<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#backgrounds"><h5>Jump to Backgrounds</h5></a>

<a href = "https://2014.igem.org/Team:Tufts/Project#methods"><h5>Jump to Methods</h5></a>

<a href = "https://2014.igem.org/Team:Tufts/Project#methods"><h5>Jump to Methods</h5></a>

<p> <h3>Ribosponge Project Description:</h3> <br>

<p> <h3>Ribosponge Project Description:</h3> <br>

We have devised a method to introduce a DNA sequence which encodes an RNA aptamer (i.e. - an  

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  

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  

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  

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.  

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  

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.  

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  

coli into other bacteria of the same species, we plan on using an M13 phage which does not kill the  

bacteria.

bacteria.

The project has also inspired our collaboration with the Rathneau Institute and SYNENERGENE as we  

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  

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>

ethical challenges of packing it into a bacteriophage.</p>

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Monoclonal antibodies are proteins produced by the immune system to selectively bind  

Monoclonal antibodies are proteins produced by the immune system to selectively bind  

foreign molecules and induce immune response. Given  

foreign molecules and induce immune response. Given  

their high affinity and specificity of binding to their target  

their high affinity and specificity of binding to their target  

molecules, monoclonal antibodies are applied clinically  

molecules, monoclonal antibodies are applied clinically  

and in research to create detection assays, therapeutics, and  

and in research to create detection assays, therapeutics, and  

diagnostics. However, their production requires harming animals, and it  

diagnostics. However, their production requires harming animals, and it  

is costly, time consuming, and for certain molecules  

is costly, time consuming, and for certain molecules  

impossible.  

impossible.  

The goal of our research is to create an alternative detection molecule, a synthetic  

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  

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  

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  

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  

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  

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  

antibodies in a proof-of-concept experiment, in which our synthetic antibody will be used to  

detect proteins on the surface of a cell.  

detect proteins on the surface of a cell.  

The creation of synthetic antibodies in  

The creation of synthetic antibodies in  

bacteria will allow researchers to circumvent the  

bacteria will allow researchers to circumvent the  

expensive, time consuming, and arduous process  

expensive, time consuming, and arduous process  

of monoclonal antibody production. The highly  

of monoclonal antibody production. The highly  

modular “plug-and-play” aspect of our template  

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  

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  

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.

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.

<h3>Robust biofilm formation using a cyclic-di-GMP aptamer and investigating ethics and applications of engineered bactiophage</h3> <br>

<h3>Robust biofilm formation using a cyclic-di-GMP aptamer and investigating ethics and applications of engineered bactiophage</h3> <br>

A long, noncoding massively expressed regulatory RNA (merRNA) discovered in Bdellovibrio

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  

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  

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  

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  

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.  

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  

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  

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  

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  

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.

can prevent and treat infection by antibiotic-resistant bacteria.

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