Revision as of 01:11, 30 October 2017

T7 Promoter+RBS+Rs-AFP2

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

Introduction

It is a 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. NCTU_Formosa used it to be one of a training data for creating our anti-fungal 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 250 ~ 500 b.p.. Its PCR product is around 500 ~ 750 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 Sa-AFP1 was E.coli Rosetta gami strain, which can form 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.

Safety

We do the safety part which is the same as the Hv1a-lectin. See more in BBa_K2262005.

Reference

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