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8/11/2011 Demonstration of nucleic acid repair in viruses after UV disinfection using coliphages as surrogates Christian McGuire REU – University of Arkansas Motivation • Improve water treatment technology • Develop understanding of high levels of resistance for certain waterborne pathogens Acknowledgments • Connie Chan SMART – Columbia University • Mentors: • Karl Linden Ph.D – University of Colorado, Boulder • Roberto Rodriguez Ph.D – University of Colorado, Boulder • Sarah Bounty – University of Colorado, Boulder • And REU program Background • Low pressure mercury vapor lamp (monochromatic light) • 254 nm wavelength • Medium pressure mercury vapor lamp (polychromatic light) UV! • 200-600 nm wavelengths Zimmer, J. L. et al. 2002. Appl. Environ. Microbiol. 68(7):3293-3299 Background ~ UV damage and repair • Germicidal wavelength : 200-300 nm • Peak DNA absorption at 260 nm • Photolyase: photo-repairing enzyme • Activated at wavelengths 350-450 nm Objectives • Determine possible reactivation of viruses utilizing host photorepair mechanisms • Determine If MP lamps (polychromatic) are more effective than LP lamps (monochromatic) at inactivating phages. 1 8/11/2011 Experimental process Phage and Host • Phage Phase 1 Phase 2 Determine dose response for each of the three bacterial host under the LP UV lamp (Famp, CN13, & LT2). Confirm photo-repair ability and parameters of bacteria after exposure to LP UV lamp. Phase 3 Determine dose response for each phage (T1, PRD-1, PHIX174) under both LP and MP UV lamps. Phase 4 Demonstrate reactivation of virus using host repair mechanisms after exposure to LP and MP UV lamps. – PRD-1: doublestranded DNA • Bacteria – LT2 (salmonella) – T1: doublestranded DNA – phiX174: single stranded DNA ` – CN13 (E.Coli) Bacteria dose-response Results Low pressure UV collimated beam system Photo repair shop Bacteria Photorepair 2 8/11/2011 Maximum photoreactivation of the bacterial host after UV exposure 2 1.8 Log Photoreactivation 1.6 1.4 1.2 1 0.8 0.6 Phage Dose-response 0.4 0.2 0 Salmonella (LT2) 3 hrs Ecoli (Famp) 2hr E coli (CN13) 1 hr Spot plating • Saved time and equipment • 5 dilutions per plate opposed to three plates per dilution Titering of phage Plating of LT2 Comparison to Double agar layer method: 1st trial: DAM ~ 3.3 x 104 2nd trial: DAM ~ 5.1 x 109 spot -titer ~ 4 x 104 Spot - titer ~ 4.8 x 109 Phage Repair Experiment PRD-1 Initial Concentration Concentration after LP exposure ~ 40 mj/cm2 Phage repair using host photorepair mechanism Concentration after 6 hours of light exposure Concentration after 6 hours of dark 3 8/11/2011 Phage repair Phage photoreactivation • 6 hours under photo-repair lamp 3 ss DNA Log Photoreactivation 2.5 ds DNA 2 1.5 1 0.5 phiX174 – LP system PRD-1 – LP system 0 PhiX174-LP UV dose-response of phage PRD-1 with and without photoreactivation 6 Log (10) Inactivation 5 T1-LP PRD-1-LP PRD-1-MP Conclusion • Phage with double-stranded DNA were able to repair using bacterial host repair-mechanisms, but phage with single stranded DNA were not LP 4 MP 3 LP - Light • More experiments need to be run to determine if MP lamps are more effective at inactivating PRD-1, T1, and phiX174 2 MP - Light 1 0 0 20 40 60 Dose (mJ/cm2) 80 100 120 Future work • Develop dose response curves for Photoreactivation of phage • Further investigate efficacy of Medium pressure lamps as opposed to Low pressure lamps. Questions? 4 8/11/2011 References Rocky Mountain Laboratories, NIAID, NIH 5