Difference between revisions of "Template:Virginia/index"

 
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<h1 id="welcome-to-our-glorious-landing-page-">Welcome to our glorious Landing Page!</h1>
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* This page and wiki was built with the help of igem-wikibrick, a tool created by Virginia iGEM 2018
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<p>Heterogeneity of cell populations caused by <span class="tooltip">quorum sensing<span class="shortdef">The ability to detect and to respond to cell population density by gene regulation.</span><span class="longdef" tabindex="1">undefined</span> </span> leads to variability in gene expression that is hard to predict. During biomanufacturing, elevating quorum-induced protein expression will lead to gain of profit. Decreasing this expression can also be beneficial in situations where undesirable <span class="tooltip">biofilms<span class="shortdef">A protective, adhesive matrix of polymers typically produced after quorum activation.</span><span class="longdef" tabindex="1">undefined</span> </span> may form on medical equipment or controlling <span class="tooltip">virulence<span class="shortdef">The likelihood of a microbe to cause disease based on its phenotypic state.</span><span class="longdef" tabindex="1">undefined</span> </span> in bacteria. We will modify the existing bacterial quorum sensing system controlled by the <span class="tooltip">Lsr operon<span class="shortdef">LuxS Regulated (Lsr) operon responsive to AI-2.</span><span class="longdef" tabindex="1">undefined</span> </span> by upregulating the synthesis and excretion of <span class="tooltip">Autoinducer-2<span class="shortdef">A universal signaling molecule used by microorganisms to coordinate group behavior through quorum sensing.</span><span class="longdef" tabindex="1">undefined</span> </span>, a universal quorum molecule. This will increase population-scale AI-2 intake and <span class="tooltip">phosphorylation<span class="shortdef"> The addition of a phosphate group to an organic compound. </span><span class="longdef" tabindex="1">undefined</span> </span> after the initial AI-2 threshold concentrations have been reached to reduce variability in induced gene expression.</p>
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* @link https://github.com/Virginia-iGEM/igem-wikibrick
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<p>A model is used to predict the impacts of manipulating the expression of quorum sensing genes and guide design and experimentation. DNA assembly of a Biobricks containing <span class="tooltip">pLsr<span class="shortdef">The bidirectional promoter of the Lsr Operon.</span><span class="longdef" tabindex="1">undefined</span> </span>, <span class="tooltip">T7 RNA Polymerase<span class="shortdef">An RNA polymerase from the T7 bacteriophage is highly selective for the pT7 promoter.</span><span class="longdef" tabindex="1">undefined</span> </span>, <span class="tooltip">LsrK<span class="shortdef">AI-2 kinase, which catalyzes the phosphorylation of A1-2 to phospho-AI-2.</span><span class="longdef" tabindex="1">undefined</span> </span>, <span class="tooltip">LsrACDB<span class="shortdef">Active import protein for AI-2.</span><span class="longdef" tabindex="1">undefined</span> </span>, <span class="tooltip">LuxS<span class="shortdef">An enzyme closely linked to the production of AI-2.</span><span class="longdef" tabindex="1">undefined</span> </span>, <span class="tooltip">YdgG<span class="shortdef">Active export protein for AI-2.</span><span class="longdef" tabindex="1">undefined</span> </span>, and superfolding Green Fluorescent Protein (<span class="tooltip">sfGFP<span class="shortdef">superfolding Green Fluorescent Protein</span><span class="longdef" tabindex="1">undefined</span> </span>) enables both the enhancement of natural quorum sensing and the quantification of protein activation among the bacteria in a colony. This device will improve the viability of <span class="tooltip">autoinduction<span class="shortdef">The activation of a phenotype without external stimuli.</span><span class="longdef" tabindex="1">undefined</span> </span> as an induction method in industries such as biomanufacturing by decreasing the variability of a cell phenotypes and increasing expression within a single culture to reduce costs leading to an increase in profits. In addition, by insertion of T7 polymerase into a quorum-sensitive region of the genome of E. coli, we will create a chassis with specific, customizable quorum response for engineering and scientific applications.</p>
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* @license MIT
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<article class="banner">
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  <section>
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    <img src="https://static.igem.org/mediawiki/2018/3/3c/T--Virginia--2018_dark.svg" alt="logo">
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    <h3>Microbial Symphony</h3>
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  </section>
 +
</article>
 +
 
