Difference between revisions of "Team:TCU Taiwan/Modeling"

 
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<tr><td align="center"><h1><span style="font-family:Arial Black;"><font size="7"><font-weight: 700;> About our modeling</font></span></h1></td></tr>
 
<tr><td align="center"><h1><span style="font-family:Arial Black;"><font size="7"><font-weight: 700;> About our modeling</font></span></h1></td></tr>
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To increase efficiency in isolating our AMPs, we introduced a signal peptide upstream of the N-terminal of mature antimicrobial peptides. This signal peptide is obtained from chitinase C of S.lividans (MGFRHKAAALAATLALPLAGLVGLASPAQA). When the premature peptides enter the periplasmic space, peptidase will identify the cleavage site Ala-Gln-Ala and cut at the double Ala between the signal and mature peptide. </br></br>
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We had collected the data of disinfection experiments and <I>in vitro</I> wound healing assay. Than we analyzed it into charts to represent our experimental achievement.
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To ensure and verify this process, we have attached an Ala at the N-terminal of AMPs. When applying a protein secondary structure prediction software base on the known peptide structure, we can analyze whether the attached Ala may have an effect on the peptide folding process.
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<tr><td align="left"><h1><span style="font-family:Arial Black;"><font size="7"><font-weight: 700;> Signiferin peptide structure analysis</font></span></h1></td></tr>
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<tr><td align="center"><h1><span style="font-family:Arial Black;"><font size="6"><font-weight: 700;>Disinfection Efficiency</font></span></h1></td></tr>
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    <td width="90%" align="center"><img src="https://static.igem.org/mediawiki/2015/e/ee/2015tcutaiwanModelingwithoutA1.jpg" align=center width="80%"  title="Result 1"></td>
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We had tested the disinfection ability of our AMPs and proved our system is really worked.First, we prepared 1 ml LB medium of <I>S. aureus</I> concentration of OD 0.01 at 600 nm. Than we added 1 ml sample into tube and measured the OD at 600 nm once in two hours.The experiments were carried out for 24 hours, and the result were collected at 0, 2nd, 4th, 6th and 24th hours. We had analyzed the data and made it into chart.
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     <td width="45%" align="center"><img src="https://static.igem.org/mediawiki/2015/f/f8/2015tcutaiwanModelingwithA1.jpg" align=center width="80%"  title="Result 1"></td>
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     <td width="50%" align="center"><img src="https://static.igem.org/mediawiki/2015/a/a7/2015tcutaiwanThe_peptides_efficiency_of_disinfection.JPG" align=center width="100%"  title="Result 1"></td>
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<td width="50%" align="center"><img src="https://static.igem.org/mediawiki/2015/f/f3/2015tcutaiwanThe_peptides_efficiency.JPG" align=center width="100%"  title="Result 2"></td>
 
