Difference between revisions of "Team:Marburg/Demonstrate"

 
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         alt="Syntex Logo">
 
         alt="Syntex Logo">
 
     </div>
 
     </div>
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  <div>
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    <div style="margin-top: 11vh;">
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    <section class="section">
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      <section class="section">
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    </section>
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        <figure style="text-align: center; margin-left: 25px;">
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    <section class="section" style="padding-top: 1;">
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          <img style="height: 200px; width: 1000px;"
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      <div class="container">
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            src="https://static.igem.org/mediawiki/2019/f/fd/T--Marburg--Timeline_UTEX.svg "
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      <h1 class="title">Demonstrate</h1>
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            alt="Timeline_UTEX">
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         <div class="columns is-desktop">
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          <figcaption style="text-align: center;max-width: 600px; margin-left: 25px;margin: auto;">
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           <div class="column">
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            Fig.1: Time improvements in workflow with cyanobacteria. With our engineered UTEX and our Toolbox we
−
             <p style="margin-bottom: 1rem;">
+
            are able to clone and test constructs in under one week.
−
<br>
+
          </figcaption>
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<br>
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         </figure>
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<b> Strain Engineering</b>
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        <figure style="text-align: center; margin-left: 25px;">
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<br>
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           <img style="height: 560px; width: 1000px;"
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<i>“Started from the bottom, now we here”</i> - Drake
+
             src="https://static.igem.org/mediawiki/2019/a/a0/T--Marburg--Growthcurve_UTEXvsPCC.svg "
−
<br>
+
            alt="Growthcurve_UTEX">
−
<br>
+
          <figcaption style="text-align: center;max-width: 600px; margin-left: 25px;margin: auto;">
−
Our project has three goals. First, we want to establish the cyanobacteria <i>Synechococcus elongatus</i> UTEX 2973 and restore its natural competence. Secondly, we wanted to show the best growing conditions with the goal of standardization for working with UTEX 2973. The final goal is to create a suitable Golden Gate toolbox for our strain and share it with other iGEM teams and researchers. <br>
+
            Fig.2: Growth-curve of <i>Synechococcus elongatus</i> UTEX 2973 in comparison to PCC 7942.
−
 
+
          </figcaption>
−
In our iGEM year we achieved to restore the natural competence of <i>Synechococcus elongatus</i> UTEX 2973, analyzed different <a href="https://2019.igem.org/Team:Marburg/Model">cultivation parameters</a> to accelerate the growth speed including finding the best conditions. Besides that we expanded the Marburg Collection from 2018 and are able to deliver a toolbox of 55 parts with free access to the world. On top of that, we <a href="https://2019.igem.org/Team:Marburg/Labautomation">automated plasmid purification</a> and colony picking via the Opentron OT-2. <br>
+
        </figure>
−
<br>
+
      </section>
−
<b> Toolbox </b>
+
      <section class="section">
−
<br>
+
        <h2 class="subtitle">Strain Engineering</h2>
−
<i>"Great things are not done by impulse, but by a series of small things brought together."</i> – Vincent van Gogh
+
        <p>
−
<br>
+
          <i>“Started from the bottom, now we're here”</i> - <b>Drake</b>
−
<br>
+
        </p>
−
This year we expanded the Marburg Collection from 2018 with 55 new parts to the Marburg Collection 2.0. With our developed workflow we could characterize our <a href="https://2019.igem.org/Team:Marburg/Parts">parts</a> and compare them with a second measurement method: FACS. We added two new features for genome engineering of cyanobacteria: a <a href="https://2019.igem.org/Team:Marburg/Parts">CRISPR/Cpf1</a> guided knockout system as well as a modularized assembly of repair templates for the knock in of genes (M.E.G.A. expansion). This includes integration sites that target conventional neutral sites in cyanobacteria but we also rationally designed two novel integration sites based on RNA-seq data. Additionally, we offer the first MoClo compatible <a href="https://2019.igem.org/Team:Marburg/Parts">shuttle vector</a> for cyanobacteria and characterized gene expression based on that origin of replication. <br>We used our new shuttle vector to build standardized devices for the characterization of BioBricks in cyanobacterial chassis to improve the reproducibility of results and to simplify large scale assemblies. For this we used placeholders, a novel part type that aids in the construction of a larger set of parts by reducing the involved cost and workload significantly. Additionally, we tested our toolbox with PCC 7942 to show that the Marburg Collection 2.0 is also working with similar cyanobacteria. <br> We offer free access to the data of our characterization, enabling the iGEM community and scientists to choose the parts based on this data. To improve the measurement method applicable for cyanobacteria we focused on measurements via luminescence reporters over fluorescence reporters, because of the fact that cyanobacteria emit autofluorescence. This way our results are way more accurate, because of the reduced background noise. The higher accuracy is obviously visible during the measurement of our parts, where we could see a difference of 5x10^5 between the background noise and the signal, which implements that already a small amount of sample has a more intensive signal.  
+
        <p style="margin-top: 1em;">
−
<br>
+
          Our project has three goals. First, we want to establish the cyanobacteria <i>Synechococcus
−
Hereby, we want to encourage the community of young scientists to work with the fastest phototrophic organism <i>Synechococcus elongatus</i> UTEX 2973 because of its high relevance for biotechnological applications.  
+
            elongatus</i> UTEX 2973 and restore its natural competence. Secondly, we wanted to show the best
−
            </p>
+
          growing conditions with the goal of standardization for working with UTEX 2973. The final goal is to
−
          </div>      
+
          create a suitable Golden Gate toolbox for our strain and share it with other iGEM teams and
−
         </div>
+
          researchers.
−
      </div>
+
        </p>
−
    </section>
+
        <p style="margin-top: 1em;">
−
  </div>
+
          In our iGEM year we worked to restore the natural competence of <i>Synechococcus elongatus</i> UTEX
−
 