 +
<article class="call-to-action">
 +
 
 +
  <section>
 +
    <div class="spacer" style="height: 40vh"></div>
 +
    <div class="card">
 +
      <h2>1.1</h2>
 +
      <p>By transforming these cells with our own synthetic genes, we can produce proteins of interest. This is the foundational goal of biomanufacturing.</p>
 +
      <img src="https://static.igem.org/mediawiki/2018/9/92/T--Virginia--2018_Plasmid-Gallery.svg">
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    </div>
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  </section>
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 +
  <section class="scroll-action">
 +
    <div class="card" id="first-card">
 +
      <h1>1. Background</h1>
 +
      <p>These are normal <em>Escherichia coli.</em> cells. Given nutrients and space, they will grow, producing various proteins.</p>
 +
    </div>
 +
    <div class="spacer" style="height: 10vh"></div>
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    <div class="petri" id="petri-normal">
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    </div>
 +
    <p>
 +
      <strong>Strain:</strong> <span>Unmodified <em>E. coli</em></span>
 +
    </p>
 +
    <p>
 +
      <strong>Division Time:</strong> <span>1 hour</span>
 +
    </p>
 +
    <p>
 +
      <strong>Yield:</strong> <span>N/A</span>
 +
    </p>
 +
    <div class="spacer" style="height: 20vh"></div>
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  </section>
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 +
  <section>
 +
    <div class="spacer" style="height: 40vh"></div>
 +
    <div class="card">
 +
      <h2>1.2</h2>
 +
      <p>
 +
        An important component of these genes is their promoter, which determines how and when they are expressed. There are two common ways genes are induced in biomanufacturing; constitutive and inducible expression.
 +
      </p>
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    </div>
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  </section>
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</article>
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 +
<article id="constitutive-article">
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 +
  <section>
 +
    <div class="spacer" style="height: 50vh"></div>
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    <div class="card" id="constitutive-card">
 +
      <h3>2.1 Constitutive Promoters</h3>
 +
      <!-- <img src="/images/landing_page/lp_wildtype.svg" alt="constitutive expression"></img> -->
 +
      <p>Constitutive promoters result in constant expression. The primary benefit of this "system" is its inherent simplicity - expression will occur with no intervention. A cell will always be producing the protein of interest, from its first division to its last.</p>
 +
      <!-- <button class="constitutive_button">Click to see it in action</button> -->
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    </div>
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    <div class="spacer" style="height: 50vh"></div>
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    <div class="card">
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      <h3>2.3</h3>
 +
      <p>
 +
        Very little protein of interest is produced because the colony isn't given the chance to mature - so many of the cells' resources are committed towards producing the protein that the colony's growth rate is reduced.
 +
      </p>
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    </div>
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    <div class="spacer" style="height: 50vh"></div>
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  </section>
 +
 
 +
  <section class="scroll-action">
 +
    <div class="card">
 +
      <h1>2. Constitutive Expression</h1>
 +
      <p>These <em>E. coli</em> have been modified with a constitutively promoted gene of interest.</p>
 +
    </div>
 +
    <div class="sticky" tabindex="-1">
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      <div class="spacer" style="height: 10vh"></div>
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      <div class="petri" id="petri-constitutive">
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      </div>
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      <p>
 +
        <strong>Strain:</strong> <span><em>E. coli</em> with constitutive expression</span>
 +
      </p>
 +
      <p>
 +
        <strong>Division Time:</strong> <span>3 hours</span>
 +
      </p>
 +
      <p>
 +
        <strong>Yield:</strong> <span>Low</span>
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      </p>
 +
      <div class="spacer" style="height: 20vh"></div>
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    </div>
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    <div class="spacer" style="height: 50vh"></div>
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  </section>
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 +
  <section>
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    <div class="spacer" style="height: 100vh"></div>
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    <div class="card">
 +
      <h3>2.2</h3>
 +
      <p>
 +
        While this is appropriate for experimentation, when optimizing for biomanufacturing, constitutive promoters begin to show their flaws:
 +
        Low yields.
 +
      </p>
 +
    </div>
 +
  </section>
 +
</article>
 +
 
