Team:Freiburg/Content/Results/Light system

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<section id="Light system">
 
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<h1>The light system</h1>
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<a href="https://2014.igem.org/Team:Freiburg/Results/The_combination">See the Results of the Combination of our Systems</div>
 +
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<section id="Light-system">
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<h1>The Light System</h1>
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<p>One advantage of our system, the spatial resolution by infecting only distinct areas in a mammalian cell culture with our viral vector, was archived by the use of different light systems. We induced the expression of the mCAT-1 receptor in non-murine cell lines by illumination them with light of a special wave length. Since, the viral vector only infect cells that express the mCAT-1 receptor on their surfaces, we transferred genes into distinct areas of an otherwise homogenous cell layer using this principle.
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<p> Light inducible systems allow spatial and temporal control over gene expression.
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</p>
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    Our goal is the expression of the mCAT-1 receptor through a light inducible
 +
    system to allow spatiotemporal control of the virus entry into the target cells.
 +
    We used two different systems: expression is induced by light of the blue and
 +
    red wavelength range. To test the performance of the light systems, we used an
 +
    assay where the cells express secreted embryonic alkaline phosphatase (SEAP).
 +
    The amount of produced SEAP can be measured by its enzymatic activity. It
 +
    breaks down the substrate para-nitrophenylphosphate to the yellow para-nitrophenol,
 +
    which can be measured with a spectrophotometer.
 +
</p>
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<h2>Function of the light systems</h2>
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<h2 id="Results-LightSystem-Function">Function of the Light System</h2>
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<p> As the function of the light system, we use, is really important for the function of our whole system, we tested two different light systems. The red light system is based on…. whereas the blue light system uses… blubb
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<div class="row category-row">
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</p>
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  <div class="col-sm-6">
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<p>Since, it is known that the red light system works better in chinese hamster ovary (CHO) cells, we used this cell line to test the function of this light system. In order to investigate the functionality of the blue light system, we tested both, CHO cells and human embryonic kidney (HEK293) cells. As reporter the gene seap was transfected together with the genes for the light systems (red light system: PKM006, PKM078; blue light system: PKM292, PKM297, PKM084). A SEAP-assay was performed after illumination. Regarding SEAP concentrations in the supernatant of each sample two important findings were archived. First, the blue light system works much more efficient than the red light system; and the blue light system works better with HEK cells than with CHO cells.  
+
    <p> Because the proper function of the light system is important for our project,
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</p>
+
      we tested two different light systems. The red light system is based on the protein
 +
      phytochrome B (PhyB) from Arabidopsis thaliana and is activated by light around 660 nm,
 +
      whereas the blue light system contains a protein from Avena sativa (AsLOV2) which
 +
      requires 465 nm light for activation.
 +
    </p>
 +
    <p> To test the function of the red light system, we used Chinese hamster ovary cells (CHO)
 +
      that are known to work better for this purpose than other cell lines. For the blue
 +
      light system, we tested CHO cells and human embryonic kidney cells (HEK-293T).
 +
      As reporter the gene seap was transfected together with the genes required for
 +
    the implementation of the blue and red light inducible systems.
 +
      A SEAP-assay was performed after illumination. Measuring SEAP concentrations in the
 +
      supernatant lead us two two important conclusions: First, the blue light system works
 +
      more efficient than the red light system; second, the blue light system works better
 +
      with HEK-293T cells than with CHO cells (Fig. 1).
 +
    </p>
 +
    <p>
 +
      The plasmids required for the implementation of the light inducible SEAP expression are:
 +
    </p>
 +
    Red light system:
 +
    <ul>
 +
      <li>pKM022: SV40_PhyB-VP16_IRES_TetR-PIF16<br/ > constitutive expression of PhyB-VP16 and TetR-PIF6</li>
