Difference between revisions of "Team:UGent Belgium/Demonstrate"

 
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<h2 id="measurements">Functional assay</h2>
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<h1>Demonstration</h1>
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<p>The goal of or project is to provide a working product that can collect a substantial amount of water. To this end, we performed some experiments to measure the exact quantity of water collected by our shape, coated with polylactic acid (PLA) with biotin and treated with the Ice Nucleating Protein (INP)-streptavidin complex.</p>
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<p>We used a mixture of PLA and biotin dissolved in dichloremethane. These were used to either coat water collectors or to dip microscope slides. The coated objects are left to dry overnight in a laminar flow cabinet, allowing the solvent to evaporate.</p>
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<p>The goal of our project is to provide a working product that can collect a substantial amount of water. To this end, we performed some experiments to measure the exact quantity of water collected by our 3D-printed shape, coated with polylactic acid (PLA) and biotin, and treated with the Ice Nucleating Protein (INP)-streptavidin complex. A mixture of PLA and biotin, dissolved in dichloromethane, was used to coat the water collectors. The coated objects were left to dry overnight in a laminar flow cabinet, allowing the solvent to evaporate.</p>
 
<p>We considered four biological treatments:</p>
 
<p>We considered four biological treatments:</p>
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<ol>  
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<ol style="list-style-type: decimal">
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<li> <strong>mGFPuv-mSA2</strong> </li>
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<li><strong>INP + INP_NC-mSA2</strong>: whole cells expressing both full length INP and membrane-bound monomeric streptavidin (mSA2)</li>
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<li> <strong>RFP + INP-NC-mSA2</strong></li>
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<li><strong>RFP + INP_NC-mSA2</strong>: Control, cells expressing membrane-bound mSA2 and a red fluorescent protein</li>
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<li> <strong>inaZ + INP-NC-mSA2</strong> </li>
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<li><strong>INP_RC-mSA2</strong>: protein extract: INP nucleating domain - mSA2 fusion protein</li>
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<li> <strong>INP-mSA</strong> </li>
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<li><strong>mGFPuv2-mSA2</strong>: Control, mGFPuv2-mSA2 fusion protein</li>
 