 +
<article id="inducible-article">
 +
  <section>
 +
    <div class="spacer" style="height: 50vh"></div>
 +
    <div class="card">
 +
      <h3>3.1 Inducible Promoters</h3>
 +
      <!-- <img src="/images/landing_page/lp_quorus.svg" alt="induced expression"></img> -->
 +
      <p>Inducible promoters are an improvement over constitutive expression because they give the manufacturer a degree of control over the gene.</p>
 +
    </div>
 +
    <div class="spacer" style="height: 50vh"></div>
 +
    <div class="card">
 +
      <h3>3.3</h3>
 +
      <!-- <img src="/images/landing_page/lp_quorus.svg" alt="induced expression"></img> -->
 +
      <p>As you can see, this results in similar levels of expression to constitutive promoters, given a high enough concentration of inducer. Furthermore, because the colony does not express the gene before the inducer is added, growth happens as quickly as possible; gene expression can be delayed until the colony reaches full size.</p>
 +
    </div>
 +
  </section>
 +
  <section class="scroll-action">
 +
    <div class="card">
 +
      <h1>3. Inducible Expression</h1>
 +
      <p>These <em>E. coli</em> have been modified with an artificially inducible gene of interest.</p>
 +
    </div>
 +
    <div class="sticky" tabindex="-1">
 +
      <div class="spacer" style="height: 10vh"></div>
 +
      <div class="petri" id="petri-inducible">
 +
      </div>
 +
      <p>
 +
        <strong>Strain:</strong> <span>Inducible <em>E. coli</em></span>
 +
      </p>
 +
      <p>
 +
        <strong>Division Time:</strong> <span>1 hours</span>
 +
      </p>
 +
      <p>
 +
        <strong>Yield:</strong> <span>High</span>
 +
      </p>
 +
      <div class="spacer" style="height: 20vh"></div>
 +
    </div>
 +
    <div class="spacer" style="height: 50vh"></div>
 +
  </section>
 +
  <section>
 +
    <div class="spacer" style="height: 100vh"></div>
 +
    <div class="card">
 +
      <h3>3.2 Inducer</h3>
 +
      <p>
 +
        These promoters will only express the protein of interest in the presence of some inducer - such as the artificial inducer IPTG - is added to the culture or bioreactor.
 +
      </p>
 +
      <button class="inducible_button">Add some IPTG</button>
 +
    </div>
 +
    <div class="spacer" style="height: 50vh"></div>
 +
    <div class="card">
 +
      <h3>3.4</h3>
 +
      <p>However, this requires intervention of the biomanufacturer or experimenter during the production cycle, and adds the cost of the inducer to production.</p>
 +
    </div>
 +
    <div class="spacer" style="height: 50vh"></div>
 +
  </section>
 +
</article>
 +
 
