The screening of microbial growth phenotype is a rate-limiting step in the fields of industrial breeding, enzyme directed evolution and synthetic biology. Accurate measurement of single cell growth phenotype is the key to break through the above bottlenecks. Recently, the single-cell center of Qingdao Institute of Biological Energy and process of the Chinese Academy of Sciences has developed a low-cost, unmarked micro-droplet microfluidic platform. The platform can complete the screening of microbial growth phenotype at single-cell level through the steps of single-cell microdroplet culture, droplet autofluorescence detection and target microdroplet automatic sorting, and demonstrate the accuracy and reliability of this method in Escherichia coli. It provides a powerful means for rapid screening of industrial strains. The relevant research results are published in <Sensors and Actuators B: chemistry> (Sensors and Actuators B: Chemical).
The breeding of high quality strains should meet the characteristics of “fast growth” and “high yield”. The traditional macroscopic system obtains the phenotypic characteristics of growth and metabolism by measuring biomass and product concentration, but the flux is low and it is difficult to truly reflect the state of the strain. Single-cell microbial phenotypic screening can accurately evaluate the growth status and metabolic characteristics of microorganisms, and then realize the breeding of dominant strains. At present, high-throughput phenotypic screening at single-cell level is limited to metabolic phenotype, such as obtaining “high-yield” strains by fluorescence activated droplet sorting (FADS). There is still a lack of high-throughput phenotypic screening programs at single-cell level, so it is difficult to achieve high-throughput breeding of “fast-growing” strains.
In this study, a low-cost and unlabeled droplet microfluidic screening platform was developed based on the autofluorescence of microorganisms in droplets. The platform can complete the high-throughput screening of microbial growth phenotype. The specific process is as follows: the microbial single cell is wrapped in microdroplets and cultured separately by droplet microfluidic technology; the cultured droplets are injected into the microfluidic chip, and the self-fluorescence of microdroplets irradiated by 365 nm laser is collected in the detection area; the optical signal is transmitted to PMT through optical fiber, and the automatic sorting of target droplets is realized by programming software and high frequency voltage control.
The research system evaluates the core performance of the above platform. In the aspect of verification of separation flux, the highest signal acquisition frequency of droplet autofluorescence is 1000 Hz, and the highest frequency of droplet separation is 200 Hz. In terms of separation reliability, if the separation threshold is set to distinguish whether the droplets contain microorganisms or not, the percentage of microbial droplets in the separation channel is 95.3%, and the percentage of empty droplets in the waste channel is 91.7%. If the sorting threshold is set to distinguish whether it is a fast-growing microorganism, only 1.58% of the droplets in the sample before separation wrap the fast-growing microorganisms, 11.46% of the droplets wrap the slow-growing microorganisms, and the rest are empty droplets. after separation, the percentage of droplets wrapped in fast-growing microbial cells in the separation channel increased to 90.78%. The percentages of droplets and empty droplets wrapped in slow-growing microorganisms were 8.25% and 0.97%, respectively.
In the previous research, a series of Raman flow single cell sorting technique FlowRACS was developed. This technique can identify and select strains with high content of single cell metabolites (“high yield”) with high throughput and high efficiency under unlabeled conditions, and by coupling the rapid screening technique of single cell horizontal growth phenotype developed in this work, the “fast-growing” strains can be further screened, so as to achieve the goal of industrial microbial breeding. Relying on the above series of innovative technologies, researchers will further develop the key technologies and equipment of the single-cell level strain selection platform to support the development of industrial biotechnology and synthetic biological industry.
