Team:Calgary/Project/HumanPractices/Platform

From 2013.igem.org

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<h1>Our Platform</h1>
<h1>Our Platform</h1>
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<p>The primary goal of our project was to develop a nucleic acid based sensor for the detection of super-shedder cattle that contain copious amounts of Enterohemmorhagic <i>E. coli</i> (EHEC) within their gastrointestinal system. Being able to detect this EHEC within cattle would allow us to limit the EHEC contamination of various products such as beef, produce and water. Throughout the process of building this project however we quickly realized that our system could be effectively applied to multiple industries and situations. The system we are building makes use of TALE proteins to bind our target DNA. These DNA binding proteins can bind different DNA sequences due to alterations in the primary structure of the proteins in regions called repeat variable di-residues (RVD). The amino acid code of these RVD regions has been unlocked however which in effect means we can easily engineer our TALE proteins for practically any DNA sequence of interest. </p>
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<p>Our team set out to develop a <a href="https://2013.igem.org/Team:Calgary/Project/OurSensor">nucleic acid biosensor</a> to monitor Enterohemorhagic <i>E. coli</i> (EHEC) in beef cattle. Though we tailored our final system to the beef industry, the molecular components forming the FerriTALE have a myriad of other applications. These include <a href="https://2013.igem.org/Team:Calgary/Project/OurSensor/Detector">detector TALEs</a> which can be readily designed to other DNA targets, a <a href="https://2013.igem.org/Team:Calgary/Project/OurSensor/Reporter/PrussianBlueFerritin">ferritin protein shelled nanoparticle</a> with interchangeable cores that impart novel function, and an <a href="https://2013.igem.org/Team:Calgary/Project/OurSensor/Linker">interchangeable coiled-coil linker system</a> to build intricate protein devices using ferritin as a scaffold. Together, these components form a customizable DNA biosensor, with individual components that could have diverse applications in future iGEM projects and beyond.</p>
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<p>Combining the power of TALE proteins with the design of our capture strip assay we have a system that could be modified to work with many different applications in industry and society. The modularity of TALEs will allow us to design our system to target DNA sequences in order to solve the problems of today. This can range from identifying the strains of bacteria present in the sputum of cystic fibrosis patients to detecting dangerous organisms that could pose a biosecurity threat (Figure 1).</p>
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<h1>Detector</h1>
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<p>We selected <a href="https://2013.igem.org/Team:Calgary/Project/OurSensor/Detector">Transcription Activator Like Effectors (TALEs)</a> to bind and detect DNA in our system. These proteins were chosen because their DNA binding domains can be engineered to bind virtually any DNA sequence (Cermak et al., 2011). This means that we have built a DNA biosensor platform, where our system can be repurposed to detection of organisms in other applications by designing TALEs to different gene sequences. By combining alternative TALE proteins into our <a href="https://2013.igem.org/Team:Calgary/Project/OurSensor/Prototype">capture strip assay</a>, our system could be relevant to other food industries, health care, bedside diagnostics, and biosecurity (Figure 1).</p>
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<img src="https://static.igem.org/mediawiki/2013/b/b3/UCalgary2013TRPLATFMORMYAY.png" alt="Platform Technology" width="800" height="434">
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<img src="https://static.igem.org/mediawiki/2013/b/b3/UCalgary2013TRPLATFMORMYAY.png" alt="Platform Technology" width="600" height="325">
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<p><b>Figure 1.</b> Our platform biosensor can be applied to many different industries and situations. Examples of this include the food industry, health applications and biosecurity applications.</p>
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<p><b>Figure 1.</b> Our platform biosensor can be applied to many different industries and situations. Examples of this include the food industry, health applications, and biosecurity applications.</p>
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<p>Beyond the potential for being a platform for industry, the individual aspects of our FerriTALE system can also be put to use in future iGEM projects. In order to help bring TALEs to a broader audience within the iGEM community we added KasI restriction cut sites to the 3’ end of the TALEs, upstream of the coils in a fusion backbone. Using this cut site future teams will be able to swap the promoter of the TALEs with ease. This allows the TALE part to have tune the expression level of the protein with greater ease. Additionally this cut site makes it easy to switch out the TALE nucleotide-binding domains allowing future iGEM teams to customize TALEs to their target sequences with greater ease. These TALE proteins are normally expressed in eukaryotes as well. Due to this the TALE proteins contain the eukaryotic Kozak consensus sequence. The presence of this sequence however interferes with the protein’s expression in bacteria. In order to make TALEs more viable as a part to be used in the iGEM registry we removed this sequence so that it can easily be expressed within bacteria. </p>
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<p>To enable the iGEM community to harness the FerriTALE to detect other DNA sequences, we added KasI restriction cut sites to the 3’ of detector Biobricks, allowing different TALEs to be introduced in the system (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189029">BBa_K1189029</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189030">BBa_K1189030</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189032">BBa_K1189032</a>, and <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189033">BBa_K1189033</a>). This cut site enables traditional restriction enzyme cloning methods to combine FerriTALE proteins with different detector TALEs (Figure 2). Additionally, we improved two TALEs (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189022">BBa_K1189022</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189023">BBa_K1189023</a>) previously submitted to iGEM to eliminate eukaryotic Kozak translation initiation sequences which prevent expression in prokaryotic systems. This modification serves as a template for how future teams should design TALEs so that they can use <i>E. coli</i> to manufacture custom FerriTALEs.</p>
