Establishment Of Metagenomic Screening System Based On Droplet Microfluidics

Microbial ecosystems are viewed as enormous reservoirs of genetic diversity.99% of the natural microorganisms cannot be cultured.Metagenomics analyzes microorganisms by direct extraction and cloning of their DNA.The analytical methodology includes sequencing and functional screening.Extensive sequencing efforts using DNA that was directly extracted from environmental samples have already given unprecedented insight into the make-up and genetic diversity of such ecosystems.However,extrapolation from sequence to gene function is often proved unreliable.As a result,metagenomic functional screening has advantages in exploring bioactive products in microbial ecosystems.Today,microfluidics facilitates functional screening develop in miniaturization,automation and high-throughput.Inspired by such development in metagenomics,we propose a new method to conduct functional screening on metagenomic library.The proposal consists of construction of metagenomic library from soil sample,double emulsified droplets enveloping clones and functional screening on microfluidics.In this thesis,we extracted DNA from soil sample and then constructed metagenomic library of 1200 Mbp.As for screening,stable double emulsified droplets created by microfluidic chip were successfully sorted by flow cytometry according to fluorescent signal,which implys further applications in metagenomic screening.We also established a microfluidic screening system that propels sample by negative pressure and selects fluorescent cells by the mechanism that fluorescent detection changes sample‘s flow direction by energizing solenoid valve.The application of the system will lay the foundation for miniaturatin and automation of flow cytometry.