Difference between revisions of "Team:Amsterdam/Results"

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{{Amsterdam}}
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{{Amsterdam/navbar}}
 
<html>
 
<html>
  
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<h2> Project Results</h2>
+
<!-- Overview -->
 +
<section id="carbon_prod" class="wrapper styleBlack">
 +
<header class="major">
 +
<h2>Engineering Stable Carbon Production</h2>
 +
</header>
 +
<div class="container">
 +
<div class="row">                                                
 +
                        <div class="4u">
 +
                        <section>
 +
                              <h3>Background</h3>
 +
                           
 +
                            <p>The driving force of our consortium's romance is the phototrophic carbon-sharing module: an engineered <i>Synechocystis</i> that fixes CO2 and produces compounds that can be used by a chemotroph to create desired end-products like biofuels.
 +
</p>
 +
                              <h3>Aim</h3>
 +
                           
 +
                            <p>Productive relationships need to stand the test of time. Having experienced the perils of instability firsthand, we sought to engineer carbon production in way that would last.
 +
              <h3>Approach</h3>
 +
                           
 +
                            <p> Using genetic engineering strategies guided by modelling results, we used a combination of gene knock-outs and over-expressions to target a pathway that would result in growth-coupled acetate production.
 +
</p>
 +
 +
                            </section>                 
 +
                        </div>
  
−
<p>Here you can describe the results of your project and your future plans. </p>
 
  
−
<h5>What should this page contain?</h5>
+
                        <div class="4u">
−
<ul>
+
                        <section>
−
<li> Clearly and objectively describe the results of your work.</li>
+
             
−
<li> Future plans for the project </li>
+
                              <h3>Results</h3>
−
<li> Considerations for replicating the experiments </li>
+
                           
 +
                            <ul style = "font-family: 'Montserrat', sans-serif">
 +
<li>We engineered growth-coupled acetate production by knocking out the <i>acs</i> gene, showing that this indeed leads to stable acetate production</li>  
 +
<li>We over-expressed the Pta and AckA1 proteins to increase the flux towards acetate formation, but found that the burden this puts on growth comes at a cost.</li>  
 
</ul>
 
</ul>
 +
.</p>
  
 +
                              <h3>Connections</h3>
 +
                    <ul style = "font-family: 'Montserrat', sans-serif">
 +
<li><a href="https://2015.igem.org/Team:Amsterdam/Project/Stability">Stable Romance:</a> Measure stability.</li> 
 +
<li><a href = "https://2015.igem.org/Team:Amsterdam/Software">Engineering Romance:</a> Using software tools to select targets</li>
 +
<li><a href = "https://2015.igem.org/Team:Amsterdam/Project/Phy_param/Synechocysytis">Measuring Romance:</a> Turbidostat experiments.</li>
 +
</ul>
 +
                            </section>                 
  
  
 +
                        </div>
 +
    <div class="4u">
 +
 +
           
 +
         
 +
<figure class ="image fit">
 +
  <img src="https://static.igem.org/mediawiki/2015/6/68/Amsterdam_qp_acetate.png" alt="Acetate Qp and growth">
 +
  <figcaption>Figure 1. - Growth and Q<sub>p</sub> of strain &Delta;acs over 900 hours of continuous culture showing a constant production over the duration of the experiment.</figcaption>
 +
</figure>
  
 +
 +
<figure class ="image fit">
 +
  <img src="https://static.igem.org/mediawiki/2015/3/32/Acetate_plot1.png" alt=" acetate concentrations">
 +
  <figcaption>Figure 2. - Acetate concentrations over time in the variety of engineered <i>Synechocystis</i> strains.</figcaption>
 +
</figure>
  
−
<h4> Project Achievements </h4>
 
  
−
<p>You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.</p>
+
                        </div>
 +
  
−
<ul>
+
 
−
<li>A list of linked bullet points of the successful results during your project</li>
+
                       
−
<li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li>
+
                  </div>
 +
</div>
 +
</section>
 +
 