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<img src="https://static.igem.org/mediawiki/2013/3/38/UCalgary2013TRTALEmodularbinding.png" alt="Modular TALE Proteins" width="800" height="502">
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<img src="https://static.igem.org/mediawiki/2013/3/38/UCalgary2013TRTALEmodularbinding.png" alt="Modular TALE Proteins" width="600" height="375">
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<p><b>Figure 2.</b> Using our built-in cut site the binding domain of our TALEs can be switched out with different binding domains making it easy to custom tailor TALEs to virtually any target DNA sequence.</p>
<p><b>Figure 2.</b> Using our built-in cut site the binding domain of our TALEs can be switched out with different binding domains making it easy to custom tailor TALEs to virtually any target DNA sequence.</p>
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<p>Another part of our system that we submitted to the registry that can have a wider applicability beyond our system are E- and K-coils. These coils are synthetic binding domains that heterodimerize with each other with a high affinity and high specificity. Fusing the coils with larger proteins such as ferritin our system to fold and assemble correctly without interference while still maintaining their coil binding qualities. In the context of our system this allows us to easily swap out components allowing us to customize our final system. Additionally these coils have application in other contexts such as affinity purification, capture systems, and developing self-assembling biomaterials (Apostolovic and Klok, 2008). The various applications that coils have been implicated in make these coils a useful part for future iGEM teams to make use of.</p>
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<h1>Ferritin— A protein shelled nanoparticle</h1>
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<p>The ferritin nanoparticle itself also has many uses as a part in the iGEM registry. The protein itself acts as a scaffold within our system. Using either direct fusions (if the protein is small enough) or the power of E- and K-coils ferritin can effectively act as a scaffold system. In the case of our system we were scaffolding TALE proteins. This scaffold however could be used however to create unique reporter systems such as GFP-ferritin or whatever the creative minds of iGEM can think of. Additionally the iron core of ferritin presents another opportunity as a platform system. We have already demonstrated that chemically modifying the core of ferritin can be used to make an effective reporter (Prussian blue ferritin). Another potential use would be making ferritin into magnetoferritin (we experimented with this early on in the summer) or using ferritin as a nanocage for other metals. Overall this summer we believe we have developed our system as both a platform biosensor and the individual components as useful platforms for future iGEM projects.</p>
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<p>Ferritin is an iron sequestering protein shelled nanoparticle which could benefit other teams as a platform system in how the iron core can be converted to other compounds with different functions. We demonstrated this in our own system where we chemically modified the iron core to make ferritin a <a href="https://2013.igem.org/Team:Calgary/Project/OurSensor/Reporter/PrussianBlueFerritin">robust colourmetric reporter</a>. Other intriguing applications include making ferritin’s iron core magnetically active as magnetoferritin (Jordan et al. 2013), using ferritin as a nanocage for other metals, or the incorporation of other reporters such as quantum dots (Naito et al. 2013) (Figure 3). The applications are diverse when one considers how these different cores can be combined with other proteins by expressing ferritin shell subunits as protein fusions. Check out how we combined detector TALEs with <a href="https://2013.igem.org/Team:Calgary/Project/OurSensor/Reporter/PrussianBlueFerritin">Prussian blue ferritin</a> using <a href="https://2013.igem.org/Team:Calgary/Project/OurSensor/Linker">coiled-coil linkers</a>.</p>
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<img src="https://static.igem.org/mediawiki/2013/b/b8/UCalgary2013TRFerritincorechange.png" alt="Ferritin Core Modulation" width="800" height="400">
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<p><b>Figure 3.</b> Chemically modifying the iron core of ferritin allows ferritin to be moulded to fit a wide magnitude of applications. Additionally the ferritin subunits can act as a nanocage to encapsulate completely new cores. </p>
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<h1>The ferritin scaffold and coiled-coil linkers</h1>
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<p>Apart from its core, the ferritin nanoparticle is useful for iGEM teams as a self-assembling and spherical protein scaffold. Each of the 24 subunits forming ferritin can be fused proteins of interest, such that when the nanoparticle assembles, proteins surround the ferritin sphere (Kim et al., 2011). We used this in the FerriTALE to allow variation of the number of detector TALEs per ferritin reporter, enabling us to modulate sensitivity of the system. To improve flexibility of the type and composition of proteins which can be attached to ferritin, we introduced a system of complementary coiled-coils onto ferritin (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189012">BBa_K1189012</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189013">BBa_K1189013</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189018">BBa_K1189018</a>). This makes our ferritin a platform since other teams can manufacture intricate protein nanoparticles by fusing their own proteins of interest to our coils (Figure 4). We are excited by how future teams might customize ferritin by interchanging different proteins and enzymes from the Parts Registry.</p>
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<img src="https://static.igem.org/mediawiki/2013/0/07/UCalgary2013TRCoilflexibility.png" alt="FerriTALE Scaffold Modularity" width="800" height="219" >
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<p><b>Figure 4.</b> Using the E and K coils in combination with ferritin as a scaffold system allows the creation of brand new FerriTALEs or protein scaffolds.</a></p>
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<p>Additionally, these coils dimerize with high affinity and specificity and can be repurposed for affinity purification, capture systems, and development of self-assembling biomaterials (Apostolovic and Klok, 2008).</p>
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Latest revision as of 03:17, 29 October 2013

