Rhodococcus opacus
What is the Research Question?
Armando VitalRivera High School
Brownsville ISDDr. Kung-Hui (Bella) Chu
Assistant Professor, Department of Civil Engineering (Environmental Engineering)
Texas A&M University
TYPE OF ENGINEERING
Civil Engineering LabEnvironmental research is conducted in the
CVLB lab.Working with two of Dr. Chu’s PhD students,
Myung Hee Kim and Do Gyun Lee
Center for Phage Technology (CPT) labBiology research is conducted in the Center for
Phage TechnologyWorking with Dr. Jason Gill and Dr. Ry Young
BACKGROUND OF RESEARCH PROJECT
Dr. Kung-Hui Chu WorkBiodegradation and bioremediation of priority
pollutants and emerging contaminantsMolecular quantification of microbial risk in
waterOptimization of bioenergy productionApplication of bioretention for stormwater runoff
managementAdvancing knowledge on microbial ecology of
nitrogen and carbon cycles
RELEVANCE OF THE RESEARCH
Research QuestionA tale of two phages: Is phage DNA sequence
highly conserved over time and space?
Significance: Phage Genome Evolution
THE LAB WORK
Techniques used in
molecular biology for working
with the phages.Applied microbiology, virology, and
environmental engineering.
RESEARCH OBJECTIVE
Isolate and characterize phages that infect Rhodococcus opacus.
Collect activated sludge and soil samples that may contain phage.
EXPLORATION
What do we know about these phages?A phage, Ropa 4, was isolated in Germany over
20 years agoThree R. opacus phages were recently isolated in
Chu’s laboratory The genomes of all four phages were sequenced
at the CPT and found to be almost identical
SURPRISE DISCOVERY!
The DNA of the newly isolated phage is almost 100% identical to that of the phage isolated from Germany.Signifies a new finding in “Phage Genome
Evolution”
Approach: E3 teachers will repeat our work at other laboratory in order to rule out any cross contamination during isolation phages in Dr. Chu’s lab.
BACKGROUND ON PHAGES
Bacteriophages are viruses
that infect bacterial cells. Phages cannot reproduce on their own.The phage hijacks the cell’s machinery to
reproduce progeny.Phages are specific for their host bacteria.YouTube - T4 Virus infecting a bacteria.
Rhodococcus opacus
Rhodococcus opacus is a
specific bacteriaBelongs to the family Actinomycetes,
related to MycobacteriumR. opacus is a rod, nonmotile,
mycobacteriumThis bacteria was used as the host to
isolate phage in Chu’s laboratory
Impact of the Research
Potentially open a new research direction in phage genomics.Some Rhodococcus opacus species are
pathogens. A better understanding of phages specific to R.
opacus can enhance the development of phage therapy.
Some R. opacus are foaming bacteria in biological wastewater treatment processes.Phage treatment might be possible to minimize
common sludge bulking problem.
THE CHALLENGE FOR SCIENCE
The cell wall of the bacteria is difficult disrupt.
First step is to see which phages can actually infect R. opacus
Rhodococcus-specific phages can lyse the cell.
LYSIS PROCESS
Lysis via a 3 component
system in MycobacteriaA holin protein opens a pore in the cytoplasmic
membraneThe creation of the pore triggers the release of
endolysinAn esterase enzyme is also released that
degrades the outer mycolic acid cell wall layer
Prepare 4 litters of R2A broth
(media/nutrients)
Prepare broth with 1.5% Agar (solid)
Research Activities Phage Titration
Prepare broth with 0.8% Agar
(gel)
Prepare petri dishes
Add R. opacus to molten agar
Pour bacterial lawn to petri dishes to achieve an even
growth of bacteria
Collect soil samples
Collect phage from soil
Apply phage spots to bacterial lawn
Proceed to full-plate titration of phage.
RESEARCH ACTIVITIES
Learn laboratory aseptic techniques when working with BL1 agents.Use ethanol as sanitizer Use flame to allow clean air to move upwards
preventing bacteria from falling on the bench.
Prepare soft agar overlay platesPrepare media of R2A with 1.5% Agar
concentration and 0.8% agar concentration.
Spot Titration of phage
Pour a bacterial lawnLabel bottom surface of plate with phage
and lawn strain.In aseptic environment, transfer host
culture into molten agar and vortex.Pour contents of tube onto agar surface.Allow agar to solidify.
Spot Titration of Phage
Apply phage spots to lawn.Aspirate phage dilution and deposit over
corresponding label marked on plate.Allow plate to dry near a flame for 15 minIncubate plates, inverted, at optimal growth
temperature until plaques appear in the lawn.
Individual plaques may be counted to estimate the concentration of phage.
EXPERIMENTS
Collecting soil samplesPreparing media (broth/plates)Enriching samples for phageGrowing liquid cultures of bacteriaPlating out phageCollecting plaques of phage on petri
dishes
DATA TO BE GENERATED
Data that will help characterize the phages from the soil samples collected from a sewer facility and close to a gasoline station.
The model used will be comparing the characteristics of newly found phages to the phage currently available.
Future Implications
Is the high similarity of phage
sequences unique to Rhodococcus species?
Does the high similarity of phage sequences present concern G+ pathogens?
What is the implications of high conservation of phage sequences?
SUMMARY
Learn how to grow R. opacus bacteria.Collect soil samples from a sewer facility
and close to a gasoline station.Isolate, and characterize newly found
phages.Compare newly found phages to the
phage found in Germany.
CLASSROOM APPLICATION
Combined lesson plan with Biology and Algebra 1 classes.
Analyze and evaluate phage growth rate using functions and scatter plots.
ACKNOWLEDGMENTS
TAMU E3 ProgramNational Science FoundationNuclear Power InstituteTexas Workforce CommissionDr. Kung-Hui (Bella) ChuThe Center for Phage TechnologyDr. Jason Gill and Dr. Ry YoungMyung Hee Kim and Do Gyun LeeAndy Hernandez (partner)
THANK YOU!