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

 
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                     <h2> - Brevinin-1E </h2>
 
                     <h2> - Brevinin-1E </h2>
                     <h3>Table 3: The result of inhibition zone with the different concentration of Brevinin-1E</h3>
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                     <h3>Table 3: The result of inhibition zone with the different concentrations of Brevinin-1E</h3>
 
                     <img src="https://static.igem.org/mediawiki/2017/3/30/Inz_photo2.png" width="80%" style="display: block; margin: 0 auto 20px auto"> <img src="https://static.igem.org/mediawiki/2017/b/b3/Inz_photo3.png" width="40%" style="display: block; margin: auto"
 
                     <img src="https://static.igem.org/mediawiki/2017/3/30/Inz_photo2.png" width="80%" style="display: block; margin: 0 auto 20px auto"> <img src="https://static.igem.org/mediawiki/2017/b/b3/Inz_photo3.png" width="40%" style="display: block; margin: auto"
 
                         width="60%" style="display: block; margin: auto">
 
                         width="60%" style="display: block; margin: auto">
 
                     <h4>Figure 2: The result of inhibition zone which used the peptide - Brevinin-1E</h4>
 
                     <h4>Figure 2: The result of inhibition zone which used the peptide - Brevinin-1E</h4>
 
                     <img src="https://static.igem.org/mediawiki/2017/0/0f/Inz_photo4.png" width="60%" style="display: block; margin: auto" width="60%" style="display: block; margin: auto">
 
                     <img src="https://static.igem.org/mediawiki/2017/0/0f/Inz_photo4.png" width="60%" style="display: block; margin: auto" width="60%" style="display: block; margin: auto">
                     <h4>Figure 3: The comparison of partial magnification of the Figure 4</h4>
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                     <h4>Figure 3: The comparison of partial magnification of the Figure 2</h4>
  
 
                     <!----------------------------------------------------------------------------->
 
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                     <h2>- T-SN2</h2>
 
                     <h2>- T-SN2</h2>
                     <h3>Table 4: The result of inhibition zone with the different concentration of Temporin-SN2 </h3>
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                     <h3>Table 4: The result of inhibition zone with the different concentrations of Temporin-SN2 </h3>
 
                     <img src="https://static.igem.org/mediawiki/2017/0/02/Inz_photo5.png" width="80%" style="display: block; margin: 0 auto 20px auto"> <img src="https://static.igem.org/mediawiki/2017/3/3a/Inz_photo6.png" width="40%" style="display: block; margin: auto">
 
                     <img src="https://static.igem.org/mediawiki/2017/0/02/Inz_photo5.png" width="80%" style="display: block; margin: 0 auto 20px auto"> <img src="https://static.igem.org/mediawiki/2017/3/3a/Inz_photo6.png" width="40%" style="display: block; margin: auto">
 
                     <h4>Figure 4: The result of inhibition zone which  used the peptide - Temporin-SN2</h4>
 
                     <h4>Figure 4: The result of inhibition zone which  used the peptide - Temporin-SN2</h4>
 
                     <img src="https://static.igem.org/mediawiki/2017/a/a2/Inz_photo7.png" width="60%" style="display: block; margin: auto">
 
                     <img src="https://static.igem.org/mediawiki/2017/a/a2/Inz_photo7.png" width="60%" style="display: block; margin: auto">
                     <h4>Figure 5: The comparison of partial magnification of the Figure 6</h4>
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                     <h4>Figure 5: The comparison of partial magnification of the Figure 4</h4>
  
 
                     <!----------------------------------------------------------------------------->
 
                     <!----------------------------------------------------------------------------->
 
                     <h2>- Mastoparan-C</h2>
 
                     <h2>- Mastoparan-C</h2>
                     <h3>Table 5: The result of inhibition zone with the different concentration of Mastoparan-C</h3>
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                     <h3>Table 5: The result of inhibition zone with the different concentrations of Mastoparan-C</h3>
 
                     <img src="https://static.igem.org/mediawiki/2017/4/47/Inz_photo8.png" width="80%" style="display: block; margin: 0 auto 20px auto"> <img src="https://static.igem.org/mediawiki/2017/b/bf/Inz_photo9.png" width="40%" style="display: block; margin: auto">
 
                     <img src="https://static.igem.org/mediawiki/2017/4/47/Inz_photo8.png" width="80%" style="display: block; margin: 0 auto 20px auto"> <img src="https://static.igem.org/mediawiki/2017/b/bf/Inz_photo9.png" width="40%" style="display: block; margin: auto">
 
