Part:BBa_K2262013

T7 Promoter+RBS+Pe-4

Figure 1. P_T7+RBS+Pe-4+terminator

Introduction

      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.

Scoring Card Predict

      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.

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

      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.

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

(2) Spore Germination

      To know whether the anti-fungal peptides can inhibit the germination of spores, we conducted the spore germination experiment.

(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 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.

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+Sa-AFP2 is around 100 ~ 200 b.p.. Its PCR product is around 250 ~ 500 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..

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]