Presentor: Jesus A. Hernandez Gregory-Portland High School Dr. Kung-Hui Chu Assistant Professor, Department of Civil Engineering

The TEAM Working along side Armando Vital, Brownsville, TX Working under Dr. Kung-Hui Chu, Environmental Engineering/Civil Engineering Working in Dr. Ry Young’s Lab, Center for Phage Technology (CPT) under direction of Dr. Jason Gill Working with assistance from Myunghee Kim and Do Gyun Lee, both PhD. Students in Dr. Chu’s Lab

Dr. Kung-Hui Chu 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

The Laboratory Work Techniques used in molecular biology for working with the phages This is a collaborative effort in the disciplines of applied microbiology, virology, and environmental engineering

Research Question A tale of two phages: Does high degree of phage gene homology exist? Significance: Phage Genome Evolution

Background on Phages Phages or bacteriophages are viruses that infect bacterial cells. They are found everywhere. Phages, like any other virus, cannot reproduce on their own. The phage hijacks the cell’s machinery to reproduce progeny. Phages are specific for their host bacteria.

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 YouTube - T4 Virus infecting a bacteria.

What do we know about these two phages? A phage, Phage Ropa N4, was isolated in Germany 20 years ago Three phages (Ropa1, Ropa2, Ropa3) were recently isolated from Chu’s laboratory To our surprise, we found that 2 of 3 of these phages have high similarity in their DNA sequences to N % Identical!!! The other was very closely related.

Discovery => If this is true, it would be a new finding in “Phage Genome Evolution” Approach: new personnel to repeat our work at other laboratory in order to rule out any cross contamination during isolation phages in Dr. Chu’s laboratory.

Primary Objective Isolate and characterize phages that infect Rhodococcus opacus. Collect activated sludge and soil samples that may contain phage. Secondary Objective: Collect some DNA

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

Relevance/Impact of the Research Potentially open a new research direction in phage genomics. Some Rhodococcus species are pathogens. A better understanding of phages specific to Rhodococcus can enhance the development of phage therapy. Phage treatment might be possible to minimize common sludge bulking problem in treatment plants.

The Challenge for Science The cell wall of the bacteria is difficult to disrupt. First step is to see which phages can actually infect R. opacus Rhodococcus-specific phages can lyse the cell.

Research Work Collecting soil samples Learn aseptic techniques Preparing media (broth/food) Enriching samples for phage Growing liquid cultures of bacteria Plating out phage Collecting plaques of phage on petri dishes

Flow Chart of Lab Work

Data to be Generated Data that will help characterize the phages from our collection sites. Comparing the characteristics of new phages to the ones currently available. Finding phages that will successfully infect R. opacus

Future Implications Does the high similarity of phage sequences unique to Rhodococcus species? Does the high similarity of phage sequences present concern G+ pathogens? What are the implications of high conservation of phage sequences?

Summary Phages infect bacterial cells Focus on phage that infect Rhodococcus opacus Isolate and characterize phages Further exploration

Classroom Lesson/Activities Physics Chemistry & Biology

Acknowledgements TAMU E3 Program National Science Foundation (NSF) Nuclear Power Institute (NPI) Texas Workforce Comission Dr. Kung-Hui Chu and her lab Dr. Ry Young and CPT Dr. Jason Gill Myunghee Kim and Do Gyun Lee Armando Vital (partner)