Part:BBa_K2262011

T7 Promoter+RBS+B-1E

Figure 1. P_T7+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. P_T7 + RBS + B-1E
The DNA sequence length of P_T7 + 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]