Team:Glasgow

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<h1>DISColi: Bio-photolithography in Device Engineering Using Different Wavelengths of Light</h1>
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<h2><center><b>DISColi: Bio-photolithography - A new 3D manufacturing platform for modular product synthesis</b></center></h2>
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<p>Light is ubiquitous.  Systems that respond to light exist throughout nature, from unicellular photosynthetic organisms to the phototropism response in plants.  Light is an abundantly available resource, offering precise control over a system even in remote locations.  <br/><br/>
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</br>
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This is why we chose to work with light. DISColi is a novel bio-photolithographic system for engineering biofilms and the use of light allows the precision control needed to create functional 2D and 3D structures and devices. Such technology already has applications in fields as microfluidics, nanotechnology and tissue engineering. <br/><Br/>
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<p>The DISColi project aims to design and construct a novel bio-photolithographic system for the engineering of biofilms into functional 2D and 3D structures for use as a novel bio-manufacturing platform. In order to precisely sculpt the structure of the biofilms we designed a series of light-responsive promoters linked to proteins which can either disperse the biofilm or cement it. We aim to use this novel biofilm platform for a variety of manufacturing applications.
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Our work with light responsive constructs has allowed us to fully take advantage of the modular nature of synthetic biology.  The main aims of our project can be separated into three light-controlled components: the designed sculpting of biofilms; 3D printing for encapsulation of cells; and the controlled modular synthesis of a variety of products.  We expect our technology to have applications for material synthesis and compound manufacture in remote locations, for example outer space.
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<p>The main aims of our project can be separated into four areas: <p>
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<br/><br/><b>
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o biofilm characterisation <br>
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1.) The sculpting of Biofilms</b><br/><Br/>
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o novel reporters for biofilm analysis <br>
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o light-controlled 3D sculpting of biofilms <br>
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o the controlled modular synthesis of a variety of products</p><br/>
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The complex 3D structures formed by microorganisms provide an ideal model for sculpting. However, biofilms are also medically significant and can cause problems for human health. Our first priority therefore was researcher safety.  We also had to deal with the problem of a lack of shuttle vector between E.coli and common biofilm forming lab strains, such as Pseudomonas sp.  For this reason, we have worked with a novel biofilm forming synthetic biology chassis, E.coli Nissle 1917.  We intend to make this available to the Registry to allow future teams who wish to have a safer alternative that is still compatible with existing parts on the registry, to work with biofilms..<br/><Br/>
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<img src="https://static.igem.org/mediawiki/2011/2/2d/Simplediagramglasgow.jpg" width="100%" />
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In order to sculpt shapes into the biofilm, it is necessary to disperse some of the microorganisms.  We have developed three novel biobricks designed to cause dispersal of the biofilm.  Under control of the light responsive promoters, we can use light to cause targeted dispersal of the biofilm. <br/><br/>
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<br>
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<h2>Applications</h2>
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Phosphodiesterase, an enzyme isolated from Pseudomonas aeruginosa PA01, breaks down the signalling molecule cyclic diguanylate (c-di-GMP). This molecule is extremely important in biofilm formation and motility, and so the targeted overexpression of phosphodiesterase is designed to interfere with biofilm formation. <br/><br/>
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<p>
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<b>Build your own nano-biofactory with modular product synthesis pathways</b> - Use the DISColi system to form a biofilm then use different colours of light to select from a range cellular products from a single strain. Make an array of functionally diverse products from a common precursor, the products you want will only be in the biofilm cells allows for easy purification of the products in the amount you require.
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</br></br>
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<b>Personalised nutritional supplements and pharmaceuticals<a href="http://syntheticbiology.arc.nasa.gov/"> IN SPACE!</a></b> - In remote locations which are difficult/costly to resupply, the DISColi system can be used. This means that a single culture can selectively produce a range of useful compounds,such as opioids and isoprenoids, tailored to exactly what is needed, from a common precursor.
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</br></br> 
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<b>Microfluidics</b> - Grow a microfluidics platform using biofilm to form the channels. These channels can be dispersed, resetting the system and allowing the formation of new channels, or alter the channels during experiments to create dynamic environments.
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</br></br>
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<b>Tissue Engineering</b> - Use precise laser light to form tiny 3D structures out of biofilm that can be used as a scaffold for tissue culture.  
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</br></br>
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<b>Light Controlled Multicellularity</b> - Use different colours of light to control dispersal of different species of microbes allowing the precise construction of a mixed community biofilm.
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</br></br>
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<b>Clean Bioreactors</b> - Biofilms are costly to clean from pipes and bioreactors. Using the DISColi system of light based dispersal of biofilms can stop the build up and even break down existing biofilms in pipes and bioreactors. Simply shining light will start production of surfactant proteins that have been shown to disperse biofilm.
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</p>
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We have also created two biobricks of novel surfactant proteins.   These are Latherin and Ranaspumin, which are surfactant proteins with antimicrobial properties. Both these proteins are studied for their antibiofilm activity.  <br/><br/>
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<h2> Highlights!</h2>
