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bioRxiv · 10.1101/2021.05.13.443940

Microfluidic Streptomyces Cultivation for Whole Lifecycle Characterization and Phenotypic Assays Enabled by Nanogap-stabilized Air-Water Interface

Chen, D.·Nie, M.·Tang, W.·Zhang, Y.·Qiao, Y.·Yun, J.·Wang, J.·Lan, Y.·Ma, L.·Chen, Y.·Du, W.
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Abstract

Streptomyces is a model filamentous prokaryote to study multicellular differentiation and a rich reservoir for antibiotics discovery. In their natural conditions, Streptomyces grows at the interface of porous soil, air, and water. The morphological development of Streptomyces is traditionally performed on agar plates and mostly studied at the population levels. However, the detailed lifecycle of Streptomyces has not been well studied due to its complexity and lack of research tools which can mimic their natural conditions in the soil. Here, we developed a simple assembled microfluidic device for cultivation and the entire lifecycle observation of Streptomyces development from single-cell level. The microfluidic device composed of a microchannel for loading samples and supplying nutrients, microwell arrays for seeding and growth of single spores, and air-filled chambers aside of the microwells that facilitate growth of aerial hyphae and spores. A unique feature of this device is that each microwell is surrounded by a 1.5 {micro}m gap connected to an air-filled chamber which provide stabilized water-air interface. We used this device to observe the development of single Streptomyces spores and found that unlike those in bulk liquid culture, Streptomyces can differentiate at water-air interfaces in microscale liquid culture. Finally, we demonstrated that phenotypic A-Factor assay can be performed at defined time point of its lifecycle. This microfluidic device could become a robust tool for studying Streptomyces multi-cellular differentiation and interaction at single cell level. IMPORTANCEWe describe a microfluidic device that mimics the natural porous environment for the growth and development of Streptomyces, the model system for bacterial multicellularity. The microfluidic device is used for cultivation and the entire lifecycle observation of Streptomyces development from single-cell level, including growth of aerial filaments. The aerial hyphae development of Streptomyces at the water-air interface was observed at real time in the microfluidic device. The early growth, opportunistic transformation (in the gap), and merging of aerial hyphae of Streptomyces in the microfluidic device were observed for the first time. It will play an important role in finding single-cell heterogeneity to study secondary metabolites related to the complex lifecycle of Streptomyces.

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Chen, D., Nie, M., Tang, W., Zhang, Y., Qiao, Y., Yun, J., Wang, J., Lan, Y., Ma, L., Chen, Y., Du, W.. 2021-05-13. Microfluidic Streptomyces Cultivation for Whole Lifecycle Characterization and Phenotypic Assays Enabled by Nanogap-stabilized Air-Water Interface. https://doi.org/10.1101/2021.05.13.443940

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