Difference between revisions of "Team:Virginia/Implementation"

 
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               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia">Main</a>
 
               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia">Main</a>
               <a class="hvr-sweep-to-right" href="#abstract">Abstract</a>
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               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia#abstract">Abstract</a>
              <a class="hvr-sweep-to-right" href="#problem">Problem</a>
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              <a class="hvr-sweep-to-right" href="#solution">Solution</a>
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             <a class="mainitem" href="#project">PROJECT</a>
 
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              <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Inspiration">Inspiration</a>
 
 
               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Description">Description</a>
 
               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Description">Description</a>
 
               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Design">Design</a>
 
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               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Results">Results</a>
 
               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Results">Results</a>
 
               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Model">Modeling</a>
 
               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Model">Modeling</a>
               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Device">Device</a>
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               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Engineering">Engineering</a>
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              <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Implementation">Implementation</a>
 
               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Notebook">Notebook</a>
 
               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Notebook">Notebook</a>
 
               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Safety">Safety</a>
 
               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Safety">Safety</a>
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             <a class="mainitem" href="#parts">PARTS</a>
 
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               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/New_parts">New Parts</a>
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               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/New_parts"> Parts</a>
              <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Utilized_parts">Utilized Parts</a>
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               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Public_engagement">Public Engagement</a>
 
               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Public_engagement">Public Engagement</a>
 
               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Collaborations">Collaborations</a>
 
               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Collaborations">Collaborations</a>
               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Meetups">Meet Ups</a>
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               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Contribution">Contributions</a>
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              <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Entrepreneurship">Entrepreneurship</a>
 
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               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Members">Members</a>
 
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               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Attributions">Attributions</a>
 
               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Attributions">Attributions</a>
              <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Gallery">Gallery</a>
 
 
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             <a class="mainitem" href="#about">RESOURCES</a>
 
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               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Papers">Sources</a>
 
               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/nucleic_acids">Nucleic Acids</a>
 
               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/nucleic_acids">Nucleic Acids</a>
 
               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Protocols">Protocols</a>
 
               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Protocols">Protocols</a>
              <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Software">Software</a>
 
 
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             <div>General Template Page</div>
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             <div>Implementation</div>
 
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           <div class="sectionTitle" id="Section 1">Section 1</div>
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           <div class="sectionTitle" id="Section 1">Testing Efficacy</div>
 
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                The lack of a versatile and reliable way to improve metabolic flux channeling, pathway orthogonality, and product yields is a major impediment to the expanded utilization of biosynthesis for the production of drugs and industrially valuable chemicals. Manifold, a platform technology that addresses this problem, consists of <div class="dict">bacterial microcompartments<span><img src="https://upload.wikimedia.org/wikipedia/commons/thumb/2/25/Carboxysome_and_bacterial_microcompartments.jpg/800px-Carboxysome_and_bacterial_microcompartments.jpg"/>Bacterial microcompartments (BMCs) are organelle-like structures, consisting of a protein shell that encloses enzymes and other proteins. BMCs are typically about 40–200 nanometers in diameter and are entirely made of proteins. The shell functions like a membrane, as it is selectively permeable.</span></div> (BMCs) with encapsulated dsDNA scaffolds <div class="ref">[1]<span>Elbaz, J., Yin, P., &amp; Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7(1), 1-11.</span></div> that sequester and spatially organize, at fixed concentrations, biosynthetic enzymes presented as zinc-finger fusion proteins. Here we deliver the designs for an E. coli cell capable of synthesizing resveratrol using the Manifold platform. The Manifold platform will help lower costs and expand the applications of chemical biosynthesis. The lack of a versatile and reliable way to improve metabolic flux channeling, pathway orthogonality, and product yields is a major impediment to the expanded utilization of biosynthesis for the production of drugs and industrially valuable chemicals. Manifold, a platform technology that addresses this problem, consists of bacterial microcompartments (BMCs) with encapsulated dsDNA scaffolds that sequester and spatially organize, at fixed concentrations, biosynthetic enzymes presented as zinc-finger fusion proteins. Here we deliver the designs for an E. coli cell capable of synthesizing resveratrol using the Manifold platform. The Manifold platform will help lower costs and expand the applications of chemical biosynthesis.
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                Beyond a proof of concept to determine the success of Manifold, further tests will be required to determine the effectiveness of our design and its scalability. The primary test for efficacy will be an HPLC analysis of the culture broth in order to identify the concentration of resveratrol being produced. To see if the Manifold system adequately increases the flux through the pathway, the final ratios of resveratrol to 4-coumaric acid can be compared. If there is no flux leakage, then this ratio will be equal to 1. In order to see if an increase in overall product is observed, the ratio of resveratrol concentrations between the experimental group and control groups can be used. Finally, to see if the rate of resveratrol is increased by the system, a linear regression can be fit to each concentration vs time plot for resveratrol and the slopes can be compared. If our yields prove that Manifold nearly eliminates flux leakage, we have the data required to move on to testing other enzyme pathways.  
 
