Desmonstration

Demonstration

For the whole circuit construct, we did following experiments:

1. Gel electrophoresis

2. Expression of LL-37 in bacteria screened by dot blotting

Fig. 2: Result of dot blot. The left light ring shows the protein was produced and the right dark region stands for the non-induced (negative control). The result shows that the whole circuit can be induced by IPTG

3. Inhibition Discs

This experiment was to qualitatively evaluate the effectivity of Anti-Microbial Peptides (AMPs).

Fig. 3: Inhibition discs of anti-microbial peptides on Staphylococcus aureus. The concentration of peptides is 1mg/ml and volume is 20ul. (A) LL-37 (B) Grammistin-Pp1 (C) GF-17 (D) three peptides (LL-37, Grammistin-Pp1, and GF-17) mix.

Fig. 3 shows the AMPs have effectivity on Staphylococcus aureus.

Aimed at examining the efficacy of the antimicrobial peptides on killing S. aureus, each experiment below were repeated 3 times.

4. Determination of the minimal bactericidal concentration (MBC) of the three AMPs.

Both single peptide of the three AMPs and the mixture of them had conspicuous lethal effect on S. aureus.

5. Absorbance (cell density) of the biofilm of S. aureus against different concentrations of the three individual AMPs and a mixture of them

The biofilm formation of S. aureus was to a great extent inhibited by these three AMPs.

Experience and conclusions:

Peptides has shown their antimicrobial activity on S. aureus. And they can successfully expressed in E. coli.
AgrA, which is an essential protein in quorum sensing construct, signifies toxicity to cells during growth.
We have trouble in obtaining enough plasmid DNA extracted from L. lactis after transformation, probably due to either low copy number or low yield from the extraction protocol.

Collaborators and Supporters

Team:XJTLU-CHINA/Demonstrate