Difference between revisions of "Part:BBa K2262013"
 
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It has been known as an antimicrobial peptide. Its grade given by scoring card, which was created by NCTU_Formosa, shows it may also have an anti-fungal function.
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Pe-4 is an antimicrobial peptide [1]. The score of SCM analysis shows its promising potential to be antifungal.
  
 
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Pe-4 got 408.83 points from the Anti-fungal Scoring Card of Parabase System, which was created by NCTU_Formosa. The grade is over 353, the threshold to divide whether a peptide has an anti-fungal function probability, that means Pe-4 has a high probability to have an anti-fungal function.
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Pe-4 got 408.83 from the Antifungal Scoring Card of Parabase System. The grade is score 353, the threshold to divide whether a peptide has an antifungal function probability, that means Pe-4 has a high probability to have an antifungal function.
 
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We put some mycelium on a Potato dextrose agar plate, PDA plate, and cultivated it at the point of 20℃ for culturing Botrytis cinerea, the fungi we used, for 2 days. Ensuring the mycelial colony has developed, we dug holes and the different concentrations of Pe-4 and HEPES, as a negative control,  were added respectively. The place where peptide been but will appear a hole after 12 ~ 18 hours if the peptide will affect the growth of mycelium.  The result shows that the place we put Pe-4 does have obviously different with negative control. It means the peptide has no obvious function on affecting the growth of mycelium.
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The result shows that the place we put Pe-4 has no different with negative control. It means the peptide has no obvious function on affecting the growth of mycelium.
  
  
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<p style="padding:1px;font-size:14px"><b>(2) Spore Germination </b></p>
 
<p style="padding:1px;font-size:14px"><b>(2) Spore Germination </b></p>
 
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To know whether the anti-fungal peptides can inhibit the germination of spores, we conducted the spore germination experiment.
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The result shows that the spore germination percentage of non-germinating spores adding Pe-4 with a concentration of 1000 μg/mL is higher than adding the negative control, which means the spore germination is inhibited by peptides and proves the peptides is effective. And the percentage of score_5 for Pe-4 is much less than the percentage of score_5 for the negative control.
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[[File:Spore Germination.png|900px|thumb|center|'''Figure 4.''' The result shows that Pe-4 might affect the germination of spore. ]]
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<p style="padding:1px;font-size:14px"><b>(3) Botany Experiment </b></p>
 
<p style="padding:1px;font-size:14px"><b>(3) Botany Experiment </b></p>
 
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In order to test our anti-fungal peptides, we put Pe-4 and negative control, Double-distilled water, on the flower. The negative control on the left hand and BPe-4 on the right hand. Both sides are infected with Botrytis cinerea. We will check the flower 3 days later whether the flower was affected or not. The result shows that the right side, which was spread on Pe-4, is not infected. It means Pe-4 has an anti-fungal function.
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In order to test our anti-fungal peptides, we put Pe-4 and negative control, double-distilled water, on the flower. The negative control is on the left and Pe-4 is on the right. Both sides are infected with Botrytis cinerea. We checked the flower 3 days later whether the flower was affected or not. The result shows that the right side, which was spread on Pe-4, is not infected. It means Pe-4 has an anti-fungal function in real plant organism.
  
  
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<p style="padding:1px;font-size:16px"><b>2. Cloning </b></p>
 
<p style="padding:1px;font-size:16px"><b>2. Cloning </b></p>
 
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We put T7 promoter, RBS, and Pe-4 together with pSB1C3 as a backbone. Then we conducted Taq PCR to check the size of the DNA sequence was right. The length of T7 promoter+RBS+Sa-AFP2 is around 100 ~ 200 b.p.. Its PCR product is around 250 ~ 500 b.p..
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We put T7 promoter, RBS, and Pe-4 together with pSB1C3 as a backbone. Then we conducted Taq PCR to check the size of the DNA sequence was right. The length of T7 promoter+RBS+Pe-4 is 120 b.p. Its PCR product is 432 b.p.
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[[File:Agarose gel Pe-4.png|200px|thumb|center|'''Figure 6.''' P<sub>T7</sub> + RBS + Pe-4 <br>
 
[[File:Agarose gel Pe-4.png|200px|thumb|center|'''Figure 6.''' P<sub>T7</sub> + RBS + Pe-4 <br>
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The DNA sequence length of P<sub>T7</sub> + RBS + Pe-4 is around 100 ~ 200 b.p.. The size of its PCR product should be close to 450 ~ 500 b.p..]]
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The DNA sequence length of P<sub>T7</sub> + RBS + Pe-4 is around 100 ~ 200 b.p. The size of its PCR product should be close to 450 ~ 500 b.p.]]
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<h1>'''Reference'''</h1>
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[1] Bai RL; Pettit GR; Hamel E. “Binding of dolastatin 10 to tubulin at a distinct site for peptide antimitotic agents near the exchangeable nucleotide and vinca alkaloid sites.” J Biol Chem. 1990 Oct 5;265(28):17141-9.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 15:07, 7 December 2017


T7 Promoter+RBS+Pe-4


Figure 1. PT7+RBS+Pe-4+terminator



Introduction


      Pe-4 is an antimicrobial peptide [1]. The score of SCM analysis shows its promising potential to be antifungal.


Scoring Card Predict

      Pe-4 got 408.83 from the Antifungal Scoring Card of Parabase System. The grade is score 353, the threshold to divide whether a peptide has an antifungal function probability, that means Pe-4 has a high probability to have an antifungal function.

Figure 2. The sequence of Pe-4.
The colors of each amino acids represent the probability of anti-fungal dipeptides.



Experiment


1. Fungal Test


(1) Inhibition Zone

      The result shows that the place we put Pe-4 has no different with negative control. It means the peptide has no obvious function on affecting the growth of mycelium.


Figure 3. The result shows that Pe-4 has no obvious function on affecting the growth of mycelium.


(2) Spore Germination

      The result shows that the spore germination percentage of non-germinating spores adding Pe-4 with a concentration of 1000 μg/mL is higher than adding the negative control, which means the spore germination is inhibited by peptides and proves the peptides is effective. And the percentage of score_5 for Pe-4 is much less than the percentage of score_5 for the negative control.


Figure 4. The result shows that Pe-4 might affect the germination of spore.



(3) Botany Experiment

      In order to test our anti-fungal peptides, we put Pe-4 and negative control, double-distilled water, on the flower. The negative control is on the left and Pe-4 is on the right. Both sides are infected with Botrytis cinerea. We checked the flower 3 days later whether the flower was affected or not. The result shows that the right side, which was spread on Pe-4, is not infected. It means Pe-4 has an anti-fungal function in real plant organism.


Figure 5. The result shows that Pe-4 will affect fungal infection.



2. Cloning

      We put T7 promoter, RBS, and Pe-4 together with pSB1C3 as a backbone. Then we conducted Taq PCR to check the size of the DNA sequence was right. The length of T7 promoter+RBS+Pe-4 is 120 b.p. Its PCR product is 432 b.p.

Figure 6. PT7 + RBS + Pe-4
The DNA sequence length of PT7 + RBS + Pe-4 is around 100 ~ 200 b.p. The size of its PCR product should be close to 450 ~ 500 b.p.



Reference

[1] Bai RL; Pettit GR; Hamel E. “Binding of dolastatin 10 to tubulin at a distinct site for peptide antimitotic agents near the exchangeable nucleotide and vinca alkaloid sites.” J Biol Chem. 1990 Oct 5;265(28):17141-9.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]