 +
 
 +
    <!-- Overview -->
 +
<section id="algorithms" class="wrapper style6">
 +
<header class="major">
 +
<h3>Algorithms</h3>
 +
</header>
 +
<div class="container">
 +
<div class="row">                                                
 +
                        <div class="3u">
 +
 
 +
                        <section>
 +
                              <h3>Background</h3>
 +
                           
 +
                            <p>In our project we focussed on the <a href="https://2015.igem.org/Team:Amsterdam/Project/Stability">stability </a> in different ways. One way is genetic stability, which we needed to engineer a stable producing strain. Another was the stability of our consortium itself. We needed an auxotrophic organism in order to be able to create a strong interdependent consortium.
 +
</p>
 +
                              <h3>Aim</h3>
 +
                           
 +
                            <p>We need to find out what genes we could knock out to create a <u>stable</u> carbon producer. We also need to know what genes we should knock out in order to make an auxotrophic organism.
 +
</p>
 +
 
 +
</section>              
 +
                        </div>
 +
 
 +
 
 +
                        <div class="3u">
 +
                        <section>
 +
                              <h3>Approach</h3>
 +
 
 +
                            <p>Here we present to you two novel algorithms which work with genome-scale FBA models. They can be used for any organism for which a genome-scale FBA model is available. One algorithm, the Stable Compound Generator, searches for ways to make a strain genetically stable produce carbon compounds. The second algorithm searches for ways to create an auxotrophic strain. Therefore we called it the Auxotrophy Sniper.
 +
</p>
 +
</section>
 +
</div>
 +
<div class="3u">
 +
<section>
 +
                              <h3>Results</h3>
 +
                           
 +
                            <ul style = "font-family: 'Montserrat', sans-serif">
 +
<li>We have created the Stable Compound Generator which find ways to stably produce a carbon compound in any organism.</li>  
 +
<li>We have created the Auxotrophy Sniper, which is able to find ways to crate an auxotrophic organism.</li>
 +
<li>With the first algorithm we found a list products which could be stably produced by <i>Synechocystis</i>. We chose acetate from the list.</li>
 +
<li>With the second algorithm we found out that it is possible to make a <i>Synechocystis</i> strain dependent on argenine, thus creating an auxotroph. </li>  
 
</ul>
 
</ul>
 +
</p>
 +
</section>   
 +
                                       
 +
                        </div>
 +
                            <div class="3u">
 +
                                <section>
 +
                                        <h3>Connections</h3>
 +
                                    <p>
 +
                                          <h3></h3>
 +
                    <p>Sometimes modellers tend to be the lone wolfs in a project. We didn't want this to happen, so there are some clear connections between the tools we created with modelling and the wet lab. Initially the need to search for compounds which could be produced genetically stable, came from the wet lab, where we saw that most producing strains are <a href="https://2015.igem.org/Team:Amsterdam/Project/Phy_param/Synechocysytis">unstable</a>. Before we even started <a href="https://2015.igem.org/Team:Amsterdam/Project/Eng_rom/Photosyn_car"> engineering <i>Synechocystis</i></a>, we wanted to find out whether we could produce a compound genetically <a href="https://2015.igem.org/Team:Amsterdam/Project/Stability">stable</a>. This is where the Stable Compound Generater comes in. We also needed to <a href="https://2015.igem.org/Team:Amsterdam/Project/Eng_rom/Dependecies">engineer an auxotroph</a> in order to to use serial propagations of consortia in <a href="https://2015.igem.org/Team:Amsterdam/Project/emulsions">emulsions</a> to find a more robust consortium. Both algorithms provided information which was really used in the lab.</p>
 +
                               
 +
                                    </p>
 +
                                </section>
 +
                            </div> <!-- 6u -->
 +
               
 +
                        </div>
 +
                        </div>
 +
</div>
 +
                                </section>
  
  
  