                     <h4>Figure 6: The result of inhibition zone which  used the peptide - Mastoparan-C</h4>
 
                     <h4>Figure 6: The result of inhibition zone which  used the peptide - Mastoparan-C</h4>
 
                     <img src="https://static.igem.org/mediawiki/2017/0/01/Inz_photo10.png" width="60%" style="display: block; margin: auto">
 
                     <img src="https://static.igem.org/mediawiki/2017/0/01/Inz_photo10.png" width="60%" style="display: block; margin: auto">
                     <h4>Figure 7: The comparison of partial magnification of the Figure 8</h4>
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                     <h4>Figure 7: The comparison of partial magnification of the Figure 6</h4>
  
 
                     <!----------------------------------------------------------------------------->
 
                     <!----------------------------------------------------------------------------->
 
                     <h2>- Pelophylaxin-4</h2>
 
                     <h2>- Pelophylaxin-4</h2>
                     <h3>Table 6: The result of inhibition zone with the different concentration of Pelophylaxin-4</h3>
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                     <h3>Table 6: The result of inhibition zone with the different concentrations of Pelophylaxin-4</h3>
 
                     <img src="https://static.igem.org/mediawiki/2017/b/b3/Inz_photo11.png" width="80%" style="display: block; margin: 0 auto 20px auto"> <img src="https://static.igem.org/mediawiki/2017/4/4c/Inz_photo12.png" width="40%" style="display: block; margin: auto">
 
                     <img src="https://static.igem.org/mediawiki/2017/b/b3/Inz_photo11.png" width="80%" style="display: block; margin: 0 auto 20px auto"> <img src="https://static.igem.org/mediawiki/2017/4/4c/Inz_photo12.png" width="40%" style="display: block; margin: auto">
                     <h4>Figure 8: The result of inhibition zone with the different concentration of 1095.6 Da venom vasodilator peptide</h4>
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                     <h4>Figure 8: The result of inhibition zone with the different concentrations of Pelophylaxin-4</h4>
 
                     <!------------------------------------------------------------------------------->
 
                     <!------------------------------------------------------------------------------->
 
                     <h2>- 1095.6 Da venom vasodilator peptide</h2>
 
                     <h2>- 1095.6 Da venom vasodilator peptide</h2>
                     <h3>Table 7: The result of inhibition zone with the different concentration of 1095.6 Da venom vasodilator peptide</h3>
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                     <h3>Table 7: The result of inhibition zone with the different concentrations of 1095.6 Da venom vasodilator peptide</h3>
 
                     <img src="https://static.igem.org/mediawiki/2017/3/3e/Inz_photo13.png" width="80%" style="display: block; margin: 0 auto 20px auto">
 
                     <img src="https://static.igem.org/mediawiki/2017/3/3e/Inz_photo13.png" width="80%" style="display: block; margin: 0 auto 20px auto">
  
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                     <!----------------------------------------------------------------------------->
 
                     <!----------------------------------------------------------------------------->
 
                     <h2>- Caeridin-1.4</h2>
 
                     <h2>- Caeridin-1.4</h2>
                     <h3>Table 8: he result of inhibition zone with the different concentration of Caeridin-1.4</h3>
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                     <h3>Table 8: The result of inhibition zone with the different concentrations of Caeridin-1.4</h3>
 
                     <img src="https://static.igem.org/mediawiki/2017/8/89/Inz_photo15.png" width="80%" style="display: block; margin: 0 auto 20px auto"> <img src="https://static.igem.org/mediawiki/2017/f/fb/Inz_photo16.png" width="40%" style="display: block; margin: auto">
 
                     <img src="https://static.igem.org/mediawiki/2017/8/89/Inz_photo15.png" width="80%" style="display: block; margin: 0 auto 20px auto"> <img src="https://static.igem.org/mediawiki/2017/f/fb/Inz_photo16.png" width="40%" style="display: block; margin: auto">
 
                     <h4>Figure 10: The result of inhibition zone which  used the peptide -Caeridin-1.4</h4>
 
                     <h4>Figure 10: The result of inhibition zone which  used the peptide -Caeridin-1.4</h4>
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                 <img src="https://static.igem.org/mediawiki/2017/a/a7/Spgm_photo4.png" width="60%" style="display: block; margin: auto;">
 
                 <img src="https://static.igem.org/mediawiki/2017/a/a7/Spgm_photo4.png" width="60%" style="display: block; margin: auto;">
  
