Microfluidic platform for spatially segregated experimental evolution studies with E. coli

Seo, Seokju; Prabhakar, Ramya Ganiga; Disney-McKeethen, Saoirse; Song, Xinhao; Shamoo, Yousif

doi:10.1016/j.xpro.2022.101332

Cited by 8 publications

(7 citation statements)

References 30 publications

(37 reference statements)

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“…2a, S6) . Here, λ k represents the average number of cells per microdroplet for the corresponding cell type, for k = 1,2,… Because we use flow-focusing microfluidic devices to produce co-encapsulated populations of cells, the distribution of cell counts for each strain within the emulsion of microdroplets is assumed to follow a Poisson distribution 31,32 . The probability of a microdroplet containing n 1 Producers, and n 2 Non-Producers, and n 3 Receivers, can be calculated as follows 33 , …”

Section: Resultsmentioning

confidence: 99%

Indirect enrichment of desirable, but less fit phenotypes, from a synthetic microbial community using microdroplet confinement

Prabhakar

Fan

Alnahhas

et al. 2023

Preprint

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Spatial structure within microbial communities can provide nearly limitless opportunities for social interactions and are an important driver for evolution. As metabolites are often molecular signals, metabolite diffusion within microbial communities can affect the composition and dynamics of the community in a manner that can be challenging to deconstruct. We used encapsulation of a synthetic microbial community within microdroplets to investigate the effects of spatial structure and metabolite diffusion on population dynamics and to examine the effects of cheating by one member of the community. The synthetic community was comprised of three strains: a Producer that makes the diffusible quorum sensing molecule (N-(3-Oxododecanoyl)-L-homoserine lactone, C12-oxo-HSL) or AHL; a Receiver that is killed by AHL and a Non-Producer or cheater that benefits from the extinction of the Receivers, but without the costs associated with the AHL synthesis. We demonstrate that despite rapid diffusion of AHL between microdroplets, the spatial structure imposed by the microdroplets allow a more efficient but transient enrichment of more rare and slower growing Producer subpopulations. Eventually, the Non-Producer population drove the Producers to extinction. By including fluorescence-activated microdroplet sorting and providing sustained competition by the Receiver strain, we demonstrate a strategy for indirect enrichment of a rare and unlabeled Producer. The ability to screen and enrich metabolite Producers from a much larger population under conditions of rapid diffusion provides an important framework for the development of applications in synthetic ecology and biotechnology.

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Section: Resultsmentioning

confidence: 99%

Indirect enrichment of desirable, but less fit phenotypes, from a synthetic microbial community using microdroplet confinement

Prabhakar

Fan

Alnahhas

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Here, λ k represents the average number of cells per microdroplet for the corresponding cell type, for k = 1,2,···. Because we use flow-focusing microfluidic devices to produce co-encapsulated populations of cells, the distribution of cell counts for each strain within the emulsion of microdroplets is assumed to follow a Poisson distribution. , The probability of a microdroplet containing n 1 Producers, n 2 Non-Producers, and n 3 Receivers can be calculated as follows: p ( n 1 , n 2 , n 3 ) = ∏ 3 k = 1 normale − λ k · λ k n k n k ! We expect that ∼90% of the microdroplets will contain only Receivers, see eq S3. Since Receivers are tagged with sfGFP , if AHL does not diffuse between the microdroplets, we expect that a strong sfGFP signal will be present in that 90% microdroplet subpopulation.…”

Section: Resultsmentioning

confidence: 99%

“…25 Because we use flow-focusing microfluidic devices to produce co-encapsulated populations of cells, the distribution of cell counts for each strain within the emulsion of microdroplets is assumed to follow a Poisson distribution. 31,32 The probability of a microdroplet containing n 1 Producers, n 2 Non-Producers, and n 3 Receivers can be calculated as follows: 33…”

Section: Construction Of a Spatially Segregated Synthetic Three-strai...mentioning

confidence: 99%

Indirect Enrichment of Desirable, but Less Fit Phenotypes, from a Synthetic Microbial Community Using Microdroplet Confinement

et al. 2023

Self Cite

View full text Add to dashboard Cite

Spatial structure within microbial communities can provide nearly limitless opportunities for social interactions and are an important driver for evolution. As metabolites are often molecular signals, metabolite diffusion within microbial communities can affect the composition and dynamics of the community in a manner that can be challenging to deconstruct. We used encapsulation of a synthetic microbial community within microdroplets to investigate the effects of spatial structure and metabolite diffusion on population dynamics and to examine the effects of cheating by one member of the community. The synthetic community was composed of three strains: a “Producer” that makes the diffusible quorum sensing molecule (N-(3-oxododecanoyl)-l-homoserine lactone, C12-oxo-HSL) or AHL; a “Receiver” that is killed by AHL; and a Non-Producer or “cheater” that benefits from the extinction of the Receivers, but without the costs associated with the AHL synthesis. We demonstrate that despite rapid diffusion of AHL between microdroplets, the spatial structure imposed by the microdroplets allows a more efficient but transient enrichment of more rare and slower-growing Producer subpopulations. Eventually, the Non-Producer population drove the Producers to extinction. By including fluorescence-activated microdroplet sorting and providing sustained competition by the Receiver strain, we demonstrate a strategy for indirect enrichment of a rare and unlabeled Producer. The ability to screen and enrich metabolite Producers from a much larger population under conditions of rapid diffusion provides an important framework for the development of applications in synthetic ecology and biotechnology.

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“…Thus, they should sufficiently adhere to each other. However, we recommend using a protocol for PDMS / PDMS bonding if a plasma cleaner is available (a detailed protocol can be found in 17 ). Fill the chambers with 50 μL each of Pluronic acid solution (S2) and incubate for 2 h at room temperature.…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

Protocol for extracting live blastoderm cells from embryos of annual killifish

Vásquez-Sepúlveda,

Guerrero,

Lemus

et al. 2023

STAR Protocols

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Microfluidic platform for spatially segregated experimental evolution studies with E. coli

Cited by 8 publications

References 30 publications

Indirect enrichment of desirable, but less fit phenotypes, from a synthetic microbial community using microdroplet confinement

Indirect enrichment of desirable, but less fit phenotypes, from a synthetic microbial community using microdroplet confinement

Indirect Enrichment of Desirable, but Less Fit Phenotypes, from a Synthetic Microbial Community Using Microdroplet Confinement

Protocol for extracting live blastoderm cells from embryos of annual killifish

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