Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries

Abstract

The identification and engineering of proteins having refined or novel characteristics is an important area of research in many scientific fields. Protein modelling has enabled the rational design of unique proteins, but high-throughput screening of large libraries is still required to identify proteins with potentially valuable properties. Here we report on the development and evaluation of a novel fluorescent activated cell sorting based screening platform. Single bacterial cells, expressing a protein library to be screened, are electronically sorted and deposited onto plates containing solid nutrient growth media in a dense matrix format of between 44 and 195 colonies/cm2. We show that this matrix format is readily applicable to machine interrogation (<30 seconds per plate) and subsequent bioinformatic analysis (~60 seconds per plate) thus enabling the high-throughput screening of the protein library. We evaluate this platform and show that bacteria containing a bioluminescent protein can be spectrally analysed using an optical imager, and a rare clone (0.5% population) can successfully be identified, picked and further characterised. To further enhance this screening platform, we have developed a prototype electronic sort stream multiplexer, that when integrated into a commercial flow cytometric sorter, increases the rate of colony deposition by 89.2% to 24 colonies per second. We believe that the screening platform described here is potentially the foundation of a new generation of high-throughput screening technologies for proteins.

Double emulsion flow cytometry with high-throughput single droplet isolation and nucleic acid recovery

“… 47 Single droplet plate counts ( Fig. 5C and Table S3† ) are consistent with prior recovery efficiency estimates for single-cell deposition via FACS, 50 – 52 suggesting sdDE-FACS has reached instrument limits.…”

“…sdDE-FACS enables sensitive droplet phenotyping and single droplet isolation by several critical departures from previous droplet FACS attempts, with guidance from suggestions in both empirical and theoretical droplet sorting literature, including use of a large sort nozzle and low flow rates to reduce shear, adjustment of FACS parameters such as delay time and scatter thresholds to increase sort accuracy and target selection, and optimization of droplet surfactant formulations to reduce breakage during sorting. Using these optimizations, sdDE-FACS achieves 1) >25% increase in sorting throughput to prior work, 45 with observed droplet breakage during sorting reduced from as high as 60% in literature 7 to near-absent in our post-recovery images, 2) a 10-fold improved signal sensitivity to prior work 45 and 5 orders of magnitude of droplet signal discrimination, 3) >70% droplet sort recovery efficiency, previously undemonstrated in droplet FACS literature and equivalent to cellular isolation efficiency limits in single cell FACS, 29 , 42 , 50 – 52 4) >99% target specificity in DE variant identification, selection, and downstream isolation via FACS.…”

“…sdDE-FACS discriminates droplet fluorescence signals spanning 5 orders of magnitude and recovers droplets with ~60–70% efficiency and over 97% target specificity, equivalent to the capabilities of single cell FACS. 35 , 42 , 50 – 52 In addition, downstream qPCR measurements on bulk and individual sorted droplets establish that all droplet-encapsulated DNA can be completely recovered, with little to no evidence of well-to-well cross-contamination. To our knowledge, this represents the first demonstration of high-efficiency single DE droplet sorting and complete nucleic acid recovery via FACS.…”

“…Typical single-cell FACS sort efficiencies range from 50-90% by cellular type and size, with lower sorting efficiencies for larger particles. 35,42,[50][51][52] For the SH800, the bead-calibrated droplet delay was optimal for DE post-sort recovery efficiency for all droplet populations. By contrast, sorting efficiency on the Aria II was optimal at droplet delays significantly different from the Accudrop values, likely due to different flow metering and acoustic breakoff mechanisms between the two instruments ( Fig.…”

Development of a shear stress-free microfluidic gradient generator capable of quantitatively analyzing single-cell morphology

“…In the past two decades, high-throughput screening (HTS) and high-content screening (HCS) have become major landmarks in the field of drug discovery, leading to fast identification of new therapeutic molecules and novel genetic engineering strategies (Zhao et al 2015 ; Lovitt et al 2013 ; Carlson-Stevermer et al 2016 ; Macchi et al 2016 ). This has largely been accomplished by miniaturization and automation, for example by developing large multiwell plate-based screens (Nishihara et al 2016 ; Vrij et al 2016 ; Spencer et al 2016 ), customized biomolecule/cell arrays (Beachley et al 2015 ; Zhao et al 2015 ; Kwon et al 2011 ), cell sorting (Liu et al 2016 ; Stowe et al 2015 ; Chuang et al 2014 ) and microfluidics (Du et al 2016 ; Barata et al 2016 ). Microfluidics has made an important contribution to HTS and HCS methodologies by enabling experiments with small amounts of reagents and low cell numbers.…”