−
<h4>Inspiration</h4>
+
      <!-- Overview -->
−
<p>See how other teams presented their results.</p>
+
<section id="Syn_phys" class="wrapper styleBlack">
−
<ul>
+
<header class="major">
−
<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
+
<h2> <i>Synechocystis</i> physiological parameters</h2>
−
<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
+
</header>
−
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
+
<div class="container">
 +
<div class="row">                                                
 +
                        <div class="3u">
 +
                        <section>
 +
                              <h3>Background</h3>
 +
                           
 +
                            <p>Estimating physiological parameters is necessary for building meaningful models that can help us to rationally design our consortium. In addition, production stability can be assessed by estimating these parameters over a long cultivation periods.
 +
</p>
 +
                              <h3>Aim</h3>
 +
                           
 +
                            <p>We aim to use different cultivation strategies to characterize growth and production rates of carbon producing <i>Synechocystis</i> strains, while assessing their stability.
 +
 
 +
</p>
 +
              <h3>Approach</h3>
 +
                           
 +
                            <p> We have used a turbidostat cultivation system to determine the stability of the physiological parameters
 +
</p>
 +
 +
                            </section>                  
 +
                        </div>
 +
 
 +
 
 +
                        <div class="3u">
 +
                        <section>
 +
             
 +
                              <h3>Results</h3>
 +
                           
 +
                            <ul style = "font-family: 'Montserrat', sans-serif">
 +
<li>We have demonstrated the instability of classical engineering strategies. As shown in figure 1 the strain SAA023 loses the ability to produce lactate after 300 hours of continuous cultivation.</li>  
 +
<li>We show that the Q<sub>p</sub> in the growth coupled producer &Delta;acs remains constant over that time (figure 2).</li>
 +
<li>We have determined Q<sub>p</sub> and growth rates for six <i>Synechocystis</i> constructs (Table 1).</li>  
 
</ul>
 
</ul>
 +
.</p>
  
−
</div>
+
                              <h3>Connections</h3>
−
</html>
+
                    <ul style = "font-family: 'Montserrat', sans-serif">
 +
<li><a href="https://2015.igem.org/Team:Amsterdam/Project/Stability">Stable Romance:</a> Measure stability.</li> 
 +
<li><a href = "https://2015.igem.org/Team:Amsterdam/Project/Eng_rom/Photosyn_car">Engineering Romance:</a> Estimate parameters of new strains.</li>
 +
<li><a href = "https://2015.igem.org/Team:Amsterdam/Project/Simulations">Simulating Romance:</a> Provide parameters.</li>
 +
</ul>
 +
                            </section>                 
 +
                        </div>
 +
 
 +
                        <div class="6u">
 +
<div class="row">                                                
 +
                        <div class="6u">
 +
<figure class ="image fit">
 +
  <img src="https://static.igem.org/mediawiki/2015/3/3b/Amsterdam_qp_lactate.png" alt="Lactate Qp and growth">
 +
  <figcaption>Figure 1. - Growth and Q<sub>p</sub> of strain SAA023 over 900 hours of continuous culture showing the drop in production.</figcaption>
 +
</figure>
 +
 
 +
 
 +
                        </div>
 +
          <div class="6u">
 +
<figure class ="image fit">
 +
  <img src="https://static.igem.org/mediawiki/2015/6/68/Amsterdam_qp_acetate.png" alt="Acetate Qp and growth">
 +
  <figcaption>Figure 2. - Growth and Q<sub>p</sub> of strain &Delta;acs over 900 hours of continuous culture showing a constant production over the duration of the experiment.</figcaption>
 +
</figure>
 +
 
 +
                        </div>
 +
<div class="8u">
 +
<figure class ="image fit">
 +
  <img src="https://static.igem.org/mediawiki/2015/f/f8/Amsterdam_table_qp.png" alt=" Qp and growth">
 +
  <figcaption>Table 1. - Estimated Growth and Q<sub>p</sub> for different <i>Synechocystis</i> strains.</figcaption>
 +
</figure>
 +
 
 +
                        </div>
 +
                        </div>
 +
 
 +
 
 +
                       
 +
                  </div>
 +
</div>
 +
</section>

Revision as of 02:17, 19 September 2015

iGEM Amsterdam 2015

Engineering Stable Carbon Production

Background

The driving force of our consortium's romance is the phototrophic carbon-sharing module: an engineered Synechocystis that fixes CO2 and produces compounds that can be used by a chemotroph to create desired end-products like biofuels.

