When the concentration of Fe3+ in the environment is high, it means that the hard skeleton of the microenvironment of the iron bacteria is destroyed and loses protection. At this time, the added bactericide will have a greater killing effect on the bacteria. The bactericide we selected was cecropin AD, a cationic chimeric antimicrobial peptide obtained from cecropin, consisting of the first 11 amino acid residues of cecropin A and the last 26 amino acid residues of cecropin D. It has strong antibacterial activity against Gram-positive and negative bacteria.
We insert fragment of Cecropin AD into vector pET-28a(Figure 1)
The plasmid was transformed to E. coli BL21 and cultured at 37 °C for 12 h. positive monoclonal bacteria were cultured and verified by PCR(Figure 2).
We verified the expression of Cecropin AD by SDS-PAGE(Figure 3).
We can see target band in SDS-PAGE. However, when we break engineered bacteria and use broken-bacteria supernatant to characterize, we find that it does not have the function of killing bacteria. So we synthesize cecropin AD from Genscript. Then antibacterial experiment is performed.
Iron bacteria grows adding with different concentration of cecropin AD, and growth curve is measured at the light absorption of 600nm.
From the figure, we can see that cecropin AD can repress growth of bacteria to a certain extent.
The figure shows that cecropin AD has powerful bacterividal activity. Only concentration of cecropin AD is low enough, can iron bacteria grow.