Difference between revisions of "Team:NCTU Formosa/Fungal Experiment"

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     <div class="fgexp_head"><img src="https://static.igem.org/mediawiki/2017/9/9b/Fgexp_head.png" width="100%"> </div>
 
     <div class="fgexp_head"><img src="https://static.igem.org/mediawiki/2017/9/9b/Fgexp_head.png" width="100%"> </div>
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    <div class="fgexp_content">
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        <div>
 +
            <h1>Fungi Experiment </h1>
 +
            <img src="https://static.igem.org/mediawiki/2017/7/7e/Fgexp_fig_1.png" width="60%" style="display: block; margin: auto;">
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        </div>
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 +
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        <!----------------------------------------------------------------------------->
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        <img id="ihzone_pic" class="subtitle" src="https://static.igem.org/mediawiki/2017/4/48/Ptp_SCM.png">
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        <div class="ihzone">
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            <div class="show">
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                <p>This block will show the visible content.</p>
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            </div>
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            <div class="hide">
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                <h1>Dual Cultures on Potato Dextrose Agar</h1>
 +
 +
                <p>
 +
                    &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;In order to know whether the peptides are effective in inhibiting hyphae and the accurate concentration of peptides for inhibition, we measured the zone of inhibition.
 +
                </p>
 +
 +
                <p>
 +
                    &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Putting the mycelium on the Petri plates of Potato dextrose agar, we cultivated plates in 20℃ for 2 days. After making sure that the mycelial colony has developed, we put the sterile blank paper which the different concentrations
 +
                    of peptides and the negative control were added respectively.
 +
                </p>
 +
 +
                <h1>Observation</h1>
 +
 +
                <p>
 +
                    &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The white part is mycelium, the holes are added different concentrations of peptides and negative control HEPES respectively.
 +
                </p>
 +
 +
                <img src="https://static.igem.org/mediawiki/2017/5/5c/Fgexp_fig_2.png" width="60%" style="display: block; margin: auto;">
 +
 +
                <p>
 +
                    &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;If the peptides can inhibit the mycelium, then there will be a range around the holes that the mycelium can not occupied. Otherwise, the mycelium will grow ignoring the holes, like the hole containing HEPES.
 +
                </p>
 +
 +
                <p>
 +
                    &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The whole scene will look like a white circle, while the edges of effective peptides concave.
 +
                </p>
 +
 +
 +
                <div><img class="hide_pic" src="https://static.igem.org/mediawiki/2017/c/cb/Ptp_show.png" style="display:block; margin:auto;"></div>
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            </div>
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 +
        <!----------------------------------------------------------------------------->
 +
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                <p>This block is going to put some words</p>
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            </div>
 +
            <div class="hide2">
 +
                <h1>Spore Germination: inhibition measurement for spores</h1>
 +
 +
                <p>
 +
                    &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;In order to know whether the antifungal peptides can inhibit the germination of spores or not and the accurate concentration of antifungal peptides for inhibition, we conducted the spore germination experiment.
 +
                </p>
 +
 +
                <h1>Preparation of Spore Suspension Solution </h1>
 +
 +
                <p>
 +
                    &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The concentration of the spore suspension solution (1.5*10^5 spores/ml) was calculated and adjusted by using a hemacytometer and a microscope. After adjusting the concentration of the solution, we mixed it with 2% glucose
 +
                    solution and each added different doses of peptides together into PCR tubes to pipet. Then we drew 15μl solution from the PCR tubes to the Petri dishes. After 20℃, 6 hours of culture, we calculated the percentage of spore germination
 +
                    afterward.
 +
                </p>
 +
 +
                <h1>Percentage of Spore Germination</h1>
 +
 +
                <p>
 +
                    &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We directly observed Petri dishes under the microscope. By calculating the amount of the germinated spores and ungerminated spores, we calculated the rate of spore germination under dose response of peptides. Then, we compared
 +
                    all the solution with the control, and we knew the effectiveness of every peptide we predicted for the spore germination. Finally, the results were made and shown the MIC and IC50.
 +
                </p>
 +
 +
 +
 +
                <div><img class="hide2_pic" src="https://static.igem.org/mediawiki/2017/c/cb/Ptp_show.png" style="display:block; margin:auto;"></div>
 +
            </div>
 +
        </div>
 +
        <!----------------------------------------------------------------------------->
 +
 +
        <div id="fgexp_result">
 +
            <h1>Results</h1>
 +
            <img src="https://static.igem.org/mediawiki/2017/8/81/Fgexp_fig_3.png" width="60%" style="display: block; margin: auto;">
 +
            <p>Sample</p>
 +
            <img src="https://static.igem.org/mediawiki/2017/e/ee/Fgexp_fig_4.png" width="60%" style="display: block; margin: auto;">
 +
            <p>
 +
                &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;HEPES buffer, which has no toxic on cells, is applied to be the negative control.
 +
            </p>
 +
 +
        </div>
 +
        <div id="fgexp_reference">
 +
            <h1>Reference</h1>
 +
            <p><small>Antifungal Mechanism of a Novel Antifungal Protein from Pumpkin Rinds against Various Fungal Pathogens.J. Agric. Food Chem. 2009, 57, 9299–9304. DOI:10.1021/jf902005g</small></p>
 +
        </div>
 +
 +
 +
 +
 +
 +
    </div>
  
 
     <div id="fut"></div>
 
     <div id="fut"></div>

Revision as of 17:33, 28 October 2017

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NCTU_Formosa: Fungal Experiment

Fungi Experiment

This block will show the visible content.

Dual Cultures on Potato Dextrose Agar

     In order to know whether the peptides are effective in inhibiting hyphae and the accurate concentration of peptides for inhibition, we measured the zone of inhibition.

     Putting the mycelium on the Petri plates of Potato dextrose agar, we cultivated plates in 20℃ for 2 days. After making sure that the mycelial colony has developed, we put the sterile blank paper which the different concentrations of peptides and the negative control were added respectively.

Observation

     The white part is mycelium, the holes are added different concentrations of peptides and negative control HEPES respectively.

     If the peptides can inhibit the mycelium, then there will be a range around the holes that the mycelium can not occupied. Otherwise, the mycelium will grow ignoring the holes, like the hole containing HEPES.

     The whole scene will look like a white circle, while the edges of effective peptides concave.

This block is going to put some words

Spore Germination: inhibition measurement for spores

     In order to know whether the antifungal peptides can inhibit the germination of spores or not and the accurate concentration of antifungal peptides for inhibition, we conducted the spore germination experiment.

Preparation of Spore Suspension Solution

     The concentration of the spore suspension solution (1.5*10^5 spores/ml) was calculated and adjusted by using a hemacytometer and a microscope. After adjusting the concentration of the solution, we mixed it with 2% glucose solution and each added different doses of peptides together into PCR tubes to pipet. Then we drew 15μl solution from the PCR tubes to the Petri dishes. After 20℃, 6 hours of culture, we calculated the percentage of spore germination afterward.

Percentage of Spore Germination

     We directly observed Petri dishes under the microscope. By calculating the amount of the germinated spores and ungerminated spores, we calculated the rate of spore germination under dose response of peptides. Then, we compared all the solution with the control, and we knew the effectiveness of every peptide we predicted for the spore germination. Finally, the results were made and shown the MIC and IC50.

Results

Sample

     HEPES buffer, which has no toxic on cells, is applied to be the negative control.

Reference

Antifungal Mechanism of a Novel Antifungal Protein from Pumpkin Rinds against Various Fungal Pathogens.J. Agric. Food Chem. 2009, 57, 9299–9304. DOI:10.1021/jf902005g

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