Aim

Productive relationships need to stand the test of time. Having experienced the perils of instability firsthand, we sought to engineer carbon production in way that would last.

Approach

Using genetic engineering strategies guided by modelling results, we used a combination of gene knock-outs and over-expressions to target a pathway that would result in growth-coupled acetate production.

Results

  • We engineered growth-coupled acetate production by knocking out the acs gene, showing that this indeed leads to stable acetate production
  • We over-expressed the Pta and AckA1 proteins to increase the flux towards acetate formation, but found that the burden this puts on growth comes at a cost.
.

Connections

Acetate Qp and growth
Figure 1. - Growth and Qp of strain Δacs over 900 hours of continuous culture showing a constant production over the duration of the experiment.
acetate concentrations
Figure 2. - Acetate concentrations over time in the variety of engineered Synechocystis strains.

Algorithms

Background

In our project we focussed on the stability in different ways. One way is genetic stability, which we needed to engineer a stable producing strain. Another was the stability of our consortium itself. We needed an auxotrophic organism in order to be able to create a strong interdependent consortium.

Aim

We need to find out what genes we could knock out to create a stable carbon producer. We also need to know what genes we should knock out in order to make an auxotrophic organism.

Approach

Here we present to you two novel algorithms which work with genome-scale FBA models. They can be used for any organism for which a genome-scale FBA model is available. One algorithm, the Stable Compound Generator, searches for ways to make a strain genetically stable produce carbon compounds. The second algorithm searches for ways to create an auxotrophic strain. Therefore we called it the Auxotrophy Sniper.

Results

  • We have created the Stable Compound Generator which find ways to stably produce a carbon compound in any organism.
  • We have created the Auxotrophy Sniper, which is able to find ways to crate an auxotrophic organism.
  • With the first algorithm we found a list products which could be stably produced by Synechocystis. We chose acetate from the list.
  • With the second algorithm we found out that it is possible to make a Synechocystis strain dependent on argenine, thus creating an auxotroph.

Connections

Sometimes modellers tend to be the lone wolfs in a project. We didn't want this to happen, so there are some clear connections between the tools we created with modelling and the wet lab. Initially the need to search for compounds which could be produced genetically stable, came from the wet lab, where we saw that most producing strains are unstable. Before we even started engineering Synechocystis, we wanted to find out whether we could produce a compound genetically stable. This is where the Stable Compound Generater comes in. We also needed to engineer an auxotroph in order to to use serial propagations of consortia in emulsions to find a more robust consortium. Both algorithms provided information which was really used in the lab.

Synechocystis physiological parameters

Background

Estimating physiological parameters is necessary for building meaningful models that can help us to rationally design our consortium. In addition, production stability can be assessed by estimating these parameters over a long cultivation periods.

Aim

We aim to use different cultivation strategies to characterize growth and production rates of carbon producing Synechocystis strains, while assessing their stability.

Approach

We have used a turbidostat cultivation system to determine the stability of the physiological parameters

Results

  • We have demonstrated the instability of classical engineering strategies. As shown in figure 1 the strain SAA023 loses the ability to produce lactate after 300 hours of continuous cultivation.
  • We show that the Qp in the growth coupled producer Δacs remains constant over that time (figure 2).
  • We have determined Qp and growth rates for six Synechocystis constructs (Table 1).
.

Connections

Lactate Qp and growth
Figure 1. - Growth and Qp of strain SAA023 over 900 hours of continuous culture showing the drop in production.
Acetate Qp and growth
Figure 2. - Growth and Qp of strain Δacs over 900 hours of continuous culture showing a constant production over the duration of the experiment.
Qp and growth
Table 1. - Estimated Growth and Qp for different Synechocystis strains.