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The first column shows the amino acid sequence that we predict.
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Conclusions: As the chart we can see that both Signiferin and Epinecidin-1 have ability to kill <I>S. aureus</I>. At 0 hour, there were no obvious differences between control and  experimental group. However, at 6th hours, inhibition of bacterial growth were  obvious in both signiferin and epi-1. But the effect is not significant. We speculated the concentration is not enough to see the best efficiency. 
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The second column shows that AMPs corresponding secondary structure state are still a-helix.
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The third column shows the probability of correct prediction.
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<tr><td align="center"><h1><span style="font-family:Arial Black;"><font size="6"><font-weight: 700;> Modeling for cell experiments</font></span></h1></td></tr>
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<tr><td align="left"><h1><span style="font-family:Arial Black;"><font size="7"><font-weight: 700;> Epinicedin-1 pepetide structure analysis</font></span></h1></td></tr>
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After cell migration experiments, we use ImageJ to measure the distance of the migration then compared them to interpret our results. We had collected data and analyze it to made charts.
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  <td width="45%" align="center"><img src="https://static.igem.org/mediawiki/2015/8/82/2015tcutaiwanModelingepi-1withoutA1.jpg" align=center width="80%"  title="Result 4"></td>
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<td align="center"><span style="font-family:Arial Black;"><font size="6"><font-weight: 700;>The Signiferin group with one ten dilution of crude extract</font></span></td>
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<td width="90%" align="center"><img src="https://static.igem.org/mediawiki/2015/1/10/2015tcutaiwanigemModelingepi-1withA1.jpg" align=center width="80%"  title="Result 3"></td>
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<span style="font-family:Calibri;text-align:justify;align:left;"><font size="4"></br>Fig. 1 The pictures of cell migration in the Signiferin group at 0 hour and 24 hours</font></span>
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<span style="font-family:Calibri;text-align:justify;align:left;"><font size="4"></br>Fig. 1-1 The percentage of migration distance between Signiferin group and mock</font></span>
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Conclusion: As Fig. 1 the Signiferin group with one ten dilution of crude extract, no significant differences of cell migration were observed. And we also compared with mock group (show as Fig. 1-1) there were no signify difference too. It means Signiferin doesn’t has ability to help wound healing.  </font></span></h1>
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<td align="center"><span style="font-family:Arial Black;"><font size="6"><font-weight: 700;>The Epinecidin-1 group with one ten dilution of crude extract</font></span></td>
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The first column shows the amino acid sequence that we predict.
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The second column shows that AMPs corresponding secondary structure state are still a-helix.
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The third column shows the probability of correct prediction.
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<span style="font-family:Calibri;text-align:justify;align:left;"><font size="4"></br>Fig. 2 The pictures of cell migration in the Epinecidin-1 group at 0 hour and 24 hours</font></span>
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<span style="font-family:Calibri;text-align:justify;align:left;"><font size="4"></br>Fig. 2-1 The percentage of migration distance between Epinecidin-1 group and mock</font></span>
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Conclusion: In the Epinecidin-1 group significant efficiency of migration were observed in the picture in Fig. 2. And we also compared the migration distance between Epinecidin-1 group and mock group. As Fig. 2-1 shows, wound healing time was cut in half, in other words, if a wound needs 4 weeks to recovered, now only needs 2 weeks. These results consistent with previous publications.</font></span></h1>
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<td align="center"><span style="font-family:Arial Black;"><font size="6"><font-weight: 700;>The combinational AMPs group using 1 in 500 dilution of crude extract</font></span></td>
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<span style="font-family:Calibri;text-align:justify;align:left;"><font size="4"></br>Fig. 3 The pictures of cell migration in the combinational AMPs group at 0 hour and 24 hours</font></span>
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<span style="font-family:Calibri;text-align:justify;align:left;"><font size="4"></br>Fig. 3-1 The percentage of migration distance between combinational AMPs group and mock</font></span>
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Conclusion: In the combinational AMPs group using 1 in 500 dilution of crude extract, synergetic effects were observed in the picture Fig.3 and also showed in the chart Fig. 3-1. That means, a wound used to recovered in 2 weeks, now we just need one. The patients suffering will be much lessen. </font></span></h1>
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<tr><td align="center"><h1><span style="font-family:Arial Black;"><font size="7"><font-weight: 700;> Conclusion</font></span></h1></td></tr>
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Through the analysis of the peptide secondary structure and confirmation of the -helix structure, the results show whether Ala is attached to Signiferin or Epinecidin-1, the peptide did not affect the peptide folding process. process.
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Latest revision as of 10:47, 27 October 2015

About our modeling

We had collected the data of disinfection experiments and in vitro wound healing assay. Than we analyzed it into charts to represent our experimental achievement.

Disinfection Efficiency

We had tested the disinfection ability of our AMPs and proved our system is really worked.First, we prepared 1 ml LB medium of S. aureus concentration of OD 0.01 at 600 nm. Than we added 1 ml sample into tube and measured the OD at 600 nm once in two hours.The experiments were carried out for 24 hours, and the result were collected at 0, 2nd, 4th, 6th and 24th hours. We had analyzed the data and made it into chart.

Conclusions: As the chart we can see that both Signiferin and Epinecidin-1 have ability to kill S. aureus. At 0 hour, there were no obvious differences between control and experimental group. However, at 6th hours, inhibition of bacterial growth were obvious in both signiferin and epi-1. But the effect is not significant. We speculated the concentration is not enough to see the best efficiency.

Modeling for cell experiments

After cell migration experiments, we use ImageJ to measure the distance of the migration then compared them to interpret our results. We had collected data and analyze it to made charts.

The Signiferin group with one ten dilution of crude extract

Fig. 1 The pictures of cell migration in the Signiferin group at 0 hour and 24 hours
Fig. 1-1 The percentage of migration distance between Signiferin group and mock

Conclusion: As Fig. 1 the Signiferin group with one ten dilution of crude extract, no significant differences of cell migration were observed. And we also compared with mock group (show as Fig. 1-1) there were no signify difference too. It means Signiferin doesn’t has ability to help wound healing.




The Epinecidin-1 group with one ten dilution of crude extract

Fig. 2 The pictures of cell migration in the Epinecidin-1 group at 0 hour and 24 hours
Fig. 2-1 The percentage of migration distance between Epinecidin-1 group and mock

Conclusion: In the Epinecidin-1 group significant efficiency of migration were observed in the picture in Fig. 2. And we also compared the migration distance between Epinecidin-1 group and mock group. As Fig. 2-1 shows, wound healing time was cut in half, in other words, if a wound needs 4 weeks to recovered, now only needs 2 weeks. These results consistent with previous publications.

The combinational AMPs group using 1 in 500 dilution of crude extract

Fig. 3 The pictures of cell migration in the combinational AMPs group at 0 hour and 24 hours
Fig. 3-1 The percentage of migration distance between combinational AMPs group and mock

Conclusion: In the combinational AMPs group using 1 in 500 dilution of crude extract, synergetic effects were observed in the picture Fig.3 and also showed in the chart Fig. 3-1. That means, a wound used to recovered in 2 weeks, now we just need one. The patients suffering will be much lessen.






             
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