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           <div class="sectionTitle" id="Section 2">Section 2</div>
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           <div class="sectionTitle" id="Section 2">Further Validating Manifold</div>
 
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               The invention consists of a protein shell comprising one or more proteins, one or more nucleic acid scaffolds of which there can be multiple copies, anabolic and/or catabolic enzymes specific to the desired biosynthesis pathway each containing a nucleic acid binding domain, recognition sequences for the utilized nucleic acid binding domains, nucleic acid spacers, and a linkage between the nucleic acid scaffolds and the protein shell. The protein shell (10) can take the form of any closed or open surface that comprises one or more repeating protein units (12). Examples of valid shells include bacterial microcompartments such as the Pdu, Eut, and carboxysome microcompartments, as well as modified, but not necessarily closed, surfaces composed of mutated versions of these microcompartment shell proteins. The nucleic acid scaffolds (18) comprise multiple recognition sequences (22) and spacers (32) and can be made from any form of nucleic acid, including: deoxyribonucleic acid, ribonucleic acid, and synthetic nucleic acids such as xeno nucleic acids and peptide nucleic acids among others. These scaffolds are attached to the protein shell. The pathway enzymes are biological proteins whose exact sequences are dependent on the given use case of the invention, but which all contain a nucleic acid binding domain either internal to their structure, or at their N or C terminus.  
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               If our implementation for Manifold proves successful with resveratrol, we can move onto testing other enzyme pathways in similar fashions. Proving Manifold with pathways that are applicable to more important pharmaceutical pathways will be the priority, and we have explored options relating to statins, medications meant to reduce cholesterol levels, in order to extend our system to the production of more diverse compounds. Doing so will demonstrate the scalability of Manifold, and show that our platform technology in the space of biologics has the potential to increase yields and bolster efficiency. 
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          <div class="sectionTitle" id="Section 3">Impact on the Pharmaceutical Field and Beyond</div>
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                Manifold represents a truly exciting advance in the field of synthetic biology. By channeling metabolic flux through BMCs and DNA scaffolds, Manifold fixes and confines metabolic pathways in space, allowing for dramatic increases in flux through the pathway. By localizing enzymes and substrates together within BMCs, required interactions happen more frequently. Additionally, by compartmentalizing the pathway within BMCs, reactions and intermediates are isolated from the rest of the cell which is both convenient for the researchers and the microbe. It’s difficult to understate the impact Manifold will have on the field of synthetic biology and pharmacology, but we do foresee these industries as having the greatest use of Manifold. If successful, it will revolutionize how bacteria are engineered to synthesize anything from bulk chemicals to pharmaceuticals to biofuels. The implications on the pharmaceutical world alone are astounding. The cost of production of virtually any drug synthesized in bacteria can be reduced, in turn, reducing the cost of life-saving as well as everyday drugs. While creating our proof of concept design for resveratrol, we focused mainly on the pharmaceutical industry, determining how the synthesis of this supplement will impact the surrounding field, but if the use of Manifold proves successful, any form of enzymatic compound production has the potential to be heightened. Through the combination of BMCs and DNA scaffolds to channel metabolic flux, Manifold represents an exciting advance in the field that can not be underestimated. Though there is much work to be done yet, the team is thrilled to continue crafting its approach to Manifold.  
 
