Tufts Championship Poster

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

Download Report