+
          2973, analyzed different <a href="https://2019.igem.org/Team:Marburg/Model#growth_curve_model">cultivation
 +
            parameters</a>
 +
          to accelerate the growth speed including finding the best conditions. Besides that we expanded the
 +
          Marburg Collection from 2018 and are able to deliver a toolbox of 55 parts with free access to the
 +
          world. On top of that, we <a href="https://2019.igem.org/Team:Marburg/Miniprep"
 +
            target="_blank">automated plasmid
 +
            purification</a> and <a href="https://2019.igem.org/Team:Marburg/Colony_Picking"
 +
            target="_blank">colony picking</a> via the Opentrons OT-2.
 +
        </p>
 +
      </section>
 +
      <section class="section">
 +
        <h2 class="subtitle">Toolbox</h2>
 +
        <p>
 +
          <i>"Great things are not done by impulse, but by a series of small things brought together."</i> –
 +
          <b>Vincent van Gogh</b>
 +
        </p>
 +
        <p style="margin-top: 1em;">
 +
          This year we expanded the Marburg Collection from 2018 with 55 new parts to the Marburg Collection
 +
          2.0.
 +
          With our developed workflow we could characterize our <a
 +
            href="https://2019.igem.org/Team:Marburg/Parts">parts</a> and
 +
          compare them with a second measurement
 +
          method: <a href="https://2019.igem.org/Team:Marburg/Measurement#facs"
 +
            target="_blank">FACS/flow cytometry</a>. We added two new features for genome engineering of
 +
          cyanobacteria: A <a href="https://2019.igem.org/Team:Marburg/Model#growth_curve_model">CRISPR/Cas12a</a>
 +
          guided
 +
          knockout
 +
          system as well as a
 +
          modularized assembly of repair templates for the knock in of genes
 +
          <a href="https://2019.igem.org/Team:Marburg/Description#marburg_collection">(M.E.G.A. expansion)</a>.
 +
          This includes
 +
          integration sites that target conventional neutral sites in cyanobacteria but we also rationally
 +
          designed two novel integration sites based on RNA-seq data. Additionally, we offer the first MoClo
 +
          compatible <a href="https://2019.igem.org/Team:Marburg/Parts">shuttle vector</a> for cyanobacteria and
 +
          characterized gene expression based on that origin of replication. <br>We used our new shuttle vector
 +
          to
 +
          build standardized devices for the characterization of BioBricks in cyanobacterial chassis to improve
 +
          the reproducibility of results and to simplify large scale assemblies. For this we used <a
 +
            href="https://2019.igem.org/Team:Marburg/Description#marburg_collection"
 +
            target="_blank">placeholders</a>, a
 +
          novel part type that aids in the construction of a larger set of parts by reducing the involved cost
 +
          and
 +
          workload significantly. Additionally, we tested our toolbox with PCC 7942 to show that the Marburg
 +
          Collection 2.0 is also working with similar cyanobacteria. <br> We offer free access to the data of
 +
          our
 +
          characterization, enabling the iGEM community and scientists to choose the parts based on this data.
 +
          To
 +
          improve the measurement method applicable for cyanobacteria we focused on measurements via
 +
          luminescence
 +
          reporters over fluorescence reporters, because of the fact that cyanobacteria emit autofluorescence.
 +
          This way our results are way more accurate, because of the reduced background noise. The higher
 +
          accuracy
 +
          is obviously visible during the measurement of our parts, where we could see a difference of
 +
          5x10<sup>5</sup>
 +
          between the background noise and the signal, which implements that already a small amount of sample
 +
          has
 +
          a more intensive signal.
 +
        </p>
 +
        <p style="margin-top: 1em;">
 +
          Hereby, we want to encourage the community of young scientists to work with the fastest phototrophic
 +
          organism <i>Synechococcus elongatus</i> UTEX 2973 because of its high relevance for biotechnological
 +
          applications.
 +
        </p>
 +
      </section>
 +
      <section class="section">
 +
         <h2 class="subtitle">Automation</h2>
 +
        <p>
 +
          We implemented a completely automated cloning workflow in the Opentrons OT-2. We have published our
 +
          complete work and can now provide you with the soft- and hardware to bring you the fully automated
 +
          Colony Picking Unit C.P.U. as well as the plasmid purification protocol. We made as fully accessible
 +
          and
 +
          cheap as possible to give access to as many people as possible.
 +
        </p>
 +
      </section>
 +
    </div>
 