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               <b>Fig 1.</b> Figure taken from iGEM Tainan 2019 for demo purposes. Notice how the figure is much longer than it is wide, and two images are coupled together to achive this. Try to do that as well so it looks good.
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               <b>Fig 1.</b> An example of a bioreactor in which bacteria produced through the Manifold system would be grown in order to produce pharmaceuticals.
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          <div class="sectionTitle" id="Section 4">Safety Considerations</div>
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              There arise a number of safety and ethical considerations that will require close attention. As pointed out before, we have questioned who will first benefit from our technology? Are there ways in which Manifold can be manipulated for malicious purposes? In looking at potential malpractice by an entity licensing our platform, we see little risk in negligent or even intentional release of our bacteria into the wild. Fortunately, with the high metabolic load placed on our engineered <i>E. coli</i> cells, it follows that our organism would face a significant disadvantage if released into the environment. For this reason, there arises no reason to suspect that the engineered organism is any cause for concern. Unfortunately, there exist perhaps an equal number of ways to commandeer the technology for selfish or malicious motivations as there are ways to use the technology for well-meaning adaptations. Here lies the worst case scenario regarding Manifold. In the wrong hands, this technology can be used to increase production of compounds to be used as weapons. Through Manifold, dangerous compounds that stem from enzymatic pathways can be produced far more efficiently, and as creators of this platform, we do feel a responsibility to manage the ways in which our technology is used. This sentiment became our Code of Ethical Conduct, our internal doctrine that governs our own use of the platform, while also giving guidelines about judging what outside groups can utilize Manifold. Foundational advances are powerful tools; they derive much of their impact and excitement from the possibility to adapt the advance to an array of purposes, organisms, and disciplines.  
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              Additionally, protein linkers are usually present between this nucleic acid binding domain and the enzyme structure to prevent inhibition of enzyme activity. However the exact linker(s) used,  if any, is(are) also dependent on the specific use case of the invention. These pathway enzymes are attached to the nucleic acid scaffolds via their nucleic acid binding domains. The nucleic acid recognition sequences (22) are unique or semi-unique sequences of nucleic acid monomers on the nucleic acid scaffolds to which the utilized nucleic acid binding domains have some degree of molecular complementarity. These nucleic recognition sequences comprise most of the scaffold and mark the locations to which the DNA binding domains of the pathway enzymes attach to the scaffolds. The nucleic acid spacers (32) are relatively short sequences of nucleic acid monomers that are also present on the nucleic acid scaffolds, between the recognition sequences. The linkage between the nucleic acid scaffolds (18) and protein shell (10) provides a means by which the nucleic acid scaffolds are bound to the protein shell through direct or multi-molecule complementarity. This linkage is found between the nucleic acid scaffolds and the protein shell. One example is through the addition of a nucleic acid binding domain (24) to one or more of the shell proteins forming a nucleic acid binding domain, shell protein fusion (14). Like the pathway enzymes, this nucleic acid binding-domain can be either internal to the shell protein structure or at its N or C terminus, where the exact placement depends on the shell protein being utilized. Alternatively, one or more intermediate proteins can be used to adhere the nucleic acid scaffolds to the shell, where the region of the protein interacting with the shell binds the shell via protein-protein complementarity (28) with a given shell protein, and the region of the protein interacting with the nucleic acid scaffold binds another recognition sequence on the nucleic acid scaffold through another nucleic acid binding domain (30). This forms a shell protein binding, nucleic acid domain fusion (26).<br/><br/><br/>
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Latest revision as of 03:31, 28 October 2020