                 <p> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The spores were classified into four grades according to the lengths of the germination tubes, which were spores that did not germinate, the lengths of germination tubes were same as the spore itself, germination
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                 <p> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The spores were classified into four grades according to the lengths of the germination tubes, 0 means the spores that did not germinate; 1, the lengths of germination tubes were same as the spore itself; 3 represents the germination
                     tubes three times longer than the spores and germination tubes five times longer than the spores. And then we calculated the percentage of each germination grade of each sample. </p>
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                     tubes three times longer than the spores and 5 stands for the germination tubes five times longer than the spores. And then we calculated the percentage of each germination grade of each sample. </p>
 
                 <div class="sub_text">
 
                 <div class="sub_text">
 
                     <p>- Analysis of Spore Germination Tendency</p>
 
                     <p>- Analysis of Spore Germination Tendency</p>

Latest revision as of 03:49, 2 November 2017

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NCTU_Formosa: Fungal Result
Inhibition Zone Result

     Each peptide was repeated the experiments for three times to ensure the results.

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

Sample

Table 1: The peptides which we used to conduct the experiments

Peptide vs. Concentration

Table 2:The table shows all the results of the inhibition zone.

Peptide Observation

Figure 1: The white part is mycelium, the holes were 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.

- Brevinin-1E

Table 3: The result of inhibition zone with the different concentrations of Brevinin-1E

Figure 2: The result of inhibition zone which used the peptide - Brevinin-1E

Figure 3: The comparison of partial magnification of the Figure 2

- T-SN2

Table 4: The result of inhibition zone with the different concentrations of Temporin-SN2

Figure 4: The result of inhibition zone which used the peptide - Temporin-SN2

Figure 5: The comparison of partial magnification of the Figure 4

- Mastoparan-C

Table 5: The result of inhibition zone with the different concentrations of Mastoparan-C

Figure 6: The result of inhibition zone which used the peptide - Mastoparan-C

Figure 7: The comparison of partial magnification of the Figure 6

- Pelophylaxin-4

Table 6: The result of inhibition zone with the different concentrations of Pelophylaxin-4

Figure 8: The result of inhibition zone with the different concentrations of Pelophylaxin-4

- 1095.6 Da venom vasodilator peptide

Table 7: The result of inhibition zone with the different concentrations of 1095.6 Da venom vasodilator peptide

Figure 9: The result of inhibition zone which used the peptide -1095.6 Da venom vasodilator peptide

- Caeridin-1.4

Table 8: The result of inhibition zone with the different concentrations of Caeridin-1.4

Figure 10: The result of inhibition zone which used the peptide -Caeridin-1.4

Conclusion

     According to the result, the Brevinin-1E and Temporin-SN2 were the strongest in the peptides we have selected, since their effectiveness on the mycelia were the most obvious. Mastoparan-C was not obviously, but still had effectiveness. 1095.6 Da venom vasodilator peptide, Pelophylaxin-4 and Caeridin-1.4 seemed to be no effect on the mycelia of Botrytis cinerea.

Table 9: The results of different concentration of peptides(circle: effective; triangle: less effective; X: ineffective)

Spore Germination Result

Sample

Table 9: Peptides and their sequences

Peptide vs. Concentration

Table 10: Experimental results

Calculation of Spore Germination

      Draw some spore suspension to a double concave slide and observe it under a microscope. When observing the sample, we put a plastic sheet that has small knife scratches we scratched beforehand to divide the whole sight under the slide, and made sure that we didn’t choose the same sight . The plastic sheet divides the whole sight of the microscope into equal squares, and we picked the square relatively of number 1,3,5,7,9 and counted the number of spores.

Figure 11: We divided our sight into nine squares under eyepiece 10X, objective 4X.

Figure 12: The picture shows the squares for counting spores under eyepiece 20X, objective 4X

Observation

     In the comparison with the inhibition zone and botany experiments, the spore germination was much hard to visualize the results only by the taken pictures. Because of the different lengths of germination tubes in one scene, what we would observe was the trend and tendency among different concentration and lengths.

Table 11: Illustration of four grades of spores according to the lengths of the germination tubes

     The spores were classified into four grades according to the lengths of the germination tubes, 0 means the spores that did not germinate; 1, the lengths of germination tubes were same as the spore itself; 3 represents the germination tubes three times longer than the spores and 5 stands for the germination tubes five times longer than the spores. And then we calculated the percentage of each germination grade of each sample.