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<p>In each experiment, the measurements were performed in a <a href="https://2016.igem.org/Team:UGent_Belgium/Hardware">controlled humidified chamber</a>. Both the temperature and the humidity were constantly monitored and the latter was actively controlled using on-off control.</p>
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<p>In each experiment, the measurements were performed in a <a href="https://2016.igem.org/Team:UGent_Belgium/Hardware">controlled humidified chamber</a>. Both the temperature and the humidity were constantly measured and the latter was actively controlled using <a href="https://2016.igem.org/Team:UGent_Belgium/Software">bang-bang control</a>.</p>
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<h3 id="experiment-1-effect-biological-treatments-on-the-water-collectors">Experiment 1: effect biological treatments on the water collectors</h3>
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<h2 id="effect-biological-treatments-on-the-water-collectors">Effect biological treatments on the water collectors</h2>
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<h4 id="setup">Setup</h4>
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<h3 id="setup">Setup</h3>
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<p>In a first experiment we probed to the effect of the four biological treatments described above.</p>
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<p>In this demonstrative experiment, we assesed the effect of the four biological treatments described above.</p>
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<p>The dome of four water collector were coated using the PLA-biotin solution. Subsequently each collector was sawn in half in order to have a treatment and associated control for each experimental unit.</p>
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<p>The dome of four water collectors were coated using the PLA-biotin solution. Subsequently each collector was sawn in half in order to have a treatment and associated control for each experimental unit.</p>
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<div class="figure" align="middle" >
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<div class="center">
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<img src="https://static.igem.org/mediawiki/2016/f/fb/T--UGent_Belgium--halfcollector.jpeg" width="500"/>
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<p class="caption center" align="center"> Water collector half. </p>
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<img src="https://static.igem.org/mediawiki/2016/f/fb/T--UGent_Belgium--halfcollector.jpeg" alt="Half a water collector." width="600" />
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<p class="center"><i>Half of a water collector.</i></p>
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<p>One half of each collector was treated with a different biological function (i.e. mGFPuv-mSA2, RFP + INP-NC-mSA2, inaZ + INP-NC-mSA2 and INP-mSA). After waiting five minutes, to allow biotin-streptavidin bonds to form, all half-collectors were rinsed thoroughly with a physiological solution. Each halve was placed on a petri dish.</p>
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<p>All halves were dried and weighted on an analytical scale. Finally, all petri dishes with collector halfs were randomly placed in the humidistat. They were left under a flow of humid air for one night. Afterwards, all halves were weighted again, as such that the accumulation of water could be determined.</p>
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<p>One half of each collector was treated with a different biological function (i.e. INP + INP_NC-mSA2, RFP + INP_NC-mSA2, INP_RC-mSA2 and mGFPuv2-mSA2). After waiting five minutes, to allow biotin-streptavidin bonds to form, all half-collectors were rinsed thoroughly with a physiological solution. Each halve was placed on a petri dish.</p>
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<div class="figure" align="middle">
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<p>All halves were dried and weighted on an analytical scale. Finally, all petri dishes were randomly placed in the humidified chamber. They were left under a flow of humid air for one night. Afterwards, all halves were weighed again, to measure the accumulation of water.</p>
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<img src="https://static.igem.org/mediawiki/2016/9/9c/T--UGent_Belgium--humchamb.jpeg" alt="The collectors in the humidified chamber." width="500" />
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<p class="caption center" align="center">The collectors in the humidified chamber.</p>
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</div>
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<h4 id="results">Results</h4>
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<p>The following bar chart shows the difference of in water accumulation in gram of each treatment compared to the half that was used as a control.</p>
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<div class="figure" align="middle">
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<img src="https://static.igem.org/mediawiki/2016/1/18/T--UGent_Belgium--collectedCollectors.jpeg" alt="Results first experiment. Difference in water collection between" width="500" />
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<p class="caption center" align="center">Results first experiment. Difference in water collection between</p>
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<p>The average amount of water collected by all the experimental units is <span class="math inline">3.07 ± 0.38</span> gram. The difference of the effect of the different treatments (between -0.92 and 0.6 gram difference in water collection of treatment versus control). Unfortunately, it appears that the INP treatments result in a decrease in water collection. As the effect is quite small and we could not perform any replications, it is perhaps safest to conclude that these results do not support any conclusions either way.</p>
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<h3 id="experiment-2-comparison-biological-treatments-on-microscope-slides">Experiment 2: comparison biological treatments on microscope slides</h3>
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<h4 id="setup-1">Setup</h4>
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<p>For this smaller experiment, we dipped four microscope slides in the PLA-biotin solution and subsequently applied all biological treatments to each of the slides. Afterwards, all slides were rinsed thoroughly with physiological solution.</p>
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<p>The slides were placed straight up in a falcon tube, allowing moist air to reach the treated surface. All four slides in a falcon were weighted on the analytical scale.</p>
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<div class="figure" align="middle">
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<img src="https://static.igem.org/mediawiki/2016/6/6c/T--UGent_Belgium--slideshum.jpeg" width="500" />
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<div class="center">
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<div class="figure">
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<img src="https://static.igem.org/mediawiki/2016/9/9c/T--UGent_Belgium--humchamb.jpeg" alt="The collectors in the humidified chamber." width="600"/>
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<p class="center"><i>The collectors in the humidified chamber.</i></p>
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</div></div>
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<h3 id="results">Results</h3>
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<p>The following bar chart shows the differences in water accumulation of each treatment-half compared to the control-half.</p>
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<div class="center">
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<div class="figure">
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<img src="https://static.igem.org/mediawiki/2016/4/41/Water_collection_collectors.png" alt="Difference in water collection treatment versus control for the four treatments." width="600"/>
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<p class="center"><i>Differences in water collection between treatment and control, for all four treatments.</i></p>
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</div></div>
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<p>The average water amount of water collected by all the experimental units is <span class="math">3.07 ± 0.38</span> gram. The difference in weight of the treatment-halves compared to the control-halves of the different treatments were between -0.92 and 0.6 gram. Unfortunately, it appears that the INP treatments result in a decrease in water collection. As the effect is quite small and we could not perform any replications, it is perhaps safest to conclude that these results do not support any conclusions either way.</p>
 