Manifold

Index:
Implementation
Testing Efficacy
Beyond a proof of concept to determine the success of Manifold, further tests will be required to determine the effectiveness of our design and its scalability. The primary test for efficacy will be an HPLC analysis of the culture broth in order to identify the concentration of resveratrol being produced. To see if the Manifold system adequately increases the flux through the pathway, the final ratios of resveratrol to 4-coumaric acid can be compared. If there is no flux leakage, then this ratio will be equal to 1. In order to see if an increase in overall product is observed, the ratio of resveratrol concentrations between the experimental group and control groups can be used. Finally, to see if the rate of resveratrol is increased by the system, a linear regression can be fit to each concentration vs time plot for resveratrol and the slopes can be compared. If our yields prove that Manifold nearly eliminates flux leakage, we have the data required to move on to testing other enzyme pathways.
Further Validating Manifold
If our implementation for Manifold proves successful with resveratrol, we can move onto testing other enzyme pathways in similar fashions. Proving Manifold with pathways that are applicable to more important pharmaceutical pathways will be the priority, and we have explored options relating to statins, medications meant to reduce cholesterol levels, in order to extend our system to the production of more diverse compounds. Doing so will demonstrate the scalability of Manifold, and show that our platform technology in the space of biologics has the potential to increase yields and bolster efficiency.
Impact on the Pharmaceutical Field and Beyond
Manifold represents a truly exciting advance in the field of synthetic biology. By channeling metabolic flux through BMCs and DNA scaffolds, Manifold fixes and confines metabolic pathways in space, allowing for dramatic increases in flux through the pathway. By localizing enzymes and substrates together within BMCs, required interactions happen more frequently. Additionally, by compartmentalizing the pathway within BMCs, reactions and intermediates are isolated from the rest of the cell which is both convenient for the researchers and the microbe. It’s difficult to understate the impact Manifold will have on the field of synthetic biology and pharmacology, but we do foresee these industries as having the greatest use of Manifold. If successful, it will revolutionize how bacteria are engineered to synthesize anything from bulk chemicals to pharmaceuticals to biofuels. The implications on the pharmaceutical world alone are astounding. The cost of production of virtually any drug synthesized in bacteria can be reduced, in turn, reducing the cost of life-saving as well as everyday drugs. While creating our proof of concept design for resveratrol, we focused mainly on the pharmaceutical industry, determining how the synthesis of this supplement will impact the surrounding field, but if the use of Manifold proves successful, any form of enzymatic compound production has the potential to be heightened. Through the combination of BMCs and DNA scaffolds to channel metabolic flux, Manifold represents an exciting advance in the field that can not be underestimated. Though there is much work to be done yet, the team is thrilled to continue crafting its approach to Manifold.
Fig 1. An example of a bioreactor in which bacteria produced through the Manifold system would be grown in order to produce pharmaceuticals.
Safety Considerations
There arise a number of safety and ethical considerations that will require close attention. As pointed out before, we have questioned who will first benefit from our technology? Are there ways in which Manifold can be manipulated for malicious purposes? In looking at potential malpractice by an entity licensing our platform, we see little risk in negligent or even intentional release of our bacteria into the wild. Fortunately, with the high metabolic load placed on our engineered E. coli cells, it follows that our organism would face a significant disadvantage if released into the environment. For this reason, there arises no reason to suspect that the engineered organism is any cause for concern. Unfortunately, there exist perhaps an equal number of ways to commandeer the technology for selfish or malicious motivations as there are ways to use the technology for well-meaning adaptations. Here lies the worst case scenario regarding Manifold. In the wrong hands, this technology can be used to increase production of compounds to be used as weapons. Through Manifold, dangerous compounds that stem from enzymatic pathways can be produced far more efficiently, and as creators of this platform, we do feel a responsibility to manage the ways in which our technology is used. This sentiment became our Code of Ethical Conduct, our internal doctrine that governs our own use of the platform, while also giving guidelines about judging what outside groups can utilize Manifold. Foundational advances are powerful tools; they derive much of their impact and excitement from the possibility to adapt the advance to an array of purposes, organisms, and disciplines.