MIT Department: Biological Engineering
Undergraduate Institution: University of Guam
Faculty Mentor: Bryan Bryson
Research Supervisors: Christopher Itoh, Sydney Solomon
Hafa Adai! I’m Joseph Lance Casila and I am a math, biology, and chemistry major at the University of Guam. My interests are biomedical engineering, tennis, karaoke, environmental conservation, and promoting opportunities for ethnic and minority groups. I’ve been blessed thus far with good health and many academic opportunities, which drives my ultimate goal to pay it forward by helping advance biomedical engineering for more rapid and precise disease treatment. Antibody engineering, nanotechnology, and regenerative medicine have all been exciting so far, and so my plan in the future is to lead research in therapeutic fields like those.
2018 Research Abstract
Development of Oxidative Stress Responsive Transcriptional Reporter Strains in Mycobacterium smegmatis
Joseph Lance V. Casila1, Christopher Itoh2, Sydney Solomon2, 3, and Bryan Bryson, PhD2, 3
1College of Natural and Applied Sciences, University of Guam
2Ragon Institute of MGH, MIT and Harvard
3Department of Biological Engineering, Massachusetts Institute of Technology
Tuberculosis (TB) is one of the deadliest infectious diseases affecting a third of the global population and leading to more than a million deaths per year. TB pathogenesis occurs when Mycobacterium tuberculosis (Mtb), the causative bacteria of TB, is phagocytosed by macrophages but subverts antimicrobial mechanisms elicited by host macrophages. One major antimicrobial response to Mtb infection is the generation of reactive oxygen species (ROS), which are toxic to bacteria. Recent evidence suggests that macrophage heterogeneity is correlated with variability in Mtb killing. We sought to test the hypothesis that there is variability in production of phagosomal ROS which potentially drives variability in bacterial killing. Reporter strains that fluoresce upon ROS stimulation were created to test for macrophage heterogeneity. Promoter region of six ROS-associated mycobacterial genes were each cloned upstream of mNeonGreen, a bright GFP variant, using Gibson Assembly with a pJEB vector backbone. The plasmids were then transformed into Mycobacteria smegmatis, a close relative to Mtb. The six ROS reporter strains were treated with tertbutyl hydroperoxide (TBHP) and then analyzed using flow cytometry. Growth rates revealed that 0.5mM – 1.0mM TBHP had bacteriostatic effects. Preliminary data from flow cytometry suggest that TBHP stimulation elicited positive GFP signals, however, further optimization is required.