 +
<article id="wild-type">
 +
  <section>
 +
    <div class="spacer" style="height: 50vh"></div>
 +
    <div class="card">
 +
      <h3>4.1 Quorum Sensing Genes</h3>
 +
      <p>Quorum Sensing is a natural phenomenon observed in many single-celled organisms. Quorum sensing results in gene expression once the bacterial colony has reached a certain density of cells, often called "colony size".</p>
 +
    </div>
 +
    <div class="spacer" style="height: 50vh"></div>
 +
    <div class="card">
 +
      <h3>4.3 Advantages</h3>
 +
      <p>On paper, this is fantastic. No need to add an inducer because cells just make their own inducer; no need to add an expensive aritificial inducer. There are all the benefits of constitutive and inducible expression; cells are allowed to grow to a colony size appropriate for manufacturing before they begin expressing the gene of interest.</p>
 +
      <p></p>
 +
    </div>
 +
    <div class="spacer" style="height: 30vh"></div>
 +
    <div class="card">
 +
      <h3>4.4 Additional Complications</h3>
 +
      <p>In reality, there are a multitude of problems with quorum sensing.</p>
 +
        <p>Because many quorum sensing molecules do not diffuse through the cell membrane, it is common for cells to have some method for amppfying the intracellular concentration of autoinducer. Most commonly, this is an autoinducer-specific transport protein; this protein is frequently also regulated by the autoinducer, resulting in rapid uptake once quorum is reached.</p>
 +
    </div>
 +
  </section>
 +
  <section class="scroll-action">
 +
    <div class="card">
 +
      <h1>4. Quorum Sensing</h1>
 +
      <p>These <em>E. coli</em> have been modified with a set of wild-type quorum sensing genes.</p>
 +
    </div>
 +
    <div class="sticky" tabindex="-1">
 +
      <div class="spacer" style="height: 10vh"></div>
 +
      <div class="petri" id="petri-qs">
 +
      </div>
 +
      <p>
 +
        <strong>Strain:</strong> <span>Wild-type quorum sensing <em>E. coli</em></span>
 +
      </p>
 +
      <p>
 +
        <strong>Division Time:</strong> <span>1 hour</span>
 +
      </p>
 +
      <p>
 +
        <strong>Yield:</strong> <span>Moderate</span>
 +
      </p>
 +
      <div class="spacer" style="height: 20vh"></div>
 +
    </div>
 +
    <div class="spacer" style="height: 50vh"></div>
 +
  </section>
 +
  <section>
 +
    <div class="spacer" style="height: 100vh"></div>
 +
    <div class="card">
 +
      <h3>4.2 Mechanism</h3>
 +
      <p>Fundamentally, the gene expression almost identically to inducible expression. Some small inducer molecule enters the cell and results in expression of a gene with a promoter corresponding to that inducer. The main difference is that strains that make use of quorum sensing produce their own <em>auto</em>inducer - resulting in a buildup of this autoinducer over time, and eventual, automatic gene expression.</p>
 +
    </div>
 +
    <div class="spacer" style="height: 140vh"></div>
 +
    <div class="card">
 +
        <p>The problem with this is activation frequency. Once quorum is reached, cells will very rapidly drain the extracellular medium of all autoinducer - starving a significant fraction of cells of autoinducer, and preventing them from reaching quorum.</p>
 +
    </div>
 +
    <div class="card">
 +
        <p>In nature, this is an acceptable outcome, but for biomanufacturing, this limits the use and value of quorum sensing.</p>
 +
    </div>
 +
  </section>
 +
</article>
 +
 
 +
<article id="reader-break">
 +
  <section>
 +
    <div class="spacer" style="height: 30vh"></div>
 +
    <div class="card">
 +
      <h1>Is there something better?</h1>
 +
      <p>
 +
        All of these methods for gene expression seem to have drawbracks.
 +
        </p><p>Constitutive expression results in low yields because the organism is not given time to reach a sufficient colony size.</p>
 +
        <p>Inducible expression allows precise control of this growth before expression, but requires use of an expensive inducer molecule.</p>
 +
        <p>Quorum sensing seems to be the best of both worlds - but the low wild-type activation rate again brings yields below levels that can be achieved with inducible expression.</p>
 +
        <div class="spacer" style="height: 30vh"></div>
 +
        <div class="card">
 +
          <h1 class="image">5. Introducing...</h1>
 +
          <img src="https://static.igem.org/mediawiki/2018/4/49/T--Virginia--2018_light.png">
 +
        </div>
 +
  </div></section>
 +
</article>
 +
 
