Construction And Evaluation Of An Engineered Pseudomonas Aeruginosa With Function And Targeted Protein Delivery

Synthetic biology aims to systematically design and build novel biological systems that can be used to solve energy,environmental,and health problems,and combining these biological systems with bacteria,through the control of bacterial behavior to achieve the purpose of production,medical treatment and even anti-tumor treatment.In this study,the genes of pelF,pslA-B,pscF,fabV in the PAO1 genome of Pseudomonas aeruginosa knocked out to construct a quadruple gene mutant strain PAO1?4,which was used as a chassis organism to construct expression elements and self-lysis systems inside it(mechanism of active cell lysis: fuse P_PA2069-prtN elements in the chromosome of strain PAO1?4;mechanism of passive cell lysis: knockout of Pf4 filaments in the genome of strain PAO1?4 bacteriophage complete coding gene cluster),thereby constructing P.aeruginosa engineered bacteria as a microbial tool for targeted protein delivery.In order to evaluate whether the biological system constructed in engineering bacteria can work,exopolysaccharide hydrolases PelA and PslG were pressed in its expression elements in this study,and the engineering bacteria PAO1102 was constructed.Working principle: During the growth of engineering bacteria PAO1102,the exopolysaccharide hydrolases PelA and PslG were largely expressed and accumulated under the action of overexpression system.Engineering bacterium grew in a stable growth period,when the cell density reached a threshold,P.aeruginosa quorum sensing system began to activate PA2069 promoter to express lysin-activating protein PrtN,which can activate cells to produce lysin and induce lysis of engineering bacteria,resulting in a large release of overexpressed extracellular polysaccharide hydrolase outside the cell,the cytoskeleton of the biofilm,Pel and Psl exopolysaccharides,were destroyed by enzymatic hydrolysis,thus the biofilm of P.aeruginosa was destroyed.In addition,when the engineered bacteria encountered Pf4 filamentous phage in the biofilm or in the environment,Pf4 filamentous phage would infect and lyse the engineered bacteria.Overexpressed extracellular polysaccharide hydrolases PelA and PslG were released and played a role in destroying the biofilm of P.aeruginosa.Finally,the damage and prevention effect of the engineered strain PAO1102 on the biofilm of P.aeruginosa PAO1 were tested by the damage experiment,inhibition experiment and antibiotic resistance experiment.The results show that compared to the control group,engineering bacteria PAO1102 can destroy 47.3% of the formed biofilm,and inhibition of P.aeruginosa PAO1 biofilm formation ability by 52.8%,and the sensitivity of tobramycin to cells in P.aeruginosa PAO1 biofilm has greatly increased,after treatment with PAO1102.These differences of the have reached a significant level.These results indicate that the engineered bacteria constructed in this research can be used as a microbial tool for targeted protein delivery.In the follow-up research,according to different needs,different target proteins can be expressed in engineering bacteria and the targeted delivery of functional proteins can be achieved to perform different biological functions.