Lysis gene codes for the colicin-producing bacteria and then activates the expression of colicin. Colicin is a type of bacteriocin produced by and toxic to some strains of Escherichia coli. Colicins are released into the environment to reduce competition from other bacterial strains. Colicins bind to outer membrane receptors, using them to translocate to the cytoplasm or cytoplasmic membrane, where they exert their cytotoxic effect, including depolarisation of the cytoplasmic membrane, DNase activity, RNase activity, or inhibition of murein synthesis. colicins contain at least three domains: an N-terminal translocation domain responsible for movement across the outer membrane and periplasmic space; a central domain responsible for receptor recognition; and a C-terminal cytotoxic domain responsible for channel formation in the cytoplasmic membrane. One domain regulates the target and binds to the receptor on the sensitive cell. The second is involved with translocation, co-opting the machinery of the target cell. The third is the 'killing' domain and may produce a pore in the target cell membrane, or act as a nuclease to chop up the DNA or RNA of the target cell.
In our system, we design to utilize lysin to realize autolyse of engineered bacteria. It has fur box in promoter and is under regulate of ferrous iron in cytoplasm. After cells autolyse, accumulated DspB and cecropin AD can be released and then kill iron bacteria.
In order to test the biofilm removal effect of DspB, we constructed the vector pET28a-dspB
The plasmid was transformed into E. coli BL21, cultured at 37 °C for 12 h, and the plasmid was extracted and verified by PCR.
In order to test the autolysis effect of Lysin, we constructed the vector pET28a-Lysin.