Revision as of 22:56, 30 October 2017

T7 Promoter+RBS+Sa-AFP1

Figure 1. P_T7+RBS+Sa-AFP1+terminator

Introduction

      This peptide possesses antifungal activity sensitive to inorganic cations from the defensin family and was proved to have an anti-fungal function. 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 Sa-AFP1 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-AFP1 is around 250 ~ 500 b.p.. Its PCR product is around 500 ~ 750 b.p..

Figure 2. P_T7 + RBS + Sa-AFP1
The DNA sequence length of P_T7 + RBS + SSa-AFP1 is around 200 ~ 250 b.p.. The size of its PCR product should be close to 500 ~ 550 b.p..

2. Expressing

      Because Sa-AFP1 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 Sa-AFP1 is around 5.7 kDa.

Figure 3.The mass of Sa-AFP1 is around 5.7 kDa. The result shows that NO.2, No.3, NO.4, No.5, and No.6 had made the peptide.

Safety

      To make sure the lysate will not contain any alive bacteria, we boiled the lysate for 15 minutes after breaking the bacteria. The result of SDS-PAGE shows the peptide still exist after boiling it. So the only thing we need to know was that whether the lysate would have alive bacteria after boiling. We put boiled lysate on an LB plate and cultured it at 37℃ for 16 hours. The result shows that there are no alive E. coli after boiling for 15 minutes.

Figure 2. The LB plate is divided into 8 parts. Each of parts is different time interval boiling in water. The control group is only LB solution. The other 7 ones are boiling for 0, 5, 10, 15, 20, 25, 30 minutes separately.

Reference

Terras FR1; Torrekens S; Van Leuven F; Osborn RW; Vanderleyden J; Cammue BP; Broekaert WF. "A new family of basic cysteine-rich plant antifungal proteins from Brassicaceae species." FEBS Lett. 1993 Feb 1;316(3):233-40.

Sequence and Features