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</div>
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<p>The falcons were placed randomly in the humidified chamber for six hours. Their places were changed periodically to decrease the chance of confounding errors. Afterwards, the accumulation of water was determined for each experimental unit.</p>
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<h3 id="results-1">Results</h3>
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<p>The amount of water collected of the four slides, by treatment, is shown in the bar chart below.</p>
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<div class="figure" align="middle">
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<img src="https://static.igem.org/mediawiki/2016/d/de/T--UGent_Belgium--micro.jpeg" width="500" />
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</div>
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<p>Again, the difference between the treatments is relatively small and the treatments with INP do not seem to collect more water. As for the previous experiment, this experiment could not prove that PLA bonded with INP could efficiently extract moisture from the air.</p>
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Latest revision as of 18:46, 19 October 2016

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Demonstration


The goal of our project is to provide a working product that can collect a substantial amount of water. To this end, we performed some experiments to measure the exact quantity of water collected by our 3D-printed shape, coated with polylactic acid (PLA) and biotin, and treated with the Ice Nucleating Protein (INP)-streptavidin complex. A mixture of PLA and biotin, dissolved in dichloromethane, was used to coat the water collectors. The coated objects were left to dry overnight in a laminar flow cabinet, allowing the solvent to evaporate.

We considered four biological treatments:

  1. INP + INP_NC-mSA2: whole cells expressing both full length INP and membrane-bound monomeric streptavidin (mSA2)
  2. RFP + INP_NC-mSA2: Control, cells expressing membrane-bound mSA2 and a red fluorescent protein
  3. INP_RC-mSA2: protein extract: INP nucleating domain - mSA2 fusion protein
  4. mGFPuv2-mSA2: Control, mGFPuv2-mSA2 fusion protein

In each experiment, the measurements were performed in a controlled humidified chamber. Both the temperature and the humidity were constantly measured and the latter was actively controlled using bang-bang control.

Effect biological treatments on the water collectors

Setup

In this demonstrative experiment, we assesed the effect of the four biological treatments described above.

The dome of four water collectors were coated using the PLA-biotin solution. Subsequently each collector was sawn in half in order to have a treatment and associated control for each experimental unit.

Half a water collector.

Half of a water collector.

One half of each collector was treated with a different biological function (i.e. INP + INP_NC-mSA2, RFP + INP_NC-mSA2, INP_RC-mSA2 and mGFPuv2-mSA2). After waiting five minutes, to allow biotin-streptavidin bonds to form, all half-collectors were rinsed thoroughly with a physiological solution. Each halve was placed on a petri dish.

All halves were dried and weighted on an analytical scale. Finally, all petri dishes were randomly placed in the humidified chamber. They were left under a flow of humid air for one night. Afterwards, all halves were weighed again, to measure the accumulation of water.

The collectors in the humidified chamber.

The collectors in the humidified chamber.

Results

The following bar chart shows the differences in water accumulation of each treatment-half compared to the control-half.

Difference in water collection treatment versus control for the four treatments.

Differences in water collection between treatment and control, for all four treatments.

The average water amount of water collected by all the experimental units is 3.07 ± 0.38 gram. The difference in weight of the treatment-halves compared to the control-halves of the different treatments were between -0.92 and 0.6 gram. Unfortunately, it appears that the INP treatments result in a decrease in water collection. As the effect is quite small and we could not perform any replications, it is perhaps safest to conclude that these results do not support any conclusions either way.