 +
      <li>pKM006: tetO-CMVmin_SEAP<br/ > SEAP under control of the tet operon</li>
 +
    </ul>
 +
    Blue light system:
 +
    <ul>
 +
      <li>pKM292: SV40_Gal4BD-LOV<br/ > constitutive expression of Gal4BD-LOV</li>
 +
      <li>pKM297: SV40_ePDZ-VP16<br/ > constitutive expression of ePDZ-VP16</li>
 +
      <li>pKM084: Gal4UAS-CMVmin_SEAP<br/ > SEAP under control of the Gal4 upstream activating sequence</li>
 +
    </ul>
 +
    <p>
 +
      <a href="https://2014.igem.org/Team:Freiburg/Project/The_light_system">
 +
        For more information about the light systems click here!
 +
      </a>
 +
    </p>
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<figure>
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<a href="https://static.igem.org/mediawiki/2014/e/ef/Freiburg2014_results_light_systems_compared.png"> <!-- ORGINAL -->
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<img src="https://static.igem.org/mediawiki/2014/e/ef/Freiburg2014_results_light_systems_compared.png"> <!-- Thumbnail -->
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</a>
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  </div>
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<figcaption>
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  <div class="col-sm-6">
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<p class="header">Test</p>
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    <figure>
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</figcaption>
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      <a href="https://static.igem.org/mediawiki/2014/e/ef/Freiburg2014_results_light_systems_compared.png">  
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         </figure>
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        <img src="https://static.igem.org/mediawiki/2014/e/ef/Freiburg2014_results_light_systems_compared.png">
 +
      </a>
 +
      <figcaption>
 +
        <p class="header">
 +
          Figure 1: Comparison of the blue light system in CHO cells and in HEK-293T cells and
 +
          comparison to the red light system in CHO cells.
 +
        </p>
 +
        <p class="desc">
 +
          To test the efficiency of the light system, SEAP expression after illumination was determined.
 +
          The SEAP assay was performed 24 hours after light exposure.
 +
          <a href="https://2014.igem.org/Team:Freiburg/Notebook/Labjournal#MirjaHarms_Red_light">Labjournal</a>
 +
         </p>
 +
      </figcaption>
 +
    </figure>
 +
  </div>
 +
</div>
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<p>The blue light system is very effective in activating an existing reporter, like the gene for seap, brought into the cell before illumination. As we know that the duration of illuminating the system with blue light (452 nm) is critical for its efficiency, we tested various time spans for illumination. Our results indicate that five hours of illumination lead to the highest activation level of SEAP expression.
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<div class="row category-row">
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</p>
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  <div class="col-sm-6">
 +
    <figure>
 +
      <a href="https://static.igem.org/mediawiki/2014/4/4f/Freiburg2014_results_blue_light_system_different_illuminations_seap.png">
 +
        <img src="https://static.igem.org/mediawiki/2014/4/4f/Freiburg2014_results_blue_light_system_different_illuminations_seap.png">
 +
      </a>
 +
      <figcaption>
 +
        <p class="header">
 +
          Figure 2: Efficiency of the blue light system using different time intervals of illumination.
 +
        </p>
 +
        <p class="desc">
 +
          To test the efficiency of the light system, SEAP expression after illumination was determined.
 +
          The SEAP assay was performed 24 hours after light exposure. Cells were incubated with
 +
          blue light for 1 hour, 2.5 hours and 5 hours.
 +
          <a href="https://2014.igem.org/Team:Freiburg/Notebook/Labjournal#MirjaHarms_Blue_light">Labjournal</a>
 +
        </p>
 +
      </figcaption>
 +
    </figure>
 +
  </div>
 +
  <div class="col-sm-6">
 +
    <p>
 +
      The blue light system is effective in activating the reporter SEAP which was introduced
 +
      into the cells before illumination by transfections. As we know
 +
      that the duration of illuminating the system with blue light (452 nm) is critical
 +
      for its efficiency, we tested various time intervals for illumination. Our results
 +
      indicate that five hours of illumination lead to the highest level of SEAP expression (Fig. 2).
 +
    </p>
 +
    <p>
 +
      Another important finding was the specificity of the blue light system.
 +
      In the dark controls, we found almost no activation of the SEAP reporter,
 +
      leading to a very low background level in our system.
 +
    </p>
 +
  </div>
 +
</div>
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<figure>
 
