Rhodococcus opacus
What is the Research Question?
Armando Vital
Rivera High School
Brownsville ISD
Dr. Kung-Hui (Bella) Chu
Assistant Professor,
Department of Civil Engineering (Environmental Engineering)
Texas A&M University
TYPE OF ENGINEERING
Civil Engineering Lab
Environmental 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) lab
Biology research is conducted in the Center for
Phage Technology
Working with Dr. Jason Gill and Dr. Ry Young
BACKGROUND OF
RESEARCH PROJECT
Dr. Kung-Hui Chu Work
Biodegradation and bioremediation of priority
pollutants and emerging contaminants
Molecular quantification of microbial risk in
water
Optimization of bioenergy production
Application of bioretention for stormwater runoff
management
Advancing knowledge on microbial ecology of
nitrogen and carbon cycles
RELEVANCE OF THE RESEARCH
Research Question
A 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 ago
Three 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 bacteria
Belongs to the family Actinomycetes,
related to Mycobacterium
R. opacus is a rod, nonmotile,
mycobacterium
This 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 Mycobacteria
A holin protein opens a pore in the cytoplasmic
membrane
The creation of the pore triggers the release of
endolysin
An esterase enzyme is also released that
degrades the outer mycolic acid cell wall layer
Prepare 4 litters
of R2A broth
(media/nutrients)
Research Activities
Phage Titration
Prepare broth
with 1.5%
Agar (solid)
Prepare broth
with 0.8% Agar
(gel)
Collect soil
samples
Prepare petri
dishes
Add R. opacus
to molten agar
Collect phage
from soil
Pour bacterial lawn
to petri dishes to
achieve an even
growth of bacteria
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 plates
Prepare media of R2A with 1.5% Agar
concentration and 0.8% agar concentration.
Spot Titration of phage
Pour a bacterial lawn
Label 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 min
Incubate 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 samples
Preparing media (broth/plates)
Enriching samples for phage
Growing liquid cultures of bacteria
Plating out phage
Collecting 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 Program
 National Science Foundation
 Nuclear Power Institute
 Texas Workforce Commission
 Dr. Kung-Hui (Bella) Chu
 The Center for Phage Technology
 Dr. Jason Gill and Dr. Ry Young
 Myung Hee Kim and Do Gyun Lee
 Andy Hernandez (partner)
THANK
YOU!