Difference between revisions of "Team:Northeastern/Design"

 
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                <h3>Project Design</h3>
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                <h4>Proteorhodopsin</h4>
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                    <img src="https://static.igem.org/mediawiki/2016/6/62/T--Northeastern--project_01_prdiagram.gif">
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                    <p>The proteorhodopsin (PR) plasmid originated from &nbsp;UNTN-Trento’s 2015 design. The Blh gene and PR protein were placed under the J23100 T7 constitutive promoter in the high copy pSB1C3 backbone, which confers negative selection through chloramphenicol resistance. We chose constitutive expression to attain maximum power production in the shortest amount of time, while also minimizing the cost and amount of feed components necessary. This combination will ultimately make a cell that can run off of minimally treated wastewater. Proteorhodopsin is post-transcriptionally controlled through light dependency and pH-dependent conformational changes which make its activity negatively correlated with H+ ion concentration. The Blh gene upstream of proteorhodopsin cleaves beta-carotene into two molecules of retinal, which is a cofactor required by proteorhodopsin. A second plasmid containing the beta-carotene synthesis pathway, also from UNITN-Trento, was designed to allow the cell to operate without manually inducing &nbsp;beta carotene addition after initial tests with the simpler design. &nbsp;</p>
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                <h4>NADH Oxidase</h4>
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By talking about your design work on this page, there is one medal criterion that you can attempt to meet, and one award that you can apply for. If your team is going for a gold medal by building a functional prototype, you should tell us what you did on this page.
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                    <p>An NADH oxidase circuit was designed using the H2O-forming Nox from <i>Streptococcus sanguinis SK36</i> (sequence from NCBI). A his-tagged part under the constitutive J23100 promoter &nbsp;was constructed to characterize protein concentration and oxygen scavenging. &nbsp;Nox converts electron-rich NADH into NAD+. This increases the energy needs of the cell and could cause imbalances that harm growth. We planned on placing Nox under the oxygen-activated Pndh promoter (Paris Saclay 2013) to quickly restore anoxia after oxygen sparging in the MEC without giving the facultative anaerobe E coli a competitive advantage. Both plasmids tested were placed under the high-copy pSB1C3 plasmid backbone incorporated chloramphenicol resistance. The Pndh-controlled nox plasmid was designed with the pSB1A3 ampicillin resistance backbone so that both plasmids could be inserted into the same cell and confirmed with double selection.</p>
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                    <img src="https://static.igem.org/mediawiki/2016/d/dd/T--Northeastern--project_03_noxdiagram.jpg">
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                        <a href="/Team:Northeastern/Description">Description</a>
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                        <a href="/Team:Northeastern/Experiments">Experiments</a>
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                        <a href="/Team:Northeastern/Proof">Proof</a>
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                        <a href="/Team:Northeastern/Results">Results</a>
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                        <a href="/Team:Northeastern/Notebook">Notebook</a>
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<p>This is a prize for the team that has developed a synthetic biology product to solve a real world problem in the most elegant way. The students will have considered how well the product addresses the problem versus other potential solutions, how the product integrates or disrupts other products and processes, and how its lifecycle can more broadly impact our lives and environments in positive and negative ways.</p>
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If you are working on art and design as your main project, please join the art and design track. If you are integrating art and design into the core of your main project, please apply for the award by completing this page.
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<p>Teams who want to focus on art and design should be in the art and design special track. If you want to have a sub-project in this area, you should compete for this award.</p>
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Latest revision as of 22:31, 16 October 2016

Project Design

Proteorhodopsin

The proteorhodopsin (PR) plasmid originated from  UNTN-Trento’s 2015 design. The Blh gene and PR protein were placed under the J23100 T7 constitutive promoter in the high copy pSB1C3 backbone, which confers negative selection through chloramphenicol resistance. We chose constitutive expression to attain maximum power production in the shortest amount of time, while also minimizing the cost and amount of feed components necessary. This combination will ultimately make a cell that can run off of minimally treated wastewater. Proteorhodopsin is post-transcriptionally controlled through light dependency and pH-dependent conformational changes which make its activity negatively correlated with H+ ion concentration. The Blh gene upstream of proteorhodopsin cleaves beta-carotene into two molecules of retinal, which is a cofactor required by proteorhodopsin. A second plasmid containing the beta-carotene synthesis pathway, also from UNITN-Trento, was designed to allow the cell to operate without manually inducing  beta carotene addition after initial tests with the simpler design.  

NADH Oxidase

An NADH oxidase circuit was designed using the H2O-forming Nox from Streptococcus sanguinis SK36 (sequence from NCBI). A his-tagged part under the constitutive J23100 promoter  was constructed to characterize protein concentration and oxygen scavenging.  Nox converts electron-rich NADH into NAD+. This increases the energy needs of the cell and could cause imbalances that harm growth. We planned on placing Nox under the oxygen-activated Pndh promoter (Paris Saclay 2013) to quickly restore anoxia after oxygen sparging in the MEC without giving the facultative anaerobe E coli a competitive advantage. Both plasmids tested were placed under the high-copy pSB1C3 plasmid backbone incorporated chloramphenicol resistance. The Pndh-controlled nox plasmid was designed with the pSB1A3 ampicillin resistance backbone so that both plasmids could be inserted into the same cell and confirmed with double selection.