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<a href="https://static.igem.org/mediawiki/2014/4/4f/Freiburg2014_results_blue_light_system_different_illuminations_seap.png"> <!-- ORGINAL -->
 
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<img src="https://static.igem.org/mediawiki/2014/4/4f/Freiburg2014_results_blue_light_system_different_illuminations_seap.png"> <!-- Thumbnail -->
 
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</a>
 
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<figcaption>
 
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<p class="header">Test</p>
 
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</figcaption>
 
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        </figure>
 
-
 
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<p>Another important finding was the specificity of the blue light system. As we always have a dark control in our experiments, we determined almost no activation of the SEAP reporter in these samples leading to a very low background level in our system.
 
-
</p>
 
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<h2>Light induced receptor/ blue light</h2>
 
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<p>In order to generate viral induced patterns in a homogenous cell culture, we combined the spatial resolution of the light system with the specificity of viral receptor interactions. Since our viral vector is specific for the mCAT-1 receptor that is under normal conditions only expressed in murine cells, we linked this specific receptor to the blue light system and transfected non-murine cells with this receptor combination. Mammalian cells which contain the inducible receptor expressed mCAT-1 after illumination. Cells expressing the receptor are visible by co-expression of mCherry. They were infected with our viral vector leading to EGFP expression. Cells of the same cell culture that were not illuminated were not able to express the receptor leading to no infection with our viral vector.
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<h2 id="Results-LightSystem-SEAP-as-Reporter">SEAP as a Reporter</h2>
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</p>
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+
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<figure>
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<a href="https://static.igem.org/mediawiki/2014/a/ac/Freiburg2014_Results_light_pos_neg_rez_virus.jpg"> <!-- ORGINAL -->
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<img class="img-no-pad fig-full-width" src="https://static.igem.org/mediawiki/2014/a/ac/Freiburg2014_Results_light_pos_neg_rez_virus.jpg"> <!-- Thumbnail -->
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</a>
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<figcaption>
+
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<p class="header"></p>
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</figcaption>
+
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        </figure>
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-
 
+
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<p>As we know that the transient mCAT-1 receptor that was not linked to a light system needs 24 hours for expression in HEK293 cells, we determined the expression time of the receptor that was induced by blue light after illumination for five hours. We used a receptor that was labeled with both, mCherry and an HA-tag (p14ls_003), for analysis with Western blot and fluorescence microscopy. The results show that the receptor had an expression peak at 24 hours after beginning of illumination. 
+
-
</p>
+
-
<figure>
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<p>
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<a href="https://static.igem.org/mediawiki/2014/b/bb/Freiburg2014-10-14_bluel-kinetic-microscopy.jpg"> <!-- ORGINAL -->
+
In order to test the capability of the blue light system for pattern generation, we transfected HEK-293T cells with the appropriate plasmids (PKM292 and PKM297) using seap as a reporter gene (PKM084). Cells were transfected in suspension and seeded on 96 well plates afterwards. Single wells of these plates were covered with a photo mask that prevents exposure to light. All other wells on the plate were illuminated to induce SEAP expression. SEAP that was secreted into the culture medium leads to a colour change from red to yellow after addition of pNPP (chromogenic substrate) by its phosphorylation.
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<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/b/bb/Freiburg2014-10-14_bluel-kinetic-microscopy.jpg"> <!-- Thumbnail -->
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</p>
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</a>
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<figcaption>
+
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<p class="header"></p>
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-
</figcaption>
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        </figure>
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<h2>SEAP as reporter</h2>
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<p>
 +
First we generated a coarse pattern realizing that scattered light also reached wells that were close to the illuminated ones, leading to an unclear pattern. However, the rough shape of the desired pattern is discernible (Fig. 3A, B).
 +
</p>
-
<p>In order to test the capability of the blue light system for pattern generation, we transfected HEK293 cells with the appropriate plasmids (PKM292 and PKM297) using seap as a reporter gene (PKM084). Cells were transfected in suspension and seeded on 96 well plates afterwards. Single wells of these plates were covered with a photo mask that prevents illumination with blue light. Since, all other wells on the plate were exposed to the blue light, SEAP expression was induced in these wells. SEAP that was secreted into the culture medium leads to colour changing from red to yellow after addition of pNPP (chromogenic substrate) by its phosphorylation.
+
<p>
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</p>
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Since it was essential to completely prevent light exposure on covered plates, we tested 96 well plates with optically separated wells. We could generate patterns with sharp contours (Fig. 3C, D).
 +
</p>
 +
 