 +
<article id="quorus-article">
 +
  <section>
 +
    <div class="card">
 +
      <p>Quorus is our system which combines the advantages of both inducible expression and quorum sensing - optimizing yield and activation rate without requiring an artificial inducer.</p>
 +
      <p>As you can see, Quorus, once cells reach quorum, has an activation rate nearly as high as IPTG-based expression, and a similar growth rate. Save for the cells activating themselves, they're as good as inducible cells.</p>
 +
      <p>To learn how it all works, scroll down.</p>
 +
    </div>
 +
  </section>
 +
  <section class="scroll-action">
 +
    <div class="sticky" tabindex="-1">
 +
      <div class="spacer" style="height: 10vh"></div>
 +
      <div class="petri" id="petri-qurous">
 +
      </div>
 +
      <p>
 +
        <strong>Strain:</strong> <span>Quorus</span>
 +
      </p>
 +
      <p>
 +
        <strong>Division Time:</strong> <span>1 hours</span>
 +
      </p>
 +
      <p>
 +
        <strong>Yield:</strong> <span>High</span>
 +
      </p>
 +
      <div class="spacer" style="height: 20vh"></div>
 +
    </div>
 +
    <div class="spacer" style="height: 50vh"></div>
 +
  </section>
 +
</article>
 +
 
 +
<article>
 +
<div class="row">
 +
  <div class="grid-selection">
 +
    <h2>Description</h2>
 +
    <p>Learn more about how Quorus works.</p>
 +
    <img src="https://static.igem.org/mediawiki/2018/6/60/T--Virginia--2018_writeups.svg" alt="Modular">
 +
    <a href="https://2018.igem.org/Team:Virginia/Description" class="buttonoverview">Description</a>
 +
  </div>
 +
  <div class="grid-selection">
 +
    <h2>Labwork</h2>
 +
    <p>See how we made it real.</p>
 +
    <img src="https://static.igem.org/mediawiki/2018/5/5c/T--Virginia--2018_labwork.svg" alt="Modular">
 +
    <a href="https://2018.igem.org/Team:Virginia/Overview_Labwork" class="buttonoverview">Labwork</a>
 +
  </div>
 +
  <div class="grid-selection">
 +
    <h2>Parts</h2>
 +
    <p>See our detailed population and cellular dynamics models.</p>
 +
    <img src="https://static.igem.org/mediawiki/2018/0/00/T--Virginia--2018_part_composite.svg" alt="Design">
 +
    <a href="https://2018.igem.org/Team:Virginia/Parts" class="buttonoverview">Parts</a>
 +
  </div>
 +
  <div class="grid-selection">
 +
    <h2>Model</h2>
 +
    <p>See our detailed population and cellular dynamics models.</p>
 +
    <img src="https://static.igem.org/mediawiki/2018/6/6d/T--Virginia--2018_quorus.svg" alt="Design">
 +
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Latest revision as of 03:55, 18 October 2018

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Microbial Symphony

 </section>

</article>

<article class="call-to-action"> 

 <section>

1.1

By transforming these cells with our own synthetic genes, we can produce proteins of interest. This is the foundational goal of biomanufacturing.

     <img src="https://static.igem.org/mediawiki/2018/9/92/T--Virginia--2018_Plasmid-Gallery.svg">

 </section>

 <section class="scroll-action">

1. Background

These are normal Escherichia coli. cells. Given nutrients and space, they will grow, producing various proteins.

Strain: Unmodified E. coli

Division Time: 1 hour

Yield: N/A 

 </section>

 <section>

1.2

An important component of these genes is their promoter, which determines how and when they are expressed. There are two common ways genes are induced in biomanufacturing; constitutive and inducible expression. 

 </section>

</article>

<article id="constitutive-article"> 

 <section>

2.1 Constitutive Promoters

Constitutive promoters result in constant expression. The primary benefit of this "system" is its inherent simplicity - expression will occur with no intervention. A cell will always be producing the protein of interest, from its first division to its last.

2.3

Very little protein of interest is produced because the colony isn't given the chance to mature - so many of the cells' resources are committed towards producing the protein that the colony's growth rate is reduced. 

 </section>
 
 <section class="scroll-action">

2. Constitutive Expression

These E. coli have been modified with a constitutively promoted gene of interest.

Strain: E. coli with constitutive expression

Division Time: 3 hours

Yield: Low 

 </section>

 <section>

2.2

While this is appropriate for experimentation, when optimizing for biomanufacturing, constitutive promoters begin to show their flaws: Low yields. 

