Part:BBa_K2262009

T7 Promoter+RBS+Rs-AFP2

Figure 1. P_T7+RBS+Rs-AFP2+terminator

Introduction

      It is an anti-function peptide and is from the defensin family. The mechanism of Rs-AFP2 is to interact with GlcCer, a kind of sphingolipids on the cell membrane of the pathogen. The interaction then induces a signaling cascade which ultimately causes cell death [1]. NCTU_Formosa used it to be one of a training data for forming the antifungal peptide scoring card.

Experiment

1. Cloning

      We put T7 promoter, RBS, and Rs-AFP2 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+Rs-AFP2 is around 234 b.p. Its PCR product is around 546 b.p.

Figure 2. P_T7 + RBS + Rs-AFP2
The DNA sequence length of P_T7 + RBS + Rs-AFP2 is around 200 ~ 250 b.p. The size of its PCR product should be close to 500 ~ 550 b.p.

2. Expressing

      Because Rs-AFP2 had not been expressed by E. coli before, it was a new challenge. The strain we chose to make Rs-AFP2 was E. coli Rosetta-gami strain, which forms the disulfide bonds in the cytoplasm to express the protein. To ensure the peptide was produced, after breaking the bacteria and boiling the lysate with sample buffer and β-mercaptoethanol mixture for 15 minutes, we run SDS-PAGE to check the mass of the peptide. The mass of Rs-AFP2 is around 5.6 kDa.

Figure 3.The mass of Rs-AFP2 is around 5.6 kDa. The result shows that NO.1, No.3, and No.4 had made the peptide.

Safety

      We do the safety part which is the same as the Sa-AFP1. See more in BBa_K2262005.

Reference

[1] Ariane F. Lacerda; Érico A. R. Vasconcelos; Patrícia Barbosa Pelegrini; Maria F. Grossi de Sa. "Antifungal defensins and their role in plant defense". Front Microbiol. 2014; 5: 116.

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]