Enhanced and homogeneous oxygen availability during incubation of microfluidic droplets

Mahler, Lisa; Tovar, Miguel; Weber, Thomas; Brandes, Susanne; Rudolph, Martin; Ehgartner, Josef; Mayr, Torsten; Figge, Marc Thilo; Roth, Martin; Zang, Emerson

doi:10.1039/c5ra20118g

Cited by 62 publications

(60 citation statements)

References 49 publications

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“…After 3–4 h incubation (Figure S1) to give the enzyme time to react, the droplets were reinjected and sorted, with samples taken and imaged at 1 or 2 h intervals. A representative trace of the mass signal for the droplets is shown in Figure B.…”

Section: Resultsmentioning

confidence: 99%

Mass Activated Droplet Sorting (MADS) Enables High‐Throughput Screening of Enzymatic Reactions at Nanoliter Scale

et al. 2020

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Microfluidic droplet sorting enables the high‐throughput screening and selection of water‐in‐oil microreactors at speeds and volumes unparalleled by traditional well‐plate approaches. Most such systems sort using fluorescent reporters on modified substrates or reactions that are rarely industrially relevant. We describe a microfluidic system for high‐throughput sorting of nanoliter droplets based on direct detection using electrospray ionization mass spectrometry (ESI‐MS). Droplets are split, one portion is analyzed by ESI‐MS, and the second portion is sorted based on the MS result. Throughput of 0.7 samples s−1 is achieved with 98 % accuracy using a self‐correcting and adaptive sorting algorithm. We use the system to screen ≈15 000 samples in 6 h and demonstrate its utility by sorting 25 nL droplets containing transaminase expressed in vitro. Label‐free ESI‐MS droplet screening expands the toolbox for droplet detection and recovery, improving the applicability of droplet sorting to protein engineering, drug discovery, and diagnostic workflows.

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Section: Resultsmentioning

confidence: 99%

Mass Activated Droplet Sorting (MADS) Enables High‐Throughput Screening of Enzymatic Reactions at Nanoliter Scale

et al. 2020

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“…Droplet populations were incubated for several days in the dynamic droplet incubation setup 37 to ensure homogeneous and aerobic culture conditions. After verifying sufficient growth of encapsulated bacteria by dark-field imaging, we pooled all droplets of one population by inducing droplet fusion with shock freezing.…”

Section: Resultsmentioning

confidence: 99%

Detection of antibiotics synthetized in microfluidic picolitre-droplets by various actinobacteria

Mahler

Wink

Beulig

et al. 2018

Sci Rep

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The natural bacterial diversity is regarded as a treasure trove for natural products. However, accessing complex cell mixtures derived from environmental samples in standardized high-throughput screenings is challenging. Here, we present a droplet-based microfluidic platform for ultrahigh-throughput screenings able to directly harness the diversity of entire microbial communities. This platform combines extensive cultivation protocols in aqueous droplets starting from single cells or spores with modular detection methods for produced antimicrobial compounds. After long-term incubation for bacterial cell propagation and metabolite production, we implemented a setup for mass spectrometric analysis relying on direct electrospray ionization and injection of single droplets. Even in the presence of dense biomass we show robust detection of streptomycin on the single droplet level. Furthermore, we developed an ultrahigh-throughput screening based on a functional whole-cell assay by picoinjecting reporter cells into droplets. Depending on the survival of reporter cells, droplets were selected for the isolation of producing bacteria, which we demonstrated for a microbial soil community. The established ultrahigh-throughput screening for producers of antibiotics in miniaturized bioreactors in which diverse cell mixtures can be screened on the single cell level is a promising approach to find novel antimicrobial scaffolds.

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“…In the microdroplet assay, there is additional variability between replicates because the cells are encapsulated according to the Poisson distribution, and each droplet does not start with an identical number of sensor cells. A more sophisticated incubation setup for improved aeration (e.g., 48 ) may also improve the homogeneity of the conditions experienced by each droplet. Advances in absorbance-activated droplet sorting 49 may also enable application of the droplet format to systems in which the strains are not easily made fluorescent.…”

Section: Resultsmentioning

confidence: 99%

Syntrophic co-culture amplification of production phenotype for high-throughput screening of microbial strain libraries

Saleski

Kerner

Chung

et al. 2019

Preprint

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Microbes can be engineered to synthesize a wide array of bioproducts, yet production phenotype evaluation remains a frequent bottleneck in the design-build-test cycle where strain development requires iterative rounds of library construction and testing. Here, we present Syntrophic Co-culture Amplification of Production phenotype (SnoCAP). Through a metabolic cross-feeding circuit, the production level of a target molecule is translated into highly distinguishable co-culture growth characteristics, which amplifies differences in production into highly distinguishable growth phenotypes. We demonstrate SnoCAP with the screening of Escherichia coli strains for production of two target molecules: 2-ketoisovalerate, a precursor of the drop-in biofuel isobutanol, and L-tryptophan. The dynamic range of the screening can be tuned by employing an inhibitory analog of the target molecule. Screening based on this framework requires compartmentalization of individual producers with the sensor strain. We explore three formats of implementation with increasing throughput capability: confinement in microtiter plates (10 2 -10 4 assays/experiment), spatial separation on agar plates (10 4 -10 5 assays/experiment), and encapsulation in microdroplets (10 5 -10 7 assays/experiment). Using SnoCAP, we identified an efficient isobutanol production strain from a random mutagenesis library, reaching a final titer that is 5-fold higher than that of the parent strain. The framework can also be extended to screening for secondary metabolite production using a push-pull strategy.We expect that SnoCAP can be readily adapted to the screening of various microbial species, to improve production of a wide range of target molecules. We expect the cross-feeding system to have two major advantages for high-throughput screening over traditional auxotrophic biosensor implementation: i) amplification of production improvement through the co-culture's exponential growth, and ii) a wider and more tunable dynamic range. The former lies in the co-culture's amplification of differences between library members (Section 3, Calculation). Since strain development generally proceeds through incremental improvements, it is critical to be able to discern modest increases in production.Amplification of differences through the cross-feeding metabolic circuit makes relatively small improvements conspicuous. The second advantage is achieved because, when neither of the cross-fed molecules is exogenously supplied, the production rates of the cross-fed molecules generally limit total co-culture growth, keeping the target molecule within the dynamic range of the sensor strain. This is in contrast to direct application of the sensor to the supernatant of a secretor strain or in co-culture with a non-auxotrophic secretor strain when the level of target molecule will often exceed sensor dynamic range. 6 Materials and MethodsKey elements are described here. Additional details are provided in the Supplementary Information. Strains and plasmidsStrains and plasmids used are lis...

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Enhanced and homogeneous oxygen availability during incubation of microfluidic droplets

Cited by 62 publications

References 49 publications

Mass Activated Droplet Sorting (MADS) Enables High‐Throughput Screening of Enzymatic Reactions at Nanoliter Scale

Mass Activated Droplet Sorting (MADS) Enables High‐Throughput Screening of Enzymatic Reactions at Nanoliter Scale

Detection of antibiotics synthetized in microfluidic picolitre-droplets by various actinobacteria

Syntrophic co-culture amplification of production phenotype for high-throughput screening of microbial strain libraries

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