Latest revision as of 01:27, 30 September 2014

Tufts iGEM 2014

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Summaries

Robust biofilm formation using a cyclic-di-GMP aptamer and investigating ethics and applications of engineered bactiophage

A long, noncoding massively expressed regulatory RNA (merRNA) discovered in Bdellovibrio bacteriovorus is present in high levels during its dormant phase. The merRNA is believed to sequester cyclic-di-GMP, much like a sponge. Since cyclic-di-GMP is a second messenger for various cellular functions, including motility and biofilm formation, the Tufts iGEM team introduced this merRNA sequence into E. coli. Constitutive expression of this merRNA transcript was shown to increase biofilm formation. This property can be useful in microbe-based approaches to environmental remediation. Earlier designs for phage delivery of the merRNA to disrupt biofilms inspired an investigation into the policy surrounding engineered bacteriophage. Tufts iGEM will be convening a panel of experts from various disciplines to put forth recommendations for the responsible use of phage in therapeutic and industrial applications. A proposal will be drafted for a silk bandage containing a phage cocktail which can prevent and treat infection by antibiotic-resistant bacteria.

Ribosponge Project Description:

We have devised a method to introduce a DNA sequence which encodes an RNA aptamer (i.e. - an oligonucleotide sequence which binds to a specific molecule) into a non-pathogenic E. coli strain. We have dubbed this RNA aptamer a “ribosponge” due to its unique mode of action. The ribosponge binds cyclic di-GMP, a secondary intracellular messenger which signals bacteria to enter a persistent or biofilm state. The signal is universal among many species such as E. coli, P. aeruginosa, and M. tuberculosis. Blocking the signal of c-di-GMP by binding it with an aptamer could prevent the persistent state in these and other pathogens. In order to ferry the sequence encoding the aptamer from our non-pathogenic E. coli into other bacteria of the same species, we plan on using an M13 phage which does not kill the bacteria. The project has also inspired our collaboration with the Rathneau Institute and SYNENERGENE as we look at the feasability of developing ribosponge into a product, and examine the regulatory, legal, and ethical challenges of packing it into a bacteriophage.

Backgrounds

Methods

Sponge Lab Notebook

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 <h1 style="color:white">Tufts iGEM 2014</h1>
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+<a href="https://igem.org/Team.cgi?year=2014&team_name=Tufts"style="color:#0099FF"target="_blank"> Official Team Profile </a></td>
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-<p> <h3>Ribosponge Project Description:</h3> <br>
+<a href = "https://2014.igem.org/Team:Tufts/Project#backgrounds"><h5>Jump to Backgrounds</h5></a>
+<a href = "https://2014.igem.org/Team:Tufts/Project#methods"><h5>Jump to Methods</h5></a>
+<h2> Summaries </h2>
+<tr> <td width="100%">
+<h3>Robust biofilm formation using a cyclic-di-GMP aptamer and investigating ethics and applications of engineered bactiophage</h3>
+A long, noncoding massively expressed regulatory RNA (merRNA) discovered in Bdellovibrio
+bacteriovorus is present in high levels during its dormant phase. The merRNA is believed to sequester
+cyclic-di-GMP, much like a sponge. Since cyclic-di-GMP is a second messenger for various cellular
+functions, including motility and biofilm formation, the Tufts iGEM team introduced this merRNA
+sequence into E. coli. Constitutive expression of this merRNA transcript was shown to increase biofilm
+formation. This property can be useful in microbe-based approaches to environmental remediation.
+Earlier designs for phage delivery of the merRNA to disrupt biofilms inspired an investigation into the
+policy surrounding engineered bacteriophage. Tufts iGEM will be convening a panel of experts from
+various disciplines to put forth recommendations for the responsible use of phage in therapeutic and
+industrial applications. A proposal will be drafted for a silk bandage containing a phage cocktail which
+can prevent and treat infection by antibiotic-resistant bacteria.
+</td></tr>
+<tr><td>
+<p> <h3>Ribosponge Project Description:</h3>
 We have devised a method to introduce a DNA sequence which encodes an RNA aptamer (i.e. - an
 oligonucleotide sequence which binds to a specific molecule) into a non-pathogenic E. coli strain. We
 have dubbed this RNA aptamer a “ribosponge” due to its unique mode of action. The ribosponge binds
 cyclic di-GMP, a secondary intracellular messenger which signals bacteria to enter a persistent or biofilm
 state. The signal is universal among many species such as E. coli, P. aeruginosa, and M. tuberculosis.
 Blocking the signal of c-di-GMP by binding it with an aptamer could prevent the persistent state in these
 and other pathogens. In order to ferry the sequence encoding the aptamer from our non-pathogenic E.
 coli into other bacteria of the same species, we plan on using an M13 phage which does not kill the
 bacteria.
 The project has also inspired our collaboration with the Rathneau Institute and SYNENERGENE as we
 look at the feasability of developing ribosponge into a product, and examine the regulatory, legal, and
 ethical challenges of packing it into a bacteriophage.</p>
 </td> </tr>
+<!--<tr> <td width="100%">
+<h3>Synthetic Antibody Proposal</h3>
+Monoclonal antibodies are proteins produced by the immune system to selectively bind
+foreign molecules and induce immune response. Given
+their high affinity and specificity of binding to their target
+molecules, monoclonal antibodies are applied clinically
+and in research to create detection assays, therapeutics, and
+diagnostics. However, their production requires harming animals, and it
+is costly, time consuming, and for certain molecules
+impossible.
+The goal of our research is to create an alternative detection molecule, a synthetic
+antibody, which can be produced more quickly, at a lower cost, using much simpler and more
+accessible methods. The product of our research will be a template plasmid, a synthetic DNA
+molecule that will cause bacteria to express our synthetic antibodies. The template plasmid will
+allow researchers to “plug-and-play” different detection regions onto the constant region of an
+antibody. We will also validate this approach by demonstrating the efficacy of our synthetic
+antibodies in a proof-of-concept experiment, in which our synthetic antibody will be used to
+detect proteins on the surface of a cell.
+The creation of synthetic antibodies in bacteria will allow researchers to circumvent the
+expensive, time consuming, and arduous process of monoclonal antibody production. The highly
+modular “plug-and-play” aspect of our template plasmid will make this tool simple to use and highly versatile, while the use of the constant
+region of a monoclonal antibody as the detection method will make this technology particularly powerful, as there already exists a large amount of chemistry, and protocols associated with different uses of the conserved domain. Thus, synthetic antibodies will provide yet another versatile tool for creating detection assays, diagnostics, and potentially even therapeutics for diseases such as cancers and autoimmune diseases.
+</td></tr>
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+<a name="backgrounds"> <h2> Backgrounds </h2> </a>
+</td> </tr>
+<tr><td>
+<a name="methods"><h2>Methods</h2></a>
+<!--<a href = "https://static.igem.org/mediawiki/2014/2/24/LabNotebookAntibodyiGEM2014.pdf"> Antibody Lab Notebook </a> <br>-->
+<a href="https://static.igem.org/mediawiki/2014/a/a1/LabNotebookSpongeiGEM2014.pdf" target="_blank"> Sponge Lab Notebook </a>
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