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<p>
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To further enhance biofilm dispersal, we have also used two biobricks from the registry – Colicin E2 (Part BBa_ K131000) and T4 endolysin (Part BBa_K112806). All five of these proteins have been modelled to predict their diffusion through a biofilm. This allowed us to understand how large an area would be affected by triggering dispersal using our light responsive constructs. This meant we could predict how fine a resolution we could get when sculpting with light.     <br/><br/>
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Over the course of our project we have created 21 new physical biobricks, identified a novel chassis and also made a series of very interesting discoveries. Here are our personal highlights, including our favourite biobricks, our new chassis, and our public presence. Have a look!
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</br>
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DIScoli centres on the use of three light responsive promoters – the BLUF domain (Part BBa_K238013) which is activated by blue light, OmpC (Part BBa_R0082) which is activated by green light and OmpF (Part BBa_R0084) which is inhibited by red light.  We have collaborated with the University of Edinburgh team, who shared with us the improved versions of OmpC and OmpF.  <br/><br/>
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<h3><a href="https://2011.igem.org/Team:Glasgow/Judging Criteria">Judging Criteria</a></h3>
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<p> In this section we explain why we deserve a gold medal in accordance with the iGEM judging criteria. If you have very little time, this may be exactly what you are looking for!</p>
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To characterise the light promoters, we developed two novel reporters, LOV2 and iLOV.  These reporters have numerous advantages over GFP derived fluorescent proteins, such as their small size and, in the case of iLOV, the ability to quickly recover from photobleaching.  Another key advantage of the reporters LOV2 and iLOV is their ability to function under anoxic conditions. This unique ability was particularly desirable to our team due to our work with biofilms.  We are using biofilms to engineer functional 2D and 3D structures using our light responsive constructs.  Bio-photolithography has many applications, including the creation of structures for tissue engineering and manufacturing nanodevices.  <br/><br/>
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<h3><a href="https://2011.igem.org/Team:Glasgow/Biofilm/Nissle">Novel biofilm-forming chassis - <i>E.coli</i> Nissle 1917</a></h3>  
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Our new transformable, non-pathogenic, biofilm-forming chassis!
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<b>
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</br>
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2.) 3D printing for encapsulation</b><br/><br/>
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<h3><a href="https://2011.igem.org/Team:Glasgow/LOV2">LOV2 and iLOV Reporters</a></h3>
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LOV2 and iLOV are our incredible new reporters. They are smaller, fluoresce brighter and recover from photobleaching faster than GFP, and also function in anaerobic conditions! Try tagging your favourite proteins.
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The third component of our final product is encapsulation. For this we have chosen colanic acid, which is an exopolysaccharide that is naturally produced by E.coli  in order to protect from acidic environments. Colanic acid was previously worked on by the 2009 Imperial College London team, and its production can be overexpressed using biobricks of the transcription factors RcsA and RcsB. <br/><br/><b>
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</br>
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<h3><a href="https://2011.igem.org/Team:Glasgow/PDE">c-di-GMP Phosphodiesterase</a></h3>  
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3.) The controlled modular synthesis of a variety of products</b><br/><br/>
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c-di-GMP Phosphodiesterase breaks down c-di-GMP, which is a secondary messenger which regulates many behaviours such as motility and biofilm formation. Over-expressing this phosphodiesterase should decrease the levels of c-di-GMP, increasing cellular motility and causing biofilm dispersal. c-di-GMP has many more functions making this biobrick useful in a wide range of applications.
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</br>
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A key component of DISColi is flexibility in manufacturing. Taking advantage of the modular nature of synthetic biology, our engineered bacteria will be able to produce a variety of useful compounds simply by inserting the genes for the desired compound into the system. <br/><br/>
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<h3><a href="https://2011.igem.org/Team:Glasgow/Ranaspumin2">Ranaspumin2</a></h3>
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The surfactant protein ranspumin-2 comes from foam nests Túngara Frog (Engystomops pustulosus). In DISColi we use it to disperse biofilm however its application are much broader.
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This would mean the ability to use different wavelengths of light to select your desired product from a single precursor molecule. As a proof of principle of manufacturing in response to light, we have been working with the carotenoid pathway.  In 2009, Cambridge submitted a number of biobricks involved in this pathway.  We have improved this by synthesizing the fourth gene, crtY, in the pathway and testing it under a pBAD promoter.   <br/><br/>
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<h3><a href="https://2011.igem.org/Team:Glasgow/Parts/Latherin">Latherin</a></h3>
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Latherin is another surfactant protein although this one is isolated from horse sweat.
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<h3><a href="https://2011.igem.org/Team:Glasgow/MCS">Multiple Cloning Site Biobrick</a></h3>  
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We believe it is vital to include a constant interaction with the public throughout our project. We aim to accomplish this through the use of regular Vlogs and personal Blogs. Uniquely, we have also created a daily time-lapse, with the hope that others can gain an insight into life in the lab.  Check out the rest of our wiki to find out what else we’ve been up to!><br/><br/>
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We were slowed down due to repeated rounds of restrictions and ligations to put multiple different coding regions with the same promoter, RBS and terminator combo. So we designed this handy multiple cloning site biobrick to speed up that process.
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</br></br>
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<h2>News</h2>
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  width="239" height="35" alt="Wellcome Trust" /></a><br />
  width="239" height="35" alt="Wellcome Trust" /></a><br />
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Latest revision as of 05:31, 22 September 2011

