Difference between revisions of "Part:BBa K2262011"
 
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__NOTOC__
 
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<partinfo>BBa_K2262011 short</partinfo>
 
<partinfo>BBa_K2262011 short</partinfo>
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<br>
  
 
[[File:Composite part B-1E.png|800px|thumb|center|'''Figure 1.''' P<sub>T7</sub>+RBS+B-1E+terminator  ]]
 
[[File:Composite part B-1E.png|800px|thumb|center|'''Figure 1.''' P<sub>T7</sub>+RBS+B-1E+terminator  ]]
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<br>
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<br>
  
 
<h1>'''Introduction'''</h1>
 
<h1>'''Introduction'''</h1>
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<br>
  
 
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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It has been known as an antibacterial peptide that has activity against representative Gram-negative and Gram-positive bacterial species. 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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B-1E an antibacterial peptide that has activity against representative Gram-negative and Gram-positive bacterial species [1]. The score of SCM analysis shows its promising potential to be antifungal.
  
 
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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B-1E got 402.91 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 B-1E has high probability to have an anti-fungal function.
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B-1E got 402.91 from the Antifungal Scoring Card of Parabase System. The score is over 353, the threshold to divide whether a peptide has an antifungal function probability, which means B-1E has a high probability to have an antifungal function.
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<br>
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<br>
  
 
[[File:Scoreing card B-1E.png|800px|thumb|center|'''Figure 2.''' The sequence of B-1E. <br>  
 
[[File:Scoreing card B-1E.png|800px|thumb|center|'''Figure 2.''' The sequence of B-1E. <br>  
 
The colors of each amino acids represent the probability of anti-fungal dipeptides. ]]
 
The colors of each amino acids represent the probability of anti-fungal dipeptides. ]]
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<br>
  
 
<h1>'''Experiment'''</h1>
 
<h1>'''Experiment'''</h1>
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<br>
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<p style="padding:1px;font-size:16px"><b>1. Fungal Test </b></p>
 
<p style="padding:1px;font-size:16px"><b>1. Fungal Test </b></p>
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<br>
  
 
<p style="padding:1px;font-size:14px"><b>(1) Inhibition Zone </b></p>
 
<p style="padding:1px;font-size:14px"><b>(1) Inhibition Zone </b></p>
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<br>
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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 different concentrations of B-1E 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 B-1E will have a hole. It means the peptide will affect the growth of mycelium.
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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The result shows that the place we put B-1E will have a hole. It means the peptide will affect the growth of mycelium.
  
 
[[File:Inhibition zone B-1E.png|400px|thumb|center|'''Figure 3.''' The result shows that B-1E will affect the growth of mycelium. ]]
 
[[File:Inhibition zone B-1E.png|400px|thumb|center|'''Figure 3.''' The result shows that B-1E will affect 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 B-1E 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.
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We can get the same result from adding B-1E with a concentration of 500 μg/mL and 250 μg/mL.
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[[File:Spore Germination.png|900px|thumb|center|'''Figure 4.''' The result shows that B-1E will 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 B-1E and negative control, Double-distilled water, on the flower. The negative controlis on the left hand and B-1E 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 B-1E, is not infected. It means B-1E has an anti-fungal function.
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In order to test our anti-fungal peptides, we put B-1E and negative control, double-distilled water, on the flower. The negative control is on the left and B-1E 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 B-1E, is not infected. It means B-1E has an anti-fungal function in real plant organism.
  
  
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We put T7 promoter, RBS, and B-1E 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+B-1E 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 B-1E 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+B-1E is around 153 b.p. Its PCR product is around 465 b.p.
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<br>
 
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[[File:Agarose gel B-1E.png|200px|thumb|center|'''Figure 6.''' P<sub>T7</sub> + RBS + B-1E <br>
 
[[File:Agarose gel B-1E.png|200px|thumb|center|'''Figure 6.''' P<sub>T7</sub> + RBS + B-1E <br>
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The DNA sequence length of P<sub>T7</sub> + RBS + B-1E 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 + B-1E 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] Morikawa N; Hagiwara; Nakajima T. “Brevinin-1 and -2, unique antimicrobial peptides from the skin of the frog, Rana brevipoda porsa.” Biochem Biophys Res Commun. 1992 Nov 30;189(1):184-90.
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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 15:09, 7 December 2017


T7 Promoter+RBS+B-1E


Figure 1. PT7+RBS+B-1E+terminator



Introduction


      B-1E an antibacterial peptide that has activity against representative Gram-negative and Gram-positive bacterial species [1]. The score of SCM analysis shows its promising potential to be antifungal.


Scoring Card Predict

      B-1E got 402.91 from the Antifungal Scoring Card of Parabase System. The score is over 353, the threshold to divide whether a peptide has an antifungal function probability, which means B-1E has a high probability to have an antifungal function.

Figure 2. The sequence of B-1E.
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 B-1E will have a hole. It means the peptide will affect the growth of mycelium.

Figure 3. The result shows that B-1E will affect the growth of mycelium.


(2) Spore Germination


      The result shows that the spore germination percentage of non-germinating spores adding B-1E 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. We can get the same result from adding B-1E with a concentration of 500 μg/mL and 250 μg/mL.


Figure 4. The result shows that B-1E will affect the germination of spore.



(3) Botany Experiment


      In order to test our anti-fungal peptides, we put B-1E and negative control, double-distilled water, on the flower. The negative control is on the left and B-1E 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 B-1E, is not infected. It means B-1E has an anti-fungal function in real plant organism.


Figure 5. The result shows that B-1E will affect fungal infection.



2. Cloning

      We put T7 promoter, RBS, and B-1E 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+B-1E is around 153 b.p. Its PCR product is around 465 b.p.

Figure 6. PT7 + RBS + B-1E
The DNA sequence length of PT7 + RBS + B-1E is around 100 ~ 200 b.p. The size of its PCR product should be close to 450 ~ 500 b.p.



Reference

[1] Morikawa N; Hagiwara; Nakajima T. “Brevinin-1 and -2, unique antimicrobial peptides from the skin of the frog, Rana brevipoda porsa.” Biochem Biophys Res Commun. 1992 Nov 30;189(1):184-90.

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]