Study On Separation And Screening Method Of Microdroplet Array For Antibacterial Active Actinomycetes

Actinomycetes are a kind of vital microbial resources.Actinomycete mycelium produces secondary metabolites in the middle and late stages of growth to counteract the adverse conditions of the surrounding environment to ensure its own better growth,and is widely used in human production and life.It is very important to screen the actinomycetes with antibacterial activity.Most of the screening methods currently used are based on large-volume liquid or solid culture,and the screening effect has certain limitations The development of microfluidic droplet technology has provided new experimental methods and ideas for the screening of antibacterial active actinomycetesIn this study,using microfluidic droplet technology as a guide,a screening method using microfluidic to screen interactive actinomycetes was established.In this study,Pseudomonas orientalis JCM 4235,Streptomyces hygroscopicus FXJ1.264,and the fluorescent strain Staphylococcus aureus RN4220 are the main research subjects.The following four aspects are mainly studied:(1)Use droplets of different volumes to culture and grow microorganisms.After comprehensive consideration,it was found that the actinomycete mycelium can grow normally in 5nL and 1?L droplets In a 1?L droplet,S.aureus RN4220 can keep the fluorescent plasmid intact and ensure its good fluorescence indicator.(2)In this study,a screening method for microfluidic droplets to screen interacting actinomycetes was constructed.The droplets of 1?L were used to culture the actinomycetes for 7-14 days.After the normal growth of the actinomycete hyphae was observed.After adding a 1?L concentration dilution,approximately 105 cells/mL of S.aureus RN4220 was cultured overnight(12 hours).Observation and detection of fluorescence intensity combined with the method of 16S rRNA gene sequencing.Through the combination of the two methods,it is proved that obvious positive results and negative results can be obtained,and the construction of the screening method is proved to be effective(3)Constructed a PCR system that directly uses droplets for PCR amplification,eliminating the need for genome extraction procedures,can be used with droplet micro-reactions for subsequent detection,and feedback based on experimental results.The success rate of PCR amplification of droplet PCR systems about 95%,and can successfully PCR amplification of droplets grown by actinomycetes(4)The actual soil samples were used to verify the system construction,and 96-well plates were used to generate,detect,and observe droplets during the verification process.In combination with the droplet PCR system developed during the experiment,barcode primer design was performed for the result detection process,and the 96-well plate plates and wells were labeled with primers,which was helpful for subsequent analysis and screeningIn this study,using microfluidic droplet technology as a guide,a screening method for screening interactive actinomycetes using microfluidics was established.The traditional screening process was miniaturized to make unknown resistant actinomycetes more efficient and faster.The screening work laid the foundation.