Progress 08/15/07 to 08/14/10
Outputs
OUTPUTS: This research effort focused on determining how bacteriophages (bacterial viruses) transfer genetic information, such as antibiotic resistance genes, among their bacterial hosts in municipal and dairy wastewater ecosystems. Outputs have included a genetically engineered biotinylated T4 bacteriophage that is being used to monitor gene transfer frequencies (via transduction) and wastewater bioreactors for experimental procedures. A non-toxigenic E. coli O157:H7 is being used as a bacterial host organism and its long-term survivability has been determined in wastewater. A modified protocol for phage isolation has been completed using an optimized DNase digestion procedure and is being prepared for publication as a note in Applied and Environmental Microbiology. Project highlights continue to be disseminated on the University of Tennessee Center for Environmental Biotechnology webpage (www.ceb.utk.edu), laboratory meetings, and departmental seminars. Two Master's level graduate students were mentored through the duration of this project. Two undergraduate students and two high school students also participated. Graduate students presented their research at regional meetings of the American Society for Microbiology and incorporated bacteriophage biology into presentations at local K-12 school systems. Agricultural extension efforts included routine on-site farm visits to promote best management antibiotic usage practices with relevant informational packets. PARTICIPANTS: Project participants include Drs. Steven Ripp (PI) and Alice Layton (co-PI) from the University of Tennessee Center for Environmental Biotechnology and Dr. Shawn Hawkins (co-PI) from Biosystems Engineering and Soil Science. Two Master's level graduate students from the Department of Microbiology were mentored and trained (Courtney Johnson and Thomas Mead). Two undergraduate students from the Department of Microbiology participated as part of their independent study projects (Frank Dicker and Rahsaan Overton). Two Oak Ridge high school students participated over the summer months (James Littrell and Peter Zhao). A nonformal collaboration was established with Dr. Qiang He from the Civil and Environmental Engineering Department and his graduate student (Si Chen) for assistance with dairy waste bioreactor set-ups. Training opportunities center on the inclusion of graduate, undergraduate, and high school students in the project as well as Agricultural Extension efforts to educate dairy owners and operators on proper antibiotic use. TARGET AUDIENCES: This research effort targets the biological risk assessment community by providing rates of gene transfer for input into modeling efforts focused on risk associated with antibiotic gene transfer in municipal and dairy wastewater environments. Target audiences also include dairy owners and operators to whom we deliver educational training and guidance on proper antibiotic use for cattle and calves, proper maintenance of treatment records, and implementation of best management practices, especially related to full course completion of antibiotic therapies. We have conducted 34 onsite visits during the first year, 20 during the second year, and 25 during the third year. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A change in knowledge was implemented when current bacteriophage isolation procedures failed. A new and optimized method for bacteriophage isolation was subsequently developed and tested by the graduate student with undergraduate and high school student assistance. The initial cloning methodology was also not effective and required a change in action to instigate a new gene insertion approach that relied on homologous recombination techniques.
Publications
- Ripp, S., A. L. Layton, and G. S. Sayler. 2011. Bioreporter sensing technologies for water quality monitoring. In K. Sen and N. J. Ashbolt (ed.), Environmental Microbiology: Current Technology and Water Applications. Caister Academic Press, Hethersett, Norwich, UK, in press.
- Mead, T. J., F. Dicker, A. Layton, G. Sayler, and S. Ripp. 2010. A modified DNase protocol for optimized extraction of bacteriophages for PCR ready reactions, University of Tennessee, Department of Microbiology Annual Retreat, Norris, Tennessee, August 2010.
- Mead, T. J., S. Ripp, A. Layton, S. Hawkins, and G. Sayler. 2009. Unbiased tagging, isolation, and genomic characterization of bacteriophage transduction events in wastewater ecosystems, University of Tennessee, Department of Microbiology Annual Retreat, Norris, Tennessee, August 2009.
- Ripp, S. 2009. Biotechnology risk assessment: minding and managing the threats around us (invited), 238th American Chemical Society National Meeting and Exposition, Washington, DC, August 16-20.
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Progress 08/15/08 to 08/14/09
Outputs
OUTPUTS: Outputs during this second year research effort include a genetically engineered biotinylated lytic T4 bacteriophage and continued efforts to engineer a biotinylated lysogenic lambda bacteriophage. Biotinylation of lambda phage has been problematic but we are attempting alternative cloning methods, specifically instigating homologous recombination between an Escherichia coli lambda lysogen and an engineered biotin carrying vector. Dairy waste bioreactors (5 liter volumes) have been established and preliminary background gene exchange frequencies for antibiotic resistance genes and common reporter genes (green fluorescent protein) have been determined for the bacteriophages. We have as well set up pure culture chemostats to determine transduction frequencies under best case scenario conditions. We have also inoculated bioreactors with nontoxigenic E. coli O157:H7 to determine long-term survivability in dairy waste. Results and project updates have been and will continue to be disseminated on the University of Tennessee Center for Environmental Biotechnology homepage (www.ceb.utk.edu) and via Department of Microbiology seminars. Posters have as well been presented at regional American Society for Microbiology meetings. We also continue to conduct onsite agricultural extension farm visits to promote best management antibiotic usage practices. PARTICIPANTS: Project participants include Drs. Steven Ripp (PI) and Alice Layton (co-PI) from the University of Tennessee Center for Environmental Biotechnology and Dr. Shawn Hawkins (co-PI) from Biosystems Engineering and Soil Science. A new Department of Microbiology graduate student, Thomas Mead, has been added to the project. A Department of Microbiology undergraduate student, Frank Dicker, also contributes to the research effort. A nonformal collaboration has been established with Dr. Qiang He from the Civil and Environmental Engineering Department and his graduate student for assistance with dairy waste bioreactor set-ups. Training opportunities center on the inclusion of graduate and undergraduate students in the project as well as Agricultural Extension efforts to educate dairy owners and operators on proper antibiotic use. TARGET AUDIENCES: Target audiences are mainly focused on dairy owners and operators to whom we deliver educational training and guidance on proper antibiotic use for cattle and calves, proper maintenance of treatment records, and implementation of best management practices, especially related to full course completion of antibiotic therapies. We have conducted 34 onsite visits during the first year and 20 during the second year. PROJECT MODIFICATIONS: Due to difficulties in construction of a biotinylated bacteriophage lambda, we have obtained a biotinylated bacteriophage T4. We will initiate gene transfer studies with bacteriophage T4 while continuing the development of bacteriophage lambda. This will allow us to first gather data related to a lytic T4 bacteriophage and then follow up with a lysogenic bacteriophage.
Impacts
The initial cloning strategy in this effort called for the ligational insertion of a biotin label within bacteriophage lambda. This effort failed due to exceedingly poor ligation efficiencies. This change in knowledge instigated a change in action whereupon a new insertion approach was designed using homologous recombination techniques, which continue to remain challenging. As a result, we have instigated a fully new homologous recombination method using a lambda lysogenic strain as a recombination host. We have also obtained a lytic T4 biotinylated bacteriophage that will be used in addition to the soon to be complete biotinylated bacteriophage lambda. By applying both a lytic and a lysogenic bacteriophage, we can establish gene transfer frequencies with both phage types, which will provide a much more comprehensive data set pertinent to environmental modeling of antibiotic gene transfer.
Publications
- Mead, T. J., Ripp, S., Layton, A., Hawkins, S. and Sayler, G. 2009. Unbiased tagging, isolation, and genomic characterization of bacteriophage transduction events in wastewater ecosystems, University of Tennessee, Department of Microbiology Annual Retreat, Norris, Tennessee.
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Progress 08/15/07 to 08/14/08
Outputs
OUTPUTS: Outputs during this first year research effort include experimental methods facilitating gene cloning and homologous recombination in bacteriophage. New fundamental knowledge has been obtained for the biotinylation of capsid genes in bacteriophage lambda. As well, microcosms for studying bacteriophage/host bacteria interactions in municipal wastewater and in dairy wastewater have been implemented. Results and continued project updates have been and will continue to be disseminated on the University of Tennessee Center for Environmental Biotechnology homepage (www.ceb.utk.edu) and we continue to conduct onsite agricultural extension farm visits to promote best management practices related to antibiotic usage. PARTICIPANTS: Project participants include Drs. Steven Ripp (PI) and Alice Layton (co-PI) from the UT Department of Microbiology and Dr. Shawn Hawkins (co-PI) from Biosystems Engineering and Soil Science. Courtney Johnson, a recent Master's level graduate from the Department of Microbiology, serves as the lead technician. She is accompanied by two undergraduate students, one from the Department of Microbiology (Nicholas Lopes) and the other from Biomedical Engineering (Nathan Simmons). A non-formal collaboration has been established with Dr. Qiang He from the Civil and Environmental Engineering Department and his graduate student for assistance with microcosm set-ups. Training opportunities center on the inclusion of two undergraduate students in the project as well as Agricultural Extension efforts to educate dairy owners and operators on proper antibiotic use. TARGET AUDIENCES: Target audiences are mainly focused on dairy owners and operators to whom we deliver educational training and guidance on proper antibiotic use for cattle and calves, proper maintenance of treatment records, and implementation of best management practices, especially related to full course completion of antibiotic therapies. We have conducted 34 onsite visits during this first year. PROJECT MODIFICATIONS: No major changes have been implemented. We are having difficulty with some cloning steps, which has advocated certain minor changes in molecular genetic techniques.
Impacts
The initial cloning strategy in this effort called for the ligational insertion of a biotin label within bacteriophage lambda. This effort failed due to exceedingly poor ligation efficiencies. This change in knowledge instigated a change in action whereupon a new insertion approach was designed using homologous recombination techniques. This cloning step, however, continues to remain challenging. We have therefore additionally instituted a new vector cloning strategy but are patiently waiting for the vector to be shipped from a research group in India. This may or may not occur.
Publications
- No publications reported this period
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