Team:YAU-China/Security mechanism
Security mechanism
The two cleavage mechanisms we designed before are also the safety mechanisms in the experimental design, which can effectively reduce the number of engineered bacteria after destroying the biofilm.
At the same time, in order to prevent the survival engineered bacteria from escaping into the environment, we knocked out the genes related to the formation of biofilms, pelF and pslA-B, so that the engineered bacteria themselves did not produce biofilm. And their viability is reduced after the engineered bacteria escaped.
At the same time, we knocked out the enoyl-ACP reductase fabV （an endogenous triclosan resistance gene）in Pseudomonas aeruginosa and the complete fabV gene was cloned, and the Gm resistance gene on pBBR1MCS-5 was replaced to control the horizontal transfer of the resistance gene.
Pseudomonas aeruginosa type III secretion system (T3SS) is a very important virulence factor in acute human infections, we further knocked out the related proteins in the T3SS structure as the injection needle site, which can make the toxin protein of the engineered bacteria unable to enter the host cell through T3SS, and reduce the infection ability of the engineered bacteria.
In order to completely avoid the leakage of engineering bacteria and make our products safer and more efficient. The team designed a kind of biosynthetic dressing model similar to band aid. It has a double-layer structure, the outer layer is made of polymer material, which provides a barrier function equivalent to the epidermis, and has good water absorption and permeability. The inner layer is mainly composed of gelatin, inorganic silica gel and other coated engineering bacteria dry powder, which can well retain the inhaled seepage liquid to revive the engineering bacteria.
When the "band aid" is applied on the surface of the burn wound, it can absorb the wound exudate into the inner layer, thus activating the dry powder engineering bacteria. When the engineering bacteria grow to a certain stage or encounter the phage induced autolysis mechanism in the environment, the extracellular polysaccharide hydrolase accumulated in the engineering bacteria penetrates into the burn wound and destroys the biofilm, so as to improve the drug sensitivity of Pseudomonas aeruginosa, and maintain a local moist environment imitating the physiological healing of the wound, which is conducive to the regeneration of wound granulation tissue and epithelial cells, and accelerate the wound healing.
Through these methods, we are doing our utmost to reduce the harm caused by the escape of engineered bacteria, and to make our products safer to use in the treatment of biofilms.
References:
[1] Jackson K D, Starkey M, Kremer S, Parsek M R, Wozniak D J. Identification of psl, a locus encoding a potential exopolysaccharide that is essential for Pseudomonas aeruginosa PAO1 biofilm formation[J]. Journal of Bacteriology, 2004, 186(14): 4466-4475.
[2] Chung, Young I, Choi K B, Heo Y J, Cho Y H. Effect of PEL exopol ysaccharide on the wspF mutant phenotypes in Pseudomonas aeruginosa PA14[J]. Journal of Microbiology and Biotechnology, 2008, 18 (7): 1227-1234.
[3] Galle M, Carpentier I, Beyaert R. Structure and function of the type III secretion system of Pseudomonas aeruginosa [J] . Current Protein and Peptide Science, 2012, 13: 831-842.
[4] Zhu L, Lin J S, Ma J C, Cronan J E, Wang H H. Triclosan resistance of Pseudomonas aeruginosa PAO1 is due to FabV, a triclosan-resistant enoyl-acyl carrier protein reductase[J]. Antimicrobial Agents and Chemotherapy, 2010, 54(2): 689-698.