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National Science Foundation, Research Experience for Undergraduates, Utah State University August 5, 2010

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Presentation on theme: "National Science Foundation, Research Experience for Undergraduates, Utah State University August 5, 2010"— Presentation transcript:

1 National Science Foundation, Research Experience for Undergraduates, Utah State University August 5, 2010

2  Problem  Pseudomonas Putida KT2440  Key Words  Objective  Equipment  Chemoattractants  Growth Curve Phases  Results/Data  Growth Curve Parameter  Velocity  Turn Angle  X vs. Y Displacement  Project Contribution  Conclusion  Future Work  Acknowledgements  References  Questions

3 Toxic Chemicals In SoilEffects  Due to pesticides, oil spills, landfill leaching etc; soil is contaminated with toxic chemicals:  2,4-Dichlorophenoxyacetic acid  Most widely used herbicide in the world  Major component in Agent Orange  Toluene  Naturally occurring in crude oil  Byproduct of gasoline production  Napthalene  Nitrobenzene  Water Contamination  Farmland Contamination  Environmental Damage  Health Risks

4  Chemotaxis: The observable reaction of an organism moving toward or away from the concentration of a certain chemical.  Chemoattractant: A certain chemical that the organism of interest is attracted to.  Video Microscopy: Microscopic analysis of nano sized samples to capture real time videos: 15 frames/second

5  Motile due to flagella  Non-pathogenic: Safe to be released into the environment  Chemotactic towards soil pollutants ◦ Naphthalene ◦ Toluene ◦ Certain aromatic compounds  4-hydroxybenzoate ◦ 2,4-Dichlorophenoxyacetic acid  Degrades certain toxic chemicals

6 Taken by Serena Bennett with IX71 inverted microscope at 60X magnification at 15 frames/sec.

7  Observe P. Putida characteristic reactions/indicators from exposure to two separate chemoattractants:  Succinic Acid (Succinate)  Sodium Benzoate  Compare  Growth Curve Parameters  Velocity (Individual bacterium)  T urn Angle (Individual bacterium)  Displacement (Individual bacterium)

8  Spectrophotometer ◦ Measures absorbance (optical density)  Proportional to concentration  IX71 Inverted Video Microscope ◦ (Olympus, Center Valley, PA, USA) ◦ Digital CCD camera (Olympus DP30BW  ImagePro v. 6.1 ◦ (Media Cybernetics, Bethesda, MD, USA) ◦ Image Analysis  OriginPro v. 7.5 ◦ (OriginLab Northampton, MA, USA)  PPKT2440 ◦ Lysogeny Broth asp?product=1023&p=72

9 Succinic AcidSodium Benzoate  15mM ◦ 88.5mg in 50ml LB ◦ Autoclave  25mM ◦ 180mg in 50ml LB ◦ Autoclave  Concentrations taken from Nisar Ahmed Kanhar experiment, Utah State University.

10 General Phases Phases Compared in KT2440 Project  Early Logarithmic  Mid Logarithmic  Late Logarithmic  Early Stationary

11  OriginPro ◦ Sigmoid Fit ◦ Modified Gompertz Equations 1) 2) 3) 4) OriginPro fitted growth curve of PPKT2440 with no chemoattractant.

12 OriginPro growth curve of PPKT2440 with 25mM Benzoate OriginPro growth curve of PPKT2440 with 15mM Succinate

13  A=a-asymptote-highest point in curve  k=rate coefficient  X c = Center point of inflection  µ m =Maximum specific growth rate  λ = Lag Phase


15 Early Logarithmic Phase Mid Logarithmic Phase Late Logarithmic PhaseEarly Stationary Phase

16 Smaller Turn Angle=Less Direction Change

17 Notice bimodal distribution in above succinate experiment. PPKT2440 without chemoattractant favors small angles (under 90°).

18 Mid Logarithmic PhaseLate Logarithmic Phase

19 Mid Logarithmic PhaseLate Logarithmic Phase

20 Mid Logarithmic PhaseLate Logarithmic Phase

21 With 15mM SuccinateWithout Chemoattractant With 25mM Benzoate

22  Collective analysis  Swarm Plates  Individual bacterium analysis  Video Microscopy  Growth Curve Parameter Comparison Project ContributionPrevious Research

23 15mM Succinate25mM Benzoate  Decreased velocity  Altered turn angle distribution-bimodal  Decrease in overall distance traveled  Extended lag phase  Lengthens distance traveled in mid log phase  Higher average direction change than without chemoattractant  Highest maximum absorbance

24  Find new sensitive indicators  More frames/sec in video analysis  Reactions to other chemoattractants  Research specific pseudomonas genes  Mutate genes to create custom bioremediator

25  Funds from National Science Foundation ◦ Research Experience for Undergraduates  Dr. YangQuan Chen ◦ Program Director  Dr. Anhong Zhou ◦ Molecular and Cellular Sensing and Imaging Laboratory  Dr. Charlie Miller ◦ P. Putida cells  Mike Davis ◦ Technical skills

26  Harwood, Caroline S., Kathy Fosnaugh, and Marilyn Dispensa. "Flagellation of Pseudomonas putida and Analysis of Its Motile Behavior." Journal of Bacteriology (1989):  Parales, Rebecca E., and John D Haddock. "Biocatalytic degradation of pollutants." Elsevier. 15. (2004):  Reva, Oleg N., Christian Weinel, Miryam Weinel, and Kertsin Bohm. "Functional Genomics of Stress Response in Pseudomonas putida KT2440." Journal of Bacteriology (2006):  Ford, Roseanne M., and Ronald W. Harvey. "Role of chemotaxis in the transport of bacteria through saturated porous media." Elsevier. 30. (2007):  Kim, Hye-Eun, Maiko Shitashiro, Akio Kurodo, Noboru Takiguchi, and Junichi Kato. “Ethylene Chemotaxis in Pseudomonas aeruginosa and Other Pseudomonas Species.” Microbes and Environments.22.2 (2007):  Hawkins, Andrew C, and Caroline S. Harwood. "Chemotaxis of Ralstonia eutropha JMP134(pJP4) to the Herbicide 2,4- Dichlorophenoxyacetate." Applied and Environmental Microbiology. 68(2), (2002):  Parales, Rebecca E., Ditty, Jayna L., & Harwood, Carolin S. Toluene-degrading bacteria are chemotactic towards the environmental pollutants benzene, toluene, and trichloroethylene. Applied and Environmental Microbiology. 66(9), (2000):  Zwietering, M. H., I. Jongenburger, F. M. Rombouts, and K. Van't Riet. "Modeling of the Bacterial Growth Curve." Environmental Microbiology (1990):  Kanhar, Nisar Ahmed, Charles Miller, and Anhong Zhou. "Chemotactic analysis of Pseudomonas putida KT2440 and Escherichia coli TOP10 towards environmental pollutants including methyl parathion." Manuscript, Utah State University.  Duffy, Kevin J., and Roseanne M. Ford. "Turn Angle and Run Time Distributions Characterize Swimming Behavior for Pseudomonas putida." Journal of Bacteriology (1997):

27 Questions?

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