Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Microfluidic compartmentalized directed evolution.

ABSTRACT: Directed evolution studies often make use of water-in-oil compartments, which conventionally are prepared by bulk emulsification, a crude process that generates nonuniform droplets and can damage biochemical reagents. A microfluidic emulsification circuit was devised that generates uniform water-in-oil droplets (21.9 +/- 0.8 microm radius) with high throughput (10(7)-10(8) droplets per hour). The circuit contains a radial array of aqueous flow nozzles that intersect a surrounding oil flow channel. This device was used to evolve RNA enzymes with RNA ligase activity, selecting enzymes that could resist inhibition by neomycin. Each molecule in the population had the opportunity to undergo 10(8)-fold selective amplification within its respective compartment. Then the progeny RNAs were harvested and used to seed new compartments. During five rounds of this procedure, the enzymes acquired mutations that conferred resistance to neomycin and caused some enzymes to become dependent on neomycin for optimal activity.

SUBMITTER: Paegel BM

PROVIDER: S-EPMC2912841 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Microfluidic compartmentalized directed evolution.

Paegel Brian M BM Joyce Gerald F GF

Chemistry & biology 20100701 7

Directed evolution studies often make use of water-in-oil compartments, which conventionally are prepared by bulk emulsification, a crude process that generates nonuniform droplets and can damage biochemical reagents. A microfluidic emulsification circuit was devised that generates uniform water-in-oil droplets (21.9 +/- 0.8 microm radius) with high throughput (10(7)-10(8) droplets per hour). The circuit contains a radial array of aqueous flow nozzles that intersect a surrounding oil flow channe ...[more]

PMID: 20659684

Similar Datasets

Quantifying the Performance of Micro-Compartmentalized Directed Evolution Protocols.

Project description:High-throughput, in vitro approaches for the evolution of enzymes rely on a random micro-encapsulation to link phenotypes to genotypes, followed by screening or selection steps. In order to optimise these approaches, or compare one to another, one needs a measure of their performance at extracting the best variants of a library. Here, we introduce a new metric, the Selection Quality Index (SQI), which can be computed from a simple mock experiment, performed with a known initial fraction of active variants. In contrast to previous approaches, our index integrates the effect of random co-encapsulation, and comes with a straightforward experimental interpretation. We further show how this new metric can be used to extract general protocol efficiency trends or reveal hidden selection mechanisms such as a counterintuitive form of beneficial poisoning in the compartmentalized self-replication protocol.

| S-EPMC7175308 | biostudies-literature

Compartmentalized partnered replication for the directed evolution of genetic parts and circuits.

Project description:Compartmentalized partnered replication (CPR) is an emulsion-based directed evolution method based on a robust and modular phenotype-genotype linkage. In contrast to other in vivo directed evolution approaches, CPR largely mitigates host fitness effects due to a relatively short expression time of the gene of interest. CPR is based on gene circuits in which the selection of a 'partner' function from a library leads to the production of a thermostable polymerase. After library preparation, bacteria produce partner proteins that can potentially lead to enhancement of transcription, translation, gene regulation, and other aspects of cellular metabolism that reinforce thermostable polymerase production. Individual cells are then trapped in water-in-oil emulsion droplets in the presence of primers and dNTPs, followed by the recovery of the partner genes via emulsion PCR. In this step, droplets with cells expressing partner proteins that promote polymerase production will produce higher copy numbers of the improved partner gene. The resulting partner genes can subsequently be recloned for the next round of selection. Here, we present a step-by-step guideline for the procedure by providing examples of (i) selection of T7 RNA polymerases that recognize orthogonal promoters and (ii) selection of tRNA for enhanced amber codon suppression. A single round of CPR should take ∼3-5 d, whereas a whole directed evolution can be performed in 3-10 rounds, depending on selection efficiency.

| S-EPMC6053311 | biostudies-literature

Directed co-evolution of interacting protein-peptide pairs by compartmentalized two