Difference between revisions of "Team:Santa Clara/OurDefenseSystem"

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<h1> The Problem </h1>
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<h1> Our Defense System </h1>
 
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     Bioreactor technology allows us, as synthetic biologists, to take our technology to the large scale. It denotes cell culture methods that allow for the mass production of organisms and their products by facilitating growth in various apparatuses. The goal of these systems is to create an environment for elevated cell growth to promote the production and/or degradation of various substances. These systems have been optimized to fit endless applications but one problem remains unresolved. In these cultures, cells are designed to reach high cell densities, which leads to an increased build up of metabolic acid. The increasing presence of acid in the environment adversely affects cell processes because weak acids can easily diffuse back through the cell membrane to inhibit and degrade DNA, as well as degrade proteins essential to cell survival. To offset this issue and maintain cell vitality, the culture must be supplemented with various basic solutions to neutralize the acid. This increases the running cost of the system, through the cost of the solutions themselves, as well as the production and implementation of systems to distribute the base. It also results in slowed cell growth due to the exposure of highly concentrated base.  
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     Of all of the researched mechanisms, the one that stood out as the best candidate was E. coli’s membrane composition change that incorporates cyclopropane fatty acids into the membrane. This mechanism was found to be the single most impactful acid resistant mechanism in this enterobacterium. It involves the increased expression of the cyclopropane fatty acid (CFA) synthase which will catalyze the addition of the methyl group to a monounsaturated fatty acid (UFA), using S-adenosyl methionine (SAM) as a methyl donor. The CFA synthase is a cytosolic protein that reversibly associates with the membrane. These modified phospholipids then incorporate into the membrane and lead to the acid resistant phenotype. The protruding carbon creates a more sterically hindered for weak acids attempting to pass through the membrane.  
 
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             <img alt="Sample Image 2" src="https://static.igem.org/mediawiki/2015/f/f5/AcidBuildupInCulture.png" style="width: 80%"/>
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             <img alt="Sample Image 2" src="https://static.igem.org/mediawiki/2015/e/eb/CFASAM.png" style="width: 70%"/>
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<p>Schematic of cyclopropane fatty acid defense system.</p>
 
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We aim to offset this hurdle in bioreactor technology by creating organisms that would be more resilient to these acidic conditions. Being as these cultures won’t continuously increase in acidity, as there is a constant efflux of the media that will lead to a leveling off of pH, if we can equip the organism with the necessary machinery to survive that pH we could eliminate the addition of base. At the very least, we can decrease the amount of base needed. We looked to nature’s solutions to devise a plan to accomplish this goal.
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            <img alt="Sample Image 2" src="https://static.igem.org/mediawiki/2015/2/2d/CFAFormationpng.png" style="width: 90%"/>
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<p>Chemical reactions involved in CFA formation.</p>
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        <h2>Rationale behind choosing the CFA system</h2>
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        <p> The CFA acid resistance system offers several advantages over the other systems that we identified in nature:
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<li> Requires no major alterations in metabolism </li>
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<li> Does not use precious cellular resources as transporters </li>
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<li> Only requires two cytosolic proteins </li>
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<li> Offers versatility for use in other organisms because the cell membrane is well conserved even across gram positive and negative bacterium </li>
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<li> Found to be the single most effective acid resistance system </li>
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<li> Not found in any other organism that has been documented </li>
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We sought to create a robust system that could be implemented in all bacterium so that regardless of the chassis chosen for a bioreactor, it would be able to increase its aciduricity. We also wanted to find a way to boost the acid resistance of the organism through the addition of a new system opposed to the upregulation of an endogenous system. By adding a new system, we think it should be possible to greatly increase the acid resistance of an organism.  
 
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Revision as of 08:32, 15 September 2015

Santa Clara Template for iGEM wiki site

Our Defense System

Of all of the researched mechanisms, the one that stood out as the best candidate was E. coli’s membrane composition change that incorporates cyclopropane fatty acids into the membrane. This mechanism was found to be the single most impactful acid resistant mechanism in this enterobacterium. It involves the increased expression of the cyclopropane fatty acid (CFA) synthase which will catalyze the addition of the methyl group to a monounsaturated fatty acid (UFA), using S-adenosyl methionine (SAM) as a methyl donor. The CFA synthase is a cytosolic protein that reversibly associates with the membrane. These modified phospholipids then incorporate into the membrane and lead to the acid resistant phenotype. The protruding carbon creates a more sterically hindered for weak acids attempting to pass through the membrane.

Sample Image 2

Schematic of cyclopropane fatty acid defense system.

Sample Image 2

Chemical reactions involved in CFA formation.

Rationale behind choosing the CFA system

The CFA acid resistance system offers several advantages over the other systems that we identified in nature:

  • Requires no major alterations in metabolism
  • Does not use precious cellular resources as transporters
  • Only requires two cytosolic proteins
  • Offers versatility for use in other organisms because the cell membrane is well conserved even across gram positive and negative bacterium
  • Found to be the single most effective acid resistance system
  • Not found in any other organism that has been documented
We sought to create a robust system that could be implemented in all bacterium so that regardless of the chassis chosen for a bioreactor, it would be able to increase its aciduricity. We also wanted to find a way to boost the acid resistance of the organism through the addition of a new system opposed to the upregulation of an endogenous system. By adding a new system, we think it should be possible to greatly increase the acid resistance of an organism.