National Science Foundation, Research Experience for
Undergraduates, Utah State University
August 5, 2010
serenalynn05@gmail.com
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Problem
Pseudomonas Putida
KT2440
Key Words
Objective
Equipment
Chemoattractants
Growth Curve Phases
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Results/Data
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Growth Curve Parameter
Velocity
Turn Angle
X vs. Y Displacement
Project Contribution
Conclusion
Future Work
Acknowledgements
References
Questions
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Due to pesticides, oil spills, landfill
leaching etc; soil is contaminated
with toxic chemicals:
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Water
Contamination
 2,4-Dichlorophenoxyacetic acid
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Farmland
Contamination
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Environmental
Damage
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Health Risks
 Most widely used herbicide in the world
 Major component in Agent Orange
 Toluene
 Naturally occurring in crude oil
 Byproduct of gasoline production
 Napthalene
 Nitrobenzene
Toxic Chemicals In Soil
Effects
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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
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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
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Degrades certain toxic chemicals
http://genome.jgi-psf.org/psepu/psepu.home.html
Taken by Serena Bennett with IX71 inverted microscope at 60X magnification at 15 frames/sec.
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Observe P. Putida characteristic
reactions/indicators from exposure to two
separate chemoattractants:
 Succinic Acid (Succinate)
 Sodium Benzoate
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Compare
 Growth Curve Parameters
 Velocity (Individual bacterium)
 Turn Angle (Individual bacterium)
 Displacement (Individual bacterium)
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Spectrophotometer
◦ Measures absorbance (optical density)
 Proportional to concentration
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IX71 Inverted Video Microscope
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ImagePro v. 6.1
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OriginPro v. 7.5
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PPKT2440
◦ (Olympus, Center Valley, PA, USA)
◦ Digital CCD camera (Olympus DP30BW
◦ (Media Cybernetics, Bethesda, MD, USA)
◦ Image Analysis
◦ (OriginLab Northampton, MA, USA)
◦ Lysogeny Broth
http://www.olympusamerica.com/seg_section/product.
asp?product=1023&p=72
Succinic Acid
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15mM
◦ 88.5mg in 50ml LB
◦ Autoclave
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Sodium Benzoate
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25mM
◦ 180mg in 50ml LB
◦ Autoclave
Concentrations taken from Nisar Ahmed Kanhar experiment, Utah
State University.
General Phases
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Early Logarithmic
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Mid Logarithmic
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Late Logarithmic
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Early Stationary
Phases Compared in
KT2440 Project
OriginPro
◦Sigmoid Fit
◦Modified Gompertz Equations
1)
2)
3)
4)
OriginPro fitted growth curve of PPKT2440 with no
chemoattractant.
OriginPro growth curve of PPKT2440 with
25mM Benzoate
OriginPro growth curve of PPKT2440 with
15mM Succinate
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A=a-asymptote-highest point in curve
k=rate coefficient
Xc= Center point of inflection
µm=Maximum specific growth rate
λ = Lag Phase
Early Logarithmic Phase
Late Logarithmic Phase
Mid Logarithmic Phase
Early Stationary Phase
Smaller Turn Angle=Less Direction Change
PPKT2440 without
chemoattractant favors
small angles (under 90°).
Notice bimodal distribution
in above succinate
experiment.
Mid Logarithmic Phase
Late Logarithmic Phase
Mid Logarithmic Phase
Late Logarithmic Phase
Mid Logarithmic Phase
Late Logarithmic Phase
Without
Chemoattractant
With 15mM Succinate
With 25mM Benzoate
Previous Research
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Collective analysis
Swarm Plates
Project Contribution
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Individual
bacterium analysis
Video Microscopy
Growth Curve
Parameter
Comparison
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Decreased velocity
Altered turn angle
distribution-bimodal
Decrease in overall
distance traveled
15mM Succinate
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Extended lag phase
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Lengthens distance
traveled in mid log phase
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Higher average direction
change than without
chemoattractant
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Highest maximum
absorbance
25mM Benzoate
 Find new sensitive indicators
 More frames/sec in video analysis
 Reactions to other chemoattractants
 Research specific pseudomonas genes
 Mutate genes to create custom bioremediator
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Funds from National Science Foundation
◦ Research Experience for Undergraduates
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Dr. YangQuan Chen
◦ Program Director
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Dr. Anhong Zhou
◦ Molecular and Cellular Sensing and Imaging Laboratory
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Dr. Charlie Miller
◦ P. Putida cells
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Mike Davis
◦ Technical skills
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Harwood, Caroline S., Kathy Fosnaugh, and Marilyn Dispensa. "Flagellation of Pseudomonas putida and Analysis of Its
Motile Behavior." Journal of Bacteriology. 171.7 (1989): 4063-4066.
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Parales, Rebecca E., and John D Haddock. "Biocatalytic degradation of pollutants." Elsevier. 15. (2004): 374-379.
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Reva, Oleg N. , Christian Weinel, Miryam Weinel, and Kertsin Bohm. "Functional Genomics of Stress Response in
Pseudomonas putida KT2440." Journal of Bacteriology. 188.11 (2006): 4079-4092.
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Ford, Roseanne M., and Ronald W. Harvey. "Role of chemotaxis in the transport of bacteria through saturated porous
media." Elsevier. 30. (2007): 1608-1617.
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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): 186-189.
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Hawkins, Andrew C, and Caroline S. Harwood. "Chemotaxis of Ralstonia eutropha JMP134(pJP4) to the Herbicide 2,4Dichlorophenoxyacetate." Applied and Environmental Microbiology. 68(2), (2002): 968-972.
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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):
4098-4104.
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Zwietering, M. H., I. Jongenburger, F. M. Rombouts, and K. Van't Riet. "Modeling of the Bacterial Growth Curve."
Environmental Microbiology. 56.6 (1990): 1875-1881.
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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.
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Duffy, Kevin J., and Roseanne M. Ford. "Turn Angle and Run Time Distributions Characterize Swimming Behavior for
Pseudomonas putida." Journal of Bacteriology. 179.4 (1997): 1428-1430.
Questions?