 </section>

</article>

<article id="inducible-article"> 

 <section>

3.1 Inducible Promoters

Inducible promoters are an improvement over constitutive expression because they give the manufacturer a degree of control over the gene.

3.3

As you can see, this results in similar levels of expression to constitutive promoters, given a high enough concentration of inducer. Furthermore, because the colony does not express the gene before the inducer is added, growth happens as quickly as possible; gene expression can be delayed until the colony reaches full size.

 </section>
 <section class="scroll-action">

3. Inducible Expression

These E. coli have been modified with an artificially inducible gene of interest.

Strain: Inducible E. coli

Division Time: 1 hours

Yield: High 

 </section>
 <section>

3.2 Inducer

These promoters will only express the protein of interest in the presence of some inducer - such as the artificial inducer IPTG - is added to the culture or bioreactor. 

     <button class="inducible_button">Add some IPTG</button>

3.4

However, this requires intervention of the biomanufacturer or experimenter during the production cycle, and adds the cost of the inducer to production.

 </section>

</article>

<article id="wild-type"> 

 <section>

4.1 Quorum Sensing Genes

Quorum Sensing is a natural phenomenon observed in many single-celled organisms. Quorum sensing results in gene expression once the bacterial colony has reached a certain density of cells, often called "colony size".

4.3 Advantages

On paper, this is fantastic. No need to add an inducer because cells just make their own inducer; no need to add an expensive aritificial inducer. There are all the benefits of constitutive and inducible expression; cells are allowed to grow to a colony size appropriate for manufacturing before they begin expressing the gene of interest.

4.4 Additional Complications

In reality, there are a multitude of problems with quorum sensing.

Because many quorum sensing molecules do not diffuse through the cell membrane, it is common for cells to have some method for amppfying the intracellular concentration of autoinducer. Most commonly, this is an autoinducer-specific transport protein; this protein is frequently also regulated by the autoinducer, resulting in rapid uptake once quorum is reached.

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4. Quorum Sensing

These E. coli have been modified with a set of wild-type quorum sensing genes.

Strain: Wild-type quorum sensing E. coli

Division Time: 1 hour

Yield: Moderate 

 </section>
 <section>

4.2 Mechanism

Fundamentally, the gene expression almost identically to inducible expression. Some small inducer molecule enters the cell and results in expression of a gene with a promoter corresponding to that inducer. The main difference is that strains that make use of quorum sensing produce their own autoinducer - resulting in a buildup of this autoinducer over time, and eventual, automatic gene expression.

The problem with this is activation frequency. Once quorum is reached, cells will very rapidly drain the extracellular medium of all autoinducer - starving a significant fraction of cells of autoinducer, and preventing them from reaching quorum.

In nature, this is an acceptable outcome, but for biomanufacturing, this limits the use and value of quorum sensing.

 </section>

</article>

<article id="reader-break"> 

 <section>

Is there something better?

All of these methods for gene expression seem to have drawbracks.

Constitutive expression results in low yields because the organism is not given time to reach a sufficient colony size.

Inducible expression allows precise control of this growth before expression, but requires use of an expensive inducer molecule.

Quorum sensing seems to be the best of both worlds - but the low wild-type activation rate again brings yields below levels that can be achieved with inducible expression.

5. Introducing...

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</section>

</article>

<article id="quorus-article"> 

 <section>

Quorus is our system which combines the advantages of both inducible expression and quorum sensing - optimizing yield and activation rate without requiring an artificial inducer.

As you can see, Quorus, once cells reach quorum, has an activation rate nearly as high as IPTG-based expression, and a similar growth rate. Save for the cells activating themselves, they're as good as inducible cells.

To learn how it all works, scroll down.

 </section>
 <section class="scroll-action">

Strain: Quorus

Division Time: 1 hours

Yield: High 

 </section>

</article>

<article>

Description

Learn more about how Quorus works.

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Labwork

See how we made it real.

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Parts

See our detailed population and cellular dynamics models.

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Model

See our detailed population and cellular dynamics models.

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Human Practices

All the people we learned from and gave back to.

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Team

Meet the people that put it together.

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Awards

See our achievements!

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