rate of bacteria is determined by inoculating the bacteria into an instrument:
Spectrophotometer: measures Absorbance at 546 nm
7. GENE INITIATION: Trichloroethylene (TCE), an organic liquid that stimulates the Bioluminescent gene: DNA, mRNA, protein (enzyme) to allow metabolism and produces light
8. PROCEDURE: Pseudomonas putida TVA8 cells were thawed and inoculated into a sterile liquid buffered medium in a sterile flask. TCE was added. Cells were shaken for aeration at 25-27C.
9. Samples of cells were taken at hourly intervals and measured as previously described for; Absorbance and Bioluminescence
10. Variables to be tested: What temperature was growth and Bioluminescence optimal? Do enzymes renature after heating to be able to perform their metabolic function?
11. RESULTS:
16. CONCLUSIONS: P. putida TVA8 grows best at: 30-32C Bioluminescence is best at: 25-27C
17. Washing the cells affects Bioluminescence. Enzymes for Bioluminescence are affected by heat.
18. Absorbance (cell growth) is not affected by washing cells or short high temperature exposure.
19. OTHER CONCLUSIONS: The base RLU for noninduced P. putida TVA8 is 10000. Nickel affects growth of P. putida TVA 8, but Cu and Al do not.
20. FUTURE EXPERIMENTS: The use of immobilized cells (in agar not liquid) What other organic compounds work? The use of a completely closed system.
21. LESSON DEVELOPMENT: This project is useful to Biotechnology for several reasons and can be incorporated into a Biomanufacturing Biotechnology program;
22. Reasons: Isolation of DNA Making genetically altered cells Growth of cells Growth curves Bioluminescence Sterilization techniques
23. Intrepretation of Data Design of new experiments
24. REFERENCES: Kate Ziemer, Northeastern Bruce Applegate, Perdue
25. THANKS TO MY COWORKERS: Professor Kate Ziemer Undergraduate Students: Dennis Callahan Laurel Rowse Graduate Student: Aneesha Sharma Postdoctorate: Jinyi Han
26. ADDITIONAL THANKS TO: RET people at Northeastern MassBay Deans: Ghazi Darkazalli Tala Khudairi Sally O’Connor, NIH