</html>
 
</html>
 
{{Marburg/footer}}
 
{{Marburg/footer}}

Latest revision as of 16:50, 8 December 2019

D E M O N S T R A T E

Timeline_UTEX
Fig.1: Time improvements in workflow with cyanobacteria. With our engineered UTEX and our Toolbox we are able to clone and test constructs in under one week.
Growthcurve_UTEX
Fig.2: Growth-curve of Synechococcus elongatus UTEX 2973 in comparison to PCC 7942.

Strain Engineering

“Started from the bottom, now we're here” - Drake

Our project has three goals. First, we want to establish the cyanobacteria Synechococcus elongatus UTEX 2973 and restore its natural competence. Secondly, we wanted to show the best growing conditions with the goal of standardization for working with UTEX 2973. The final goal is to create a suitable Golden Gate toolbox for our strain and share it with other iGEM teams and researchers.

In our iGEM year we worked to restore the natural competence of Synechococcus elongatus UTEX 2973, analyzed different cultivation parameters to accelerate the growth speed including finding the best conditions. Besides that we expanded the Marburg Collection from 2018 and are able to deliver a toolbox of 55 parts with free access to the world. On top of that, we automated plasmid purification and colony picking via the Opentrons OT-2.

Toolbox

"Great things are not done by impulse, but by a series of small things brought together." – Vincent van Gogh

This year we expanded the Marburg Collection from 2018 with 55 new parts to the Marburg Collection 2.0. With our developed workflow we could characterize our parts and compare them with a second measurement method: FACS/flow cytometry. We added two new features for genome engineering of cyanobacteria: A CRISPR/Cas12a guided knockout system as well as a modularized assembly of repair templates for the knock in of genes (M.E.G.A. expansion). This includes integration sites that target conventional neutral sites in cyanobacteria but we also rationally designed two novel integration sites based on RNA-seq data. Additionally, we offer the first MoClo compatible shuttle vector for cyanobacteria and characterized gene expression based on that origin of replication.
We used our new shuttle vector to build standardized devices for the characterization of BioBricks in cyanobacterial chassis to improve the reproducibility of results and to simplify large scale assemblies. For this we used placeholders, a novel part type that aids in the construction of a larger set of parts by reducing the involved cost and workload significantly. Additionally, we tested our toolbox with PCC 7942 to show that the Marburg Collection 2.0 is also working with similar cyanobacteria.
We offer free access to the data of our characterization, enabling the iGEM community and scientists to choose the parts based on this data. To improve the measurement method applicable for cyanobacteria we focused on measurements via luminescence reporters over fluorescence reporters, because of the fact that cyanobacteria emit autofluorescence. This way our results are way more accurate, because of the reduced background noise. The higher accuracy is obviously visible during the measurement of our parts, where we could see a difference of 5x105 between the background noise and the signal, which implements that already a small amount of sample has a more intensive signal.

Hereby, we want to encourage the community of young scientists to work with the fastest phototrophic organism Synechococcus elongatus UTEX 2973 because of its high relevance for biotechnological applications.

Automation

We implemented a completely automated cloning workflow in the Opentrons OT-2. We have published our complete work and can now provide you with the soft- and hardware to bring you the fully automated Colony Picking Unit C.P.U. as well as the plasmid purification protocol. We made as fully accessible and cheap as possible to give access to as many people as possible.