抗菌活性放线菌的界面微滴移液互作分离筛选方法

[背景]放线菌是天然产物的宝库,目前应用于临床的天然抗生素有70％来源于放线菌的次级代谢产物.随着细菌对传统抗生素耐药问题的日趋严重,如何从自然生境中高效筛选新型活性放线菌资源并发现新型抗生素成为当前微生物学者面临的重要挑战.通过传统方法筛选活性放线菌不仅费时费力、试剂耗材消耗量大,并且筛选通量非常有限,难以对自然样品...

Isolating and screening for antimicrobial actinobacteria in interfacial micropipetting-based droplets

[Background] Actinobacteria are a treasure trove of natural products, and 70% of the natural antibiotics currently used in clinics are derived from secondary metabolites of actinomycetes. With the increasing resistance of bacteria to traditional antibiotics, how to efficiently screen new active actinomycete resources from natural habitats and discover new antibiotics has become an important challenge for microbiologists. [Objective] However, screening active actinomycetes by traditional methods not only takes time and effort, consumes large amounts of reagent consumables, but also has a very limited screening throughput, making it difficult to analyze the complex microbial community in natural samples as a whole. This study proposes a new strategy based on micro-well plate droplet array culture, which can screen antibacterial Actinobacteria with high throughput. The study analyzed the culture characteristics and screening conditions of model actinomycetes in micro-droplets, which laid the foundation for the establishment of an ultra-high-throughput bioactive Actinobacteria screening platform based on droplet array technology. [Methods] Our new solution is to use interfacial micropipetting(IMP) technology to miniaturize the traditional multi-well high-throughput screening system to 1 μL level, and generate microliter culture droplet arrays in oil-filled micro-well plates(96-well plates), each microdroplet encapsulates an actinobacterial spore or hyphae. After a short period of cultivation, the actinobacteria complete mycelial differentiation and secretion of secondary metabolites in the microdroplets. At this time, the indicator bacteria with fluorescent markers were added through the second step of IMP and the fusion of the droplets, the active target strains were located through the antagonistic screening of the whole bacteria, and the activity spectrum was converted into quantitative fluorescence values. [Results] The model actinobacteria test was found that the mycelium can reach the optimal culture state in the microdroplets and accumulate enough biomass and metabolites, which have obvious inhibitory effects on the fluorescent indicator bacteria. [Conclusion] By establishing the above-mentioned high-throughput screening technology based on microplate droplet arrays, we can quickly screen strains with antibacterial activity from the single-cell level, which significantly saves screening costs and improves screening throughput, which provides a starting point for the discovery and study of novel functional natural products.