- Analysis of Spore Germination Tendency

Figure 13: In this bar chart above shows a comparison of spore germination between six kinds of peptides with a concentration of 1000ug/ml and the negative control.

     We calculated the percentage of each spore level that we mentioned above and turned it into this bar chart. With this chart, we were able to know that after 6 hours of incubation, the spore germination percentage of non-germinating spores adding peptides with a concentration of 1000ug/ml was higher than adding the negative control, which meant the spore germination was inhibited by peptides and proved the peptides are effective.

Figure 14: In this bar chart shows a comparison of spore germination between six kinds of peptides with a concentration of 500ug/ml and the negative control

     Except for Pe-4, the other peptides had a better effectiveness than our control. Comparing with the result to the 1000ug/ml chart, we understood that we would need a higher concentration of Pe-4 to inhibit Botrytis cinerea.

     Comparing the result to the 1000ug/ml chart, we understood that we would need a higher concentration to inhibit Botrytis cinerea.

Figure 15: In this bar chart shows a comparison of spore germination between six kinds of peptides with a concentration of 250ug/ml and the negative control

     Except for Pe-4, the other peptides had a better effectiveness than our control.

      If the percentage of ungerminated spores are more than the negative control, we defined the peptides under the concentration of 1000ug/mL as effective antifungal peptides.

      In the premise of effective antifungal peptides, we then considered the concentration of 250ug/mL. We defined the ungerminated spores more than 50% of the spores as better antifungal peptides, which presented as circles in the table below; otherwise, they were the less effective peptides, presented in triangles.

      For Pe-4, although the percentage of ungerminated spores at 1000 ug/mL was similar to that of the negative control, it was observed that Pe-4 was effective because the percentage of score_5 for Pe-4 was much less than the percentage of score_5 for the negative control.

Conclusion

      With our experiment, we not only observed the suppression effect of each concentration of peptides but also the change in length of germ tubes in a micro view.

Table 12: The effectiveness of different peptides

Botany Experiment Result

Sample

Table 13: The sequences of peptides

Peptide vs. Concentration

Table 14: This table shows all the result of plant experiment

     In order to test our antifungal peptides, we put peptides and the negative control on the flower. The negative control was on the left hand which was infected with pathogens and our peptide sprayed on the right.

Peptide Observation

Figure 16: Left: Botrytis cinerea infection on Oncidium petal; Right: uninfected Oncidium petal

- T-SN2

Figure 17: Left: Pathogens mixed with the negative control; Right: Pathogens mixed with Temporin-SN2

     This picture shows that T-SN2 has the ability to inhibit fungi growth because the control which in the left side was obviously infected with pathogens and the right side wasn’t.

- B-1E

Figure 18: Left: Pathogens mixed with the negative control; Right: Pathogens mixed with Brevinin-1E

     This picture shows that B-1E has the ability to inhibit fungi growth because the control which in the left side was obviously infected with pathogens and the right side wasn’t.

- 1095.6

Figure 19: Left: Pathogens mixed with the negative control; Right: Pathogens mixed with 1095.6 Da venom vasodilator peptide.

     The black spot in this picture formed a strong contrast with the left side so we can say that 1095.6 Da venom vasodilator peptide has antifungal mechanism.

- Pe-4

Figure 20: Left: Pathogens mixed with the negative control; Right: Pathogens mixed with Pelophylaxin-4.

     Although the right side of flower had a little bit infected, the left side was more obvious. According to the picture, we can presume that the peptides have antifungal ability.

- M-C

Figure 21: Left: Pathogens mixed with the negative control; Right: Pathogens mixed with Mastoparan-C.

     The black spots in this picture were not very clear to see but in the right side of the flower there were three little spots on it. By these three little spots, we can say that this peptide ability to inhibit fungi from multiply.

Conclusion

     According to our experiments, these peptides all have shown their antifungal characteristics.

Table 14: The result of different peptides

Final Result

Table 15: The figure shows the result of six peptides with three fungal experiments (circle: effective; triangle: less effective; X: ineffective).

      We have conducted three experiments in order to validate our Parabase system. The spore germination experiment was the measurement for inhibiting spores, while the inhibition zone for inhibiting mycelia and the botany experiment for inhibiting the disease occurrence in reality.

      There weren’t an absolute relationship among three experiments because they were conducted for different factors. As a result, we defined a peptide as an antifungal peptide if it has been proved to be antifungal in at least one experiment. Our overall result turned out to be successful, validating our Parabase as a high-level system.

Reference

     [1]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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