Single-cell activity screening in microfluidic droplets

Neun, Stefanie; Kamiński, Tomasz S.; Hollfelder, Florian

doi:10.1016/bs.mie.2019.07.009

Cited by 21 publications

(23 citation statements)

References 23 publications

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“…Fluorescein-di-β-glucuronide (FD-β-GlcA) was used as a bait substrate, making the detection exquisitely sensitive (requiring only 2500 product molecules per droplet). 18, 31 Rather than using one stringent sorting step, the library size was reduced in a stepwise manner accepting to gradually remove false positive hits (e.g. arising from enzymatic rates just above background) from one layer to another to enrich genuine hits progressively, in a strategy similar to functional metagenomic campaigns in formats that did not involve droplet screening.…”

Section: Resultsmentioning

confidence: 99%

Functional metagenomic screening in microfluidic droplets identifies a β-glucuronidase in an unprecedented sequence neighbourhood

Neun

Brear

Campbell

et al. 2022

Preprint

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The abundance of recorded protein sequence data stands in contrast to the small number of experimentally verified functional annotation. Here we screened a million-membered metagenomic library at ultrahigh throughput in microfluidic droplets for β-glucuronidase activity. We identified SN243, a genuine β-glucuronidase with little homology to previously studied enzymes of this type, as a glycoside hydrolase (GH) 3 family member. This GH family had no recorded evidence of β-glucuronidases at the outset of this study, showing that a functional metagenomic approach can shed light on assignments that are currently ‘unpredictable’ by bioinformatics. Kinetic analyses of SN243 characterised it as a promiscuous catalyst and structural analysis suggests regions of divergence from homologous GH3 members creating a wide-open active site. With a screening throughput of >107 library members per day, picolitre volume microfluidic droplets enable functional assignments that complement current enzyme database dictionaries and provide bridgeheads for the annotation of unexplored sequence space.

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Section: Resultsmentioning

confidence: 99%

Functional metagenomic screening in microfluidic droplets identifies a β-glucuronidase in an unprecedented sequence neighbourhood

Neun

Brear

Campbell

et al. 2022

Preprint

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“…Despite being predominantly charged at alkaline pH, 4-nitrophenolate is known to ''leak'' between droplets, meaning that it can transiently enter the oil phase and then move to neighbouring droplets. 58 Some dyes leak within seconds (aminocoumarin), some in minutes (rhodamine 6G), some over hours (resorufin) and some over days (fluorescein). 59,60 While adding charged groups to a dye is known to dramatically slow leaking (from seconds to days), the case of 4-nitrophenolate demonstrates that this is a complex and often counterintuitive phenomenon.…”

Section: Microfluidicsmentioning

confidence: 99%

“…59,60 While adding charged groups to a dye is known to dramatically slow leaking (from seconds to days), the case of 4-nitrophenolate demonstrates that this is a complex and often counterintuitive phenomenon. 58,60 Not only hydrophilicity but also size matters, demonstrated by the ability of water molecules to diffuse over the fluorinated oil barrier rather easily. 43 For simple hydrophobic molecules like haloalkanes there is a correlation between hydrophobicity (logP) and a tendency to partition into the oil phase.…”

Section: Microfluidicsmentioning

confidence: 99%

Recent trends in biocatalysis

et al. 2021

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“…Highest frequency Minimal droplet size FADS 2.5 nM (Colin et al, 2015) 5 kHz (Neun et al, 2019) 2 pl (Colin et al, 2015) AADS 10 µM (Gielen et al, 2016) 100 Hz (Gielen et al, 2016) 100 pl (Maceiczyk et al, 2017) ECDS 1 µM (Goto et al, 2020) 10 Hz (Goto et al, 2020) 30 nl (Goto et al, give the absorbing dye WST-1 formazan. The sorting frequency could achieve ∼1 million droplets per hour.…”

Section: Sensitivitymentioning

confidence: 99%

Recent Advances on Sorting Methods of High-Throughput Droplet-Based Microfluidics in Enzyme Directed Evolution

Zhang

et al. 2021

Front. Chem.

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Droplet-based microfluidics has been widely applied in enzyme directed evolution (DE), in either cell or cell-free system, due to its low cost and high throughput. As the isolation principles are based on the labeled or label-free characteristics in the droplets, sorting method contributes mostly to the efficiency of the whole system. Fluorescence-activated droplet sorting (FADS) is the mostly applied labeled method but faces challenges of target enzyme scope. Label-free sorting methods show potential to greatly broaden the microfluidic application range. Here, we review the developments of droplet sorting methods through a comprehensive literature survey, including labeled detections [FADS and absorbance-activated droplet sorting (AADS)] and label-free detections [electrochemical-based droplet sorting (ECDS), mass-activated droplet sorting (MADS), Raman-activated droplet sorting (RADS), and nuclear magnetic resonance-based droplet sorting (NMR-DS)]. We highlight recent cases in the last 5 years in which novel enzymes or highly efficient variants are generated by microfluidic DE. In addition, the advantages and challenges of different sorting methods are briefly discussed to provide an outlook for future applications in enzyme DE.

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Single-cell activity screening in microfluidic droplets

Cited by 21 publications

References 23 publications

Functional metagenomic screening in microfluidic droplets identifies a β-glucuronidase in an unprecedented sequence neighbourhood

Functional metagenomic screening in microfluidic droplets identifies a β-glucuronidase in an unprecedented sequence neighbourhood

Recent trends in biocatalysis

Recent Advances on Sorting Methods of High-Throughput Droplet-Based Microfluidics in Enzyme Directed Evolution

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