 +
<p>
 +
The colour change of pNPP in cell culture medium could be measured at 405 nm in a plate reader. Setting a distinct threshold made pattern recognition easier.  
 +
</p>
-
<p>First we generated a coarse pattern (fig.) realizing that by scattered light also covered wells close to illuminated wells were exposed to light. This was leading to an unclear pattern. However, we managed to give an idea of the desired pattern.
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<figure class="fig-full-width">
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</p>
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  <a href="https://static.igem.org/mediawiki/2014/3/31/Freiburg2014_Results_light_QR_96.jpg">  
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    <img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/3/31/Freiburg2014_Results_light_QR_96.jpg">
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<div class="row category-row">
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  </a>
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<div class="col-sm-6">
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  <figcaption>
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<figure>
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    <p class="header">
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<a href="https://static.igem.org/mediawiki/2014/9/92/Freiburg2014_Results_light_herzchen_rot.JPG.jpg"> <!-- ORGINAL -->
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      Figure 3: Generation of patterns on 96-well plates.
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<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/9/92/Freiburg2014_Results_light_herzchen_rot.JPG"> <!-- Thumbnail -->
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    </p>
-
</a>
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    <p class="desc">
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<figcaption>
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      HEK-293T cells were transfected with the blue light system (PKM292, PKM297) and
-
<p class="header"></p>
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      light induced SEAP as reporter (PKM084). Single wells were covered with a photo
-
</figcaption>
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      mask to prevent cell from light exposure. 24 hours after illumination with blue
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        </figure>
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      light, a SEAP assay was performed, leading to a colour change of wells containing SEAP.
-
</div>
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      (A) Photo mask in heart form, (B) pseudocoloring done at a computer,
 +
      (C) the same experiment was performed with optically separated wells,
 +
      (D) the contrast was increased and the colours of the wells were changed
 +
      at a computer. All wells together are forming the word: IGEM.
 +
      <a href="https://2014.igem.org/Team:Freiburg/Notebook/Labjournal#MirjaHarms_QR_IGEM">Labjournal 1</a>
 +
      <a href="https://2014.igem.org/Team:Freiburg/Notebook/Labjournal#MirjaHarms_QR_Herz">Labjournal 2</a>
 +
    </p>
 +
  </figcaption>
 +
</figure>
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<div class="col-sm-6">
 
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<figure>
 
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<a href="https://static.igem.org/mediawiki/2014/1/1e/Freiburg2014_Results_light_herzchen_blau.jpg.jpg"> <!-- ORGINAL -->
 
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<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/1/1e/Freiburg2014_Results_light_herzchen_blau.jpg"> <!-- Thumbnail -->
 
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</a>
 
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<figcaption>
 
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<p class="header"></p>
 
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</figcaption>
 
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        </figure>
 
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</div>
 
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</div>
 
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<p>Since, it was essential to completely prevent light exposure on covered plates we tested 96 well plates with optical separated wells. As shown in fig. we could generate patterns with sharp contours.
+
 