Our Platform

Our team set out to develop a nucleic acid biosensor to monitor Enterohemorhagic E. coli (EHEC) in beef cattle. Though we tailored our final system to the beef industry, the molecular components forming the FerriTALE have a myriad of other applications. These include detector TALEs which can be readily designed to other DNA targets, a ferritin protein shelled nanoparticle with interchangeable cores that impart novel function, and an interchangeable coiled-coil linker system to build intricate protein devices using ferritin as a scaffold. Together, these components form a customizable DNA biosensor, with individual components that could have diverse applications in future iGEM projects and beyond.

Detector

We selected Transcription Activator Like Effectors (TALEs) to bind and detect DNA in our system. These proteins were chosen because their DNA binding domains can be engineered to bind virtually any DNA sequence (Cermak et al., 2011). This means that we have built a DNA biosensor platform, where our system can be repurposed to detection of organisms in other applications by designing TALEs to different gene sequences. By combining alternative TALE proteins into our capture strip assay, our system could be relevant to other food industries, health care, bedside diagnostics, and biosecurity (Figure 1).

Platform Technology

Figure 1. Our platform biosensor can be applied to many different industries and situations. Examples of this include the food industry, health applications, and biosecurity applications.

To enable the iGEM community to harness the FerriTALE to detect other DNA sequences, we added KasI restriction cut sites to the 3’ of detector Biobricks, allowing different TALEs to be introduced in the system (BBa_K1189029, BBa_K1189030, BBa_K1189032, and BBa_K1189033). This cut site enables traditional restriction enzyme cloning methods to combine FerriTALE proteins with different detector TALEs (Figure 2). Additionally, we improved two TALEs (BBa_K1189022, BBa_K1189023) previously submitted to iGEM to eliminate eukaryotic Kozak translation initiation sequences which prevent expression in prokaryotic systems. This modification serves as a template for how future teams should design TALEs so that they can use E. coli to manufacture custom FerriTALEs.

Modular TALE Proteins

Figure 2. Using our built-in cut site the binding domain of our TALEs can be switched out with different binding domains making it easy to custom tailor TALEs to virtually any target DNA sequence.

Ferritin— A protein shelled nanoparticle

Ferritin is an iron sequestering protein shelled nanoparticle which could benefit other teams as a platform system in how the iron core can be converted to other compounds with different functions. We demonstrated this in our own system where we chemically modified the iron core to make ferritin a robust colourmetric reporter. Other intriguing applications include making ferritin’s iron core magnetically active as magnetoferritin (Jordan et al. 2013), using ferritin as a nanocage for other metals, or the incorporation of other reporters such as quantum dots (Naito et al. 2013) (Figure 3). The applications are diverse when one considers how these different cores can be combined with other proteins by expressing ferritin shell subunits as protein fusions. Check out how we combined detector TALEs with Prussian blue ferritin using coiled-coil linkers.

Ferritin Core Modulation

Figure 3. Chemically modifying the iron core of ferritin allows ferritin to be moulded to fit a wide magnitude of applications. Additionally the ferritin subunits can act as a nanocage to encapsulate completely new cores.

The ferritin scaffold and coiled-coil linkers

Apart from its core, the ferritin nanoparticle is useful for iGEM teams as a self-assembling and spherical protein scaffold. Each of the 24 subunits forming ferritin can be fused proteins of interest, such that when the nanoparticle assembles, proteins surround the ferritin sphere (Kim et al., 2011). We used this in the FerriTALE to allow variation of the number of detector TALEs per ferritin reporter, enabling us to modulate sensitivity of the system. To improve flexibility of the type and composition of proteins which can be attached to ferritin, we introduced a system of complementary coiled-coils onto ferritin (BBa_K1189012, BBa_K1189013, BBa_K1189018). This makes our ferritin a platform since other teams can manufacture intricate protein nanoparticles by fusing their own proteins of interest to our coils (Figure 4). We are excited by how future teams might customize ferritin by interchanging different proteins and enzymes from the Parts Registry.

FerriTALE Scaffold Modularity

Figure 4. Using the E and K coils in combination with ferritin as a scaffold system allows the creation of brand new FerriTALEs or protein scaffolds.

Additionally, these coils dimerize with high affinity and specificity and can be repurposed for affinity purification, capture systems, and development of self-assembling biomaterials (Apostolovic and Klok, 2008).