A Microfluidic Platform for High-Throughput Screening of Small Mutant Libraries

Abstract

The screening and isolation of target microorganisms from mutated recombinant libraries are crucial for the advancement of synthetic biology and metabolic engineering. However, conventional screening tools present several limitations in throughput, cost, and labor. Herein, we describe a novel microfluidic high-throughput screening (HTS) platform with several advantages. The platform utilizes a fluid array to compartmentalize bacterial cells in well-ordered separated microwells and allows long-term cell culture with high throughput. The platform enables the extraction of selected target cells from the fluid array for additional culture and postanalysis by using a capillary-driven sample relocation method. To confirm the feasibility of the platform, we demonstrated two different types of HTS methods based on the levels of reporter gene expression and cellular growth rate difference. For the reporter gene-based HTS, a spike recovery approach was taken to demonstrate that target cells are successfully screened out from a mixture containing nontarget cells by repeating the culture and extraction processes. Additionally, the same platform allowed us to screen and sort target cells according to their cellular growth rate difference, which seems hard in conventional screening methods. Hence, the platform could be used for various microbiological assays, including the detection of cell-excreted metabolites, microbial biosensors, and other HTS systems.

Droplet-based microfluidic analysis and screening of single plant cells

“…While microdroplet based microfluidics has previously been applied to unicellular photosynthetic eukaryotes [ 21 ] the work presented here allows for rapid screening of synthetic circuits designed for operation in multicellular plants. Although further work will be required to assess the capacity for regeneration of whole plants from individual sorted protoplasts, this work provides a fundament for the development of techniques reducing the time and cost involved in the screening of transgenic plants, by alleviating the need to maintain and cultivate large quantities of callus prior to selection.…”

High-Throughput Screening of Acyl-CoA Thioesterase I Mutants Using a Fluid Array Platform

“…The device was then flipped over to another Petri dish filled with hydrocarbon oil (hexadecane, SG: 0.76) to retain the aqueous volume of the fluid array from the dehydration, so that top 1% of selective extractions of extraordinary mutants can be performed using glass capillary tubes for post-analysis of mutant cells. Because the recovery of the isolated cells was already optimized in the previous work, 22 the isolated mutants were cultured in fresh medium. Finally, we identified the mutated region of ‘ tesA of the isolated mutants for further investigation of the effects of mutations on FFA production.…”

“…We previously described a fluid array platform that had been specifically designed for the HTS of a large, microbial random mutation library. 22 The simplicity of device fabrication and the ease of identifying extraordinary samples mean that the fluid array platform was successful in several different HTS applications that involved the growth-based and reporter-based screening of a microbial mutant library. 23,24…”

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

“…In microbiology, the selection and isolation of rare or unique microbes after observation becomes important in many applications that require a connection between genomic information and observable phenotypic information. These applications include selecting phenotypically rare strains from mutant libraries 2 , selecting keystone microorganisms from complex microbial communities 3 , 4 , and selecting phenotypically rare but important bacteria from isogenic populations. Isolation of viable but nonculturable cells (VBNC) from a bacteria population is an important example of the latter, where cells with the VBNC phenotype are often hidden in bacteria populations at 1:10 2 to 1:10 5 ratios 5 , 6 .…”