Difference between revisions of "Part:BBa K2262011"
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__NOTOC__ | __NOTOC__ | ||
<partinfo>BBa_K2262011 short</partinfo> | <partinfo>BBa_K2262011 short</partinfo> | ||
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[[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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<h1>'''Introduction'''</h1> | <h1>'''Introduction'''</h1> | ||
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+ | <br> | ||
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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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<br> | <br> | ||
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− | B-1E got 402.91 | + | 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> | <br> | ||
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<h1>'''Experiment'''</h1> | <h1>'''Experiment'''</h1> | ||
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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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<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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− | + | 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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[[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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− | + | 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. | ||
+ | |||
+ | |||
+ | [[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, | + | 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 | + | 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> | <br> | ||
[[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> | ||
− | The DNA sequence length of P<sub>T7</sub> + RBS + B-1E is around 100 ~ 200 b.p | + | 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
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.
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.
(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.
(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.
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.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]