Rhodococcus opacus What is the Research Question?

1. RhodococcusopacusWhat 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

2. TYPE OF ENGINEERING • Civil Engineering Lab • Environmental research is conducted in the CVLB lab. • Working with two of Dr. Chu’s PhD students, MyungHee 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

3. 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

4. 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

5. THE LAB WORK • Techniques used in molecular biology for working with the phages. • Applied microbiology, virology, and environmental engineering.

6. RESEARCH OBJECTIVE • Isolate and characterize phages that infect Rhodococcus opacus. • Collect activated sludge and soil samples that may contain phage.

7. 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

8. 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. 

9. 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.

10. 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

11. Impact of the Research • Potentially open a new research direction in phage genomics. • Some Rhodococcusopacus 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.

12. 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.

13. LYSIS PROCESS • Lysis via a 3 component system in Mycobacteria • A holin protein opens a pore in the cytoplasmic membrane • Thecreation of the pore triggers the release of endolysin • An esterase enzyme is also released that degrades the outer mycolic acid cell wall layer

15. 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.

16. 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.

17. 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.

18. 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

19. 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.

20. Future Implications • Is the high similarity of phage sequences unique to Rhodococcusspecies? • Does the high similarity of phage sequences present concern G+ pathogens? • What is the implications of high conservation of phage sequences?

21. 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.

22. CLASSROOM APPLICATION • Combined lesson plan with Biology and Algebra 1 classes. • Analyze and evaluate phage growth rate using functions and scatter plots.

23. 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 • MyungHee Kim and Do Gyun Lee • Andy Hernandez (partner)

24. THANK YOU!