Part:BBa_K2262013

T7 Promoter+RBS+Pe-4

Figure 1. P_T7+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. P_T7 + RBS + Pe-4
The DNA sequence length of P_T7 + 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]