DISColi: Bio-photolithography - A new 3D manufacturing platform for modular product synthesis


The DISColi project aims to design and construct a novel bio-photolithographic system for the engineering of biofilms into functional 2D and 3D structures for use as a novel bio-manufacturing platform. In order to precisely sculpt the structure of the biofilms we designed a series of light-responsive promoters linked to proteins which can either disperse the biofilm or cement it. We aim to use this novel biofilm platform for a variety of manufacturing applications.

The main aims of our project can be separated into four areas:

o biofilm characterisation
o novel reporters for biofilm analysis
o light-controlled 3D sculpting of biofilms
o the controlled modular synthesis of a variety of products



Applications

Build your own nano-biofactory with modular product synthesis pathways - Use the DISColi system to form a biofilm then use different colours of light to select from a range cellular products from a single strain. Make an array of functionally diverse products from a common precursor, the products you want will only be in the biofilm cells allows for easy purification of the products in the amount you require.

Personalised nutritional supplements and pharmaceuticals IN SPACE! - In remote locations which are difficult/costly to resupply, the DISColi system can be used. This means that a single culture can selectively produce a range of useful compounds,such as opioids and isoprenoids, tailored to exactly what is needed, from a common precursor.

Microfluidics - Grow a microfluidics platform using biofilm to form the channels. These channels can be dispersed, resetting the system and allowing the formation of new channels, or alter the channels during experiments to create dynamic environments.

Tissue Engineering - Use precise laser light to form tiny 3D structures out of biofilm that can be used as a scaffold for tissue culture.

Light Controlled Multicellularity - Use different colours of light to control dispersal of different species of microbes allowing the precise construction of a mixed community biofilm.

Clean Bioreactors - Biofilms are costly to clean from pipes and bioreactors. Using the DISColi system of light based dispersal of biofilms can stop the build up and even break down existing biofilms in pipes and bioreactors. Simply shining light will start production of surfactant proteins that have been shown to disperse biofilm.

Highlights!

Over the course of our project we have created 21 new physical biobricks, identified a novel chassis and also made a series of very interesting discoveries. Here are our personal highlights, including our favourite biobricks, our new chassis, and our public presence. Have a look!

Judging Criteria

In this section we explain why we deserve a gold medal in accordance with the iGEM judging criteria. If you have very little time, this may be exactly what you are looking for!

Novel biofilm-forming chassis - E.coli Nissle 1917

Our new transformable, non-pathogenic, biofilm-forming chassis!

LOV2 and iLOV Reporters

LOV2 and iLOV are our incredible new reporters. They are smaller, fluoresce brighter and recover from photobleaching faster than GFP, and also function in anaerobic conditions! Try tagging your favourite proteins.

c-di-GMP Phosphodiesterase

c-di-GMP Phosphodiesterase breaks down c-di-GMP, which is a secondary messenger which regulates many behaviours such as motility and biofilm formation. Over-expressing this phosphodiesterase should decrease the levels of c-di-GMP, increasing cellular motility and causing biofilm dispersal. c-di-GMP has many more functions making this biobrick useful in a wide range of applications.

Ranaspumin2

The surfactant protein ranspumin-2 comes from foam nests Túngara Frog (Engystomops pustulosus). In DISColi we use it to disperse biofilm however its application are much broader.

Latherin

Latherin is another surfactant protein although this one is isolated from horse sweat.

Multiple Cloning Site Biobrick

We were slowed down due to repeated rounds of restrictions and ligations to put multiple different coding regions with the same promoter, RBS and terminator combo. So we designed this handy multiple cloning site biobrick to speed up that process.

Sponsors

With many thanks to our generous sponsors, without whom this project would not have been possible.