-
</p>
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<p>
 +
  For generating QR codes with our method 384-well plates were used. Since we had a collaboration
 +
  with the iGEM Team Aachen 2014, they made a photo mask for a QR code for us fitting on these plates (Fig. 4).
 +
  <a href="https://2014.igem.org/Team:Freiburg/Team/Collaboration#Team-Collaboration-Aachen">Collaborations</a>.
 +
</p>
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<div class="row category-row">
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<figure class="fig-full-width">
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<div class="col-sm-6">
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  <a href="https://static.igem.org/mediawiki/2014/b/b4/Freiburg2014_Results_light_QR_384.jpg">  
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<figure>
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    <img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/b/b4/Freiburg2014_Results_light_QR_384.jpg">  
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<a href="https://static.igem.org/mediawiki/2014/7/7f/Freiburg2014-09-03-QR-iGEM-red.jpg"> <!-- ORGINAL -->
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  </a>
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<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/7/7f/Freiburg2014-09-03-QR-iGEM-red.jpg"> <!-- Thumbnail -->
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  <figcaption>
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</a>
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    <p class="header">
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<figcaption>
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      Figure 4: Generation of a QR code on a 384 well plate.
-
<p class="header"></p>
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    </p>
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</figcaption>
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    <p class="desc">
-
        </figure>
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      HEK-293T cells were transfected with the blue light system (PKM292, PKM297)
-
</div>
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      and light induced SEAP as reporter (PKM084). Single wells were covered with a photo mask
-
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      to prevent cell from light exposure using a photo mask for a QR code (made by iGEM Team Aachen 2014).
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<div class="col-sm-6">
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      24 hours after illumination with blue light a SEAP assay was performed leading to colour
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<figure>
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      changing of wells containing SEAP. Only a part of the plate was used. (A) Photo mask
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<a href="https://static.igem.org/mediawiki/2014/9/9d/2014-09-03_QR-iGEM_blue.jpg"> <!-- ORGINAL -->
+
      for QR code generation, (B) Picture of the 384-well plate after the SEAP assay, (C) the
-
<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/9/9d/2014-09-03_QR-iGEM_blue.jpg"> <!-- Thumbnail -->
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      same plate with contrast of the colours increased at a computer.
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</a>
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      <a href="https://2014.igem.org/Team:Freiburg/Notebook/Labjournal#MirjaHarms_QR_384">Labjournal</a>
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<figcaption>
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    </p>
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<p class="header"></p>
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  </figcaption>
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</figcaption>
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</figure>
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        </figure>
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</div>
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</div>
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-
+
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<p>Colour changing of pNPP in cell culture medium could be measured at 405 nm in a plate reader. Calculating the gradient of light absorption we also generated patterns using a data matrix (Excel) by setting a distinct threshold, thus making pattern recognition easier.
+
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</p>
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<h2>MuLV SEAP</h2>
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</section>
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</section>
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<div class="col-sm-6">
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<div class="container-fluid" style="float: left">
 +
<div style="position: relative; float: right; margin-top: 4px;">
 +
<a href="https://2014.igem.org/Team:Freiburg/Results/Receptor">Go back to The Receptor</div>
 +
<div style="position: relative; float: left;"> <img class="img-no-border" style="max-width: 50px; margin-top:5px;" src=" https://static.igem.org/mediawiki/2014/4/44/Freiburg2014_Navigation_Arrow_rv.png">  <!-- Pfeil rv--></a></div>
 +
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 +
</div>
 +
<div class="col-sm-6">
 +
<div class="container-fluid" style="float: right">
 +
<div style="position: relative; float: left; margin-top: 4px;">
 +
<a href="https://2014.igem.org/Team:Freiburg/Results/The_combination">See the Results of the Combination of our Systems</div>
 +
<div style="position: relative; float: right;"> <img class="img-no-border" style="max-width: 50px; margin-top:5px;" src=" https://static.igem.org/mediawiki/2014/9/95/Freibur2014_pfeilrechts.png">  <!-- Pfeil fw--></a></div>
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Latest revision as of 03:24, 18 October 2014

The AcCELLerator

The Light System

Light inducible systems allow spatial and temporal control over gene expression. Our goal is the expression of the mCAT-1 receptor through a light inducible system to allow spatiotemporal control of the virus entry into the target cells. We used two different systems: expression is induced by light of the blue and red wavelength range. To test the performance of the light systems, we used an assay where the cells express secreted embryonic alkaline phosphatase (SEAP). The amount of produced SEAP can be measured by its enzymatic activity. It breaks down the substrate para-nitrophenylphosphate to the yellow para-nitrophenol, which can be measured with a spectrophotometer.

Function of the Light System

Because the proper function of the light system is important for our project, we tested two different light systems. The red light system is based on the protein phytochrome B (PhyB) from Arabidopsis thaliana and is activated by light around 660 nm, whereas the blue light system contains a protein from Avena sativa (AsLOV2) which requires 465 nm light for activation.

To test the function of the red light system, we used Chinese hamster ovary cells (CHO) that are known to work better for this purpose than other cell lines. For the blue light system, we tested CHO cells and human embryonic kidney cells (HEK-293T). As reporter the gene seap was transfected together with the genes required for the implementation of the blue and red light inducible systems. A SEAP-assay was performed after illumination. Measuring SEAP concentrations in the supernatant lead us two two important conclusions: First, the blue light system works more efficient than the red light system; second, the blue light system works better with HEK-293T cells than with CHO cells (Fig. 1).

The plasmids required for the implementation of the light inducible SEAP expression are:

Red light system:
  • pKM022: SV40_PhyB-VP16_IRES_TetR-PIF16
    constitutive expression of PhyB-VP16 and TetR-PIF6
  • pKM006: tetO-CMVmin_SEAP
    SEAP under control of the tet operon
Blue light system:
  • pKM292: SV40_Gal4BD-LOV
    constitutive expression of Gal4BD-LOV
  • pKM297: SV40_ePDZ-VP16
    constitutive expression of ePDZ-VP16
  • pKM084: Gal4UAS-CMVmin_SEAP
    SEAP under control of the Gal4 upstream activating sequence

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Figure 1: Comparison of the blue light system in CHO cells and in HEK-293T cells and comparison to the red light system in CHO cells.

To test the efficiency of the light system, SEAP expression after illumination was determined. The SEAP assay was performed 24 hours after light exposure. Labjournal

Figure 2: Efficiency of the blue light system using different time intervals of illumination.

To test the efficiency of the light system, SEAP expression after illumination was determined. The SEAP assay was performed 24 hours after light exposure. Cells were incubated with blue light for 1 hour, 2.5 hours and 5 hours. Labjournal

The blue light system is effective in activating the reporter SEAP which was introduced into the cells before illumination by transfections. As we know that the duration of illuminating the system with blue light (452 nm) is critical for its efficiency, we tested various time intervals for illumination. Our results indicate that five hours of illumination lead to the highest level of SEAP expression (Fig. 2).

Another important finding was the specificity of the blue light system. In the dark controls, we found almost no activation of the SEAP reporter, leading to a very low background level in our system.

SEAP as a Reporter

In order to test the capability of the blue light system for pattern generation, we transfected HEK-293T cells with the appropriate plasmids (PKM292 and PKM297) using seap as a reporter gene (PKM084). Cells were transfected in suspension and seeded on 96 well plates afterwards. Single wells of these plates were covered with a photo mask that prevents exposure to light. All other wells on the plate were illuminated to induce SEAP expression. SEAP that was secreted into the culture medium leads to a colour change from red to yellow after addition of pNPP (chromogenic substrate) by its phosphorylation.

First we generated a coarse pattern realizing that scattered light also reached wells that were close to the illuminated ones, leading to an unclear pattern. However, the rough shape of the desired pattern is discernible (Fig. 3A, B).

Since it was essential to completely prevent light exposure on covered plates, we tested 96 well plates with optically separated wells. We could generate patterns with sharp contours (Fig. 3C, D).

The colour change of pNPP in cell culture medium could be measured at 405 nm in a plate reader. Setting a distinct threshold made pattern recognition easier.

Figure 3: Generation of patterns on 96-well plates.

HEK-293T cells were transfected with the blue light system (PKM292, PKM297) and light induced SEAP as reporter (PKM084). Single wells were covered with a photo mask to prevent cell from light exposure. 24 hours after illumination with blue light, a SEAP assay was performed, leading to a colour change of wells containing SEAP. (A) Photo mask in heart form, (B) pseudocoloring done at a computer, (C) the same experiment was performed with optically separated wells, (D) the contrast was increased and the colours of the wells were changed at a computer. All wells together are forming the word: IGEM. Labjournal 1 Labjournal 2

For generating QR codes with our method 384-well plates were used. Since we had a collaboration with the iGEM Team Aachen 2014, they made a photo mask for a QR code for us fitting on these plates (Fig. 4). Collaborations.

Figure 4: Generation of a QR code on a 384 well plate.

HEK-293T cells were transfected with the blue light system (PKM292, PKM297) and light induced SEAP as reporter (PKM084). Single wells were covered with a photo mask to prevent cell from light exposure using a photo mask for a QR code (made by iGEM Team Aachen 2014). 24 hours after illumination with blue light a SEAP assay was performed leading to colour changing of wells containing SEAP. Only a part of the plate was used. (A) Photo mask for QR code generation, (B) Picture of the 384-well plate after the SEAP assay, (C) the same plate with contrast of the colours increased at a computer. Labjournal

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