Using a highly expressed cyclic-di-GMP aptamer to affect biofilm formation P. Todorov, C. Ghadban, C. McBrine, P. Cavanagh, M. Gold, M. Zalesne, J. Starger, B. French, D. Rao PROBLEM HUMAN PRACTICES EXPERIMENTAL DESIGN • Biofilms are bacterial growths that form on surfaces when bacteria experience stressful conditions. • The bacteria secrete an extracellular matrix that promotes attachment, excludes immune cells, and increases antibiotic resistance. • These biofilms form in wounds, lungs of cystic fibrosis patients, chronic infections, and inserted medical devices. • The CDC estimates that 65% of all infections in developed nations are the result of biofilms • They are the leading cause of Healthcare Associated Infections. Phage Encapsulation in Silk Bandages The . merRNA transcript will bind to and sequester c-diGMP. Lower intracellular c-diGMP concentrations inhibit biofilm formation. “Tufts Synthetic Biology envisions a biomedical product consisting of a silk film embedded with a lyophilized cocktail of bacteriophage-targeting pathogens responsible for chronic wound infection.” Bacteriophage Growing antibiotic resistance perhaps most critical concern of modern medicine Deaths caused by antibiotic resistance top 23,000 per year in the U.S. alone U.S. economic cost due to antibiotic resistant infections upwards of $60 Billion per year Bacteriophages are viruses which target specific bacterial strains Approved for food, agricultural, and environmental use by FDA, USDA, and CDC Utilized therapeutically against bacterial infections in Republic of Georgia since 1930’s Face negative stigma and insufficient, credible research in Western medicine • • • • • • • Silk – Promising Platform for Bacteriophage Delivery & Wound Healing Thermo-stabilizes bacteriophages, Confers moisture resistance • Increases distributive ability for elimination of expensive and electricitybased storage for developing countries Biocompatible, Promotes wound healing, Directed Dissolution and compound release • • BACKGROUND Bandage Encapsulate bacteriophage cocktail within silk for treatment of Methicillin-resistant Staphylococcus aereus (MRSA) • Utilized against antibiotic resistant infections aids in more rapid approval and use by the medical industry. Potential for treatment of combat wounds, household wounds, burn victims, etc. • • Cyclic-di-GMP is a second messenger universal in biofilm signaling. • Increased [c-di-GMP] leads to increased biofilm formation. • Decreased [c-di-GMP] leads to increased motility and flagella expression. • B. bacteriovorus has a massively expressed regulatory RNA (merRNA) that contains a cdi-GMP aptamer. • This is hypothesized to sequester c-di-GMP and therefore promote motility. The merRNA sequence was placed under a T7 promoter and inserted into the Litmus28i+T7 RNAP ‘phagemid’ vector. It was then transformed into JM109 and ZK1056 E. coli. Biofilm Assay • Grow bacteria statically in a 96-well plate • Remove planktonic bacteria and rinse. Only cells stuck in a biofilm remain behind. • Stain with crystal violet and rinse • Dissolve crystal violet in acetone/ethanol • Record A550. Tufts iGEM hosted a national conference which brought together leaders of industry, regulation, and research to discuss the intersection of bacteriophage therapy, synthetic biology, and society. The conference consisted of two main events. The first was a professional workshop between our participating experts, held under Chatham House rules, to discuss and advance a pre-written framework for bacteriophage applications and associated safety and regulatory considerations. This was followed by a public forum to present the results and implications before shifting to guided, informal discussion. RESULTS Initial Results Fig 1. Absorbance values at 550 nm of cells with and without the merRNA construct. JM109 and ZK1056 are both known to form rather robust biofilms, whereas ELS-30 is not. As such, ELS-30 was used as a control. Cultures were were started from 1:100 dilutions of JM109 and ZK1056 (with and without the merRNA) along with the ELS-30 control. • Temperature Dependence? Contrary to expectations, cells expressing the merRNA exhibited increased biofilm formation. This was observed most consistently at lower temperatures, which tend to incur a metabolic burden and favor biofilm states. Our Hypothesis • The merRNA is expressed and binds to cdi-GMP as intended, thereby lowering the available intracellular concentration. • This increases motility, largely by flagella expression, as seen in B. bacteriovorus. • Several studies report that more motile bacteria form robust biofilms because flagella aid in the attachment of cells to surfaces and other cells. • We reason that the merRNA increases flagella expression and facilitates the attachment that makes biofilms possible. • Applications Include: Bioremediation (oil spills, waste water treatment, etc.) and further study of biofilms • • • • • • Biology of Phage – Tufts Synthetic Biology Phage History and Current Use – Anna Kuchment Current Research: Natural Phage – Dr. Andrew Camilli Current Research: Engineered Phage – Mark Mimee Distributive Justice – Christopher Ghadban Panel – Anna Kuchment, Dr. Robin Pierce, Dr. Andrew Camilli, Mark Mimee REFERENCES 1. 2. 3. 4. 5. Principles of c-di-GMP signalling in bacteria. Hengge, Regina. April 2009, Nature Reviews | Microbiology, pp. 263-273. A Global Transcriptional Switch between the Attack and Growth Forms of Bdellovibrio bacteriovorus. Karunker, Iris, et al., et al. April 16, 2013, PLOS|ONE. Microtiter dish biofilm formation assay. O'Toole, GA.,January 30, 2011.J Vis Exp. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development.O'Toole, GA and R Kolter., October 30, 1998. Mol Microbiol. Small Regulatory RNAs in the Control of Motility and Biofilm Formation in E. coli and Salmonella. Mika, F and R Hengge., February 26, 2013. Int J Mol Sci. SPONSORS Gold Bronze