Difference between revisions of "Team:WashU StLouis/Description"

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<h1>Overview</h1>
 
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<h2> A brief description of our iGEM project</h2>
 
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<p class="description">The Washington University and Penn State iGEM 2016 project is supported by the Nitrogen Project, a multi-university research initiative to enable plants to fix nitrogen independent of soil bacteria. In previous years, our iGEM team has worked on an intermediate step in this process—transforming E. coli cells (which do not fix nitrogen naturally) with nitrogenase genes from a diazotroph (an organism that fixes nitrogen). This has involved selecting genes from the nif cluster – those known to aid in nitrogenase activity. However, getting these genes from the cyanobacteria <i>Synechocystis</i> into E. coli has proven difficult in practice.</p>
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<!--<div class="action"><img  src="https://static.igem.org/mediawiki/2016/d/d9/T--WashU_StLouis--Action.png" style="width:100%;"></div>-->
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<div class = "speech"><img  src="https://static.igem.org/mediawiki/2016/0/05/T--WashU_StLouis--Speech.png" style="width:100%;"></div>
  
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<p class="description">In 2014, the WashU iGEM team reported minimal increases in nitrogenase expression from their nif plasmid. However, these results could not be replicated in 2015. The 2015 iGEM team identified a promising 14 gene subset of the nif cluster but again found <i>E. coli</i> to be an unsuitable host for <i>Synechocystis</i>' nitrogenase. The 2016 WashU / Penn State iGEM team is addressing this problem with a related yet more broadly applicable project.</p>
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<h1 class="actionwords">Overview</h1>  
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<h2 class="speechwords">A brief introduction of our iGEM project</h2>
  
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<p class="description">Nitrogenase activity in <i>E. coli</i> requires a large amount of ATP and electron donors to reduce N2 to consumable nitrates. Our goal for this year’s iGEM is to increase the production of these two cofactors. To do so, we are adding genes that produce electron donors and ATP to plasmids with inducible promoters.</p>
 
  
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<p class="description">Flavodoxin, an electron donor, is produced naturally in <i>E. coli</i>. We have added <i>fldA</i>, the gene associated with flavodoxin production to a plasmid and used this to transform E. coli. In addition, we created a plasmid containing a gene for ferredoxin, an analogous electron donor found in <i>Synechocystis</i>, from which the nif genes came. Expression of these genes will provide the nitrogenase enzyme with electrons necessary for the reduction of N2, while also helping us to understand the differences between its activity in <i>E. coli</i> and <i>Synechocystis</i>.</p>
 
  
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<p class="description">Adenosine triphosphate—or ATP—is not just a key factor in the activity of nitrogenase. It is used in active transport, protein synthesis, and many other active processes of <i>E. coli</i>’s metabolism. We have identified 2 genes or gene clusters associated with ATP production: phosphoenolpyruvate kinase (PCK) and phosphoglycerate kinase (PGK), that will allow us to increase the concentration of this molecule within the cell.</p>
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<p class="description">Increasing the concentration of intracellular ATP and electron donors may not only be beneficial for nitrogenase activity, but may help facilitate other energetic processes. Manufacturing recombinant proteins, particularly those high in sulfur, require generous amounts of intracellular ATP. Cells with higher ATP concentrations have been shown to be more resistant to toxins, and could therefore be effective in ethanol bioproduction. We plan to continue exploring the possibilities and ramifications of this project.</p>
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<h3>Motivation</h3>
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<p class="description">The growth of human civilization has been closely linked to fertilizer production for over a century. Before the development of the Haber-Bosch process in 1910, nitrogenous fertilizers were difficult to manufacture on an industrial scale. In the following century, agricultural production exploded and global population soared from 2 to 7 billion people. However, such demand for nitrate fertilizers has taken a toll on the environment. Nitrates are easily washed from farmland into tributaries and larger bodies of water, where plants and microbes are unable to consume them quickly enough. Instead, algae and other organisms proliferate rapidly in the excess of nutrients and then die off, leaving massive, oxygen-depleted 'dead zones.' In the graph below, you can see how aqueous nitrate levels in the Mississippi River have not only risen in recent years<sup>1</sup>, but are also correlated with the size of the dead zone where it empties in the Gulf of Mexico<sup>2</sup>.</p>
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<img src="https://static.igem.org/mediawiki/2016/f/fb/T--WashU_StLouis--Overiew1.png" style="width:50vw;display:block;margin: 0 auto;">
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<p class="description" style="text-align:center;"><sup><a href="http://toxics.usgs.gov/hypoxia/mississippi/oct_jun/">Source</a></sup></p>
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<p class="description">Nitrates can also leach into groundwater and aquifers. When used by people for drinking, nitrate-contaminated water can be toxic<sup>3</sup>. It is associated with “blue baby syndrome,” a potentially lethal condition.</p>
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<img src="https://static.igem.org/mediawiki/2016/f/f0/T--WashU_StLouis--Overiew2.gif" style="width:50vw;display:block;margin: 0 auto;">
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<p class="description" style="text-align:center;"><sup><a href="http://water.usgs.gov/edu/nitrogen.html">Source</a></sup></p>
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<p class="description">The Nitrogen Project was initiated to fund research that investigates ways to mitigate nitrate use in agriculture and slow the impact of runoff.</p>
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<h3>Our Project:</h3>
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<p class="description">A key area of research for the Nitrogen Project has been to get nitrogenase, the enzyme that fixes gaseous nitrogen and converts it to usable nitrates, into non-diazotrophs, which are bacteria that cannot fix their own nitrogen. Past efforts have been made to transform the <i>nif</i> gene cluster, which codes for nitrogenase, into <i>E. coli</i>, but no conclusive results were found.</p>
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<img src="https://static.igem.org/mediawiki/2016/2/28/T--WashU_StLouis--Overiew3.png" style="width:50vw;display:block;margin: 0 auto;">
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<p class="description" style="text-align:center;"><sup>PLANT PHYSIOLOGY, <i>Fourth Edition</i></sup></p>
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<p class="description"> In order to solve the nitrate problem, we propose creating a “Super Cell,” a cell that overproduces ATP and reduced electron donors in order to provide nitrogenase with a suitable environment for maximized activity. We overexpressed genes involved in ATP and electron donor production and created constructs that ultimately increased the intracellular concentrations of these co-factors. We also conducted a proof of concept experiment to show these co-factors being utilized by the cell. </p>
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<p class="description">Additionally, we improved the characterization a <a href="http://parts.igem.org/Part:BBa_K873002:Experience">Heat Shock Promoter (HSP) BioBrick</a> that is supposedly activated at high temperatures but had no previous data on the registry. Our characterization experiments, which were repeated by <a href="https://2016.igem.org/Team:Vilnius-Lithuania">Vilnius iGEM</a>, showed that this HSP was only slightly activated by high temperatures (42&deg;C) and its activity greatly decreased over time.</p>
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<p class="description">To start learning about our project, find out how we <a href="https://2016.igem.org/Team:WashU_StLouis/Design">designed</a> our Super Cells</p>
  
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        <p class="description">Additionally, we improved the characterization an HSP BioBrick that is supposed to be activated at high temperatures but had no previous data on the registry. Our characterization experiments, which were repeated by Vilnius iGEM, showed that this HSP was only slight activated by high temperatures (42C) and its activity greatly decreased over time.</p>
 
 
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<p>References:</p>
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<li>Woodside, Michael, and Lori Sprague. "Nitrate Levels Continue to Increase in the Mississippi River; Signs of Progress in the Illinois River." Nitrate Levels Continue to Increase in the Mississippi River; Signs of Progress in the Illinois River. USGS, 8 Dec. 2015. Web</li>
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<li>Lee, Casey. "Streamflow and Nutrient Delivery to the Gulf of Mexico for October 2015 to May 2016 (Preliminary)." Streamflow and Nutrient Delivery to the Gulf of Mexico for October 2015 to May 2016 (Preliminary). USGS, 1 June 2016. Web.</li>
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<li>Perlman, Howard. "Nitrogen and Water." : USGS Water Science School. USGS, 2 May 2016. Web. </li>
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Latest revision as of 01:08, 20 October 2016

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Overview

A brief introduction of our iGEM project

Motivation

The growth of human civilization has been closely linked to fertilizer production for over a century. Before the development of the Haber-Bosch process in 1910, nitrogenous fertilizers were difficult to manufacture on an industrial scale. In the following century, agricultural production exploded and global population soared from 2 to 7 billion people. However, such demand for nitrate fertilizers has taken a toll on the environment. Nitrates are easily washed from farmland into tributaries and larger bodies of water, where plants and microbes are unable to consume them quickly enough. Instead, algae and other organisms proliferate rapidly in the excess of nutrients and then die off, leaving massive, oxygen-depleted 'dead zones.' In the graph below, you can see how aqueous nitrate levels in the Mississippi River have not only risen in recent years1, but are also correlated with the size of the dead zone where it empties in the Gulf of Mexico2.

Source

Nitrates can also leach into groundwater and aquifers. When used by people for drinking, nitrate-contaminated water can be toxic3. It is associated with “blue baby syndrome,” a potentially lethal condition.

Source

The Nitrogen Project was initiated to fund research that investigates ways to mitigate nitrate use in agriculture and slow the impact of runoff.

Our Project:

A key area of research for the Nitrogen Project has been to get nitrogenase, the enzyme that fixes gaseous nitrogen and converts it to usable nitrates, into non-diazotrophs, which are bacteria that cannot fix their own nitrogen. Past efforts have been made to transform the nif gene cluster, which codes for nitrogenase, into E. coli, but no conclusive results were found.

PLANT PHYSIOLOGY, Fourth Edition

In order to solve the nitrate problem, we propose creating a “Super Cell,” a cell that overproduces ATP and reduced electron donors in order to provide nitrogenase with a suitable environment for maximized activity. We overexpressed genes involved in ATP and electron donor production and created constructs that ultimately increased the intracellular concentrations of these co-factors. We also conducted a proof of concept experiment to show these co-factors being utilized by the cell.

Additionally, we improved the characterization a Heat Shock Promoter (HSP) BioBrick that is supposedly activated at high temperatures but had no previous data on the registry. Our characterization experiments, which were repeated by Vilnius iGEM, showed that this HSP was only slightly activated by high temperatures (42°C) and its activity greatly decreased over time.

To start learning about our project, find out how we designed our Super Cells

References:

  1. Woodside, Michael, and Lori Sprague. "Nitrate Levels Continue to Increase in the Mississippi River; Signs of Progress in the Illinois River." Nitrate Levels Continue to Increase in the Mississippi River; Signs of Progress in the Illinois River. USGS, 8 Dec. 2015. Web
  2. Lee, Casey. "Streamflow and Nutrient Delivery to the Gulf of Mexico for October 2015 to May 2016 (Preliminary)." Streamflow and Nutrient Delivery to the Gulf of Mexico for October 2015 to May 2016 (Preliminary). USGS, 1 June 2016. Web.
  3. Perlman, Howard. "Nitrogen and Water." : USGS Water Science School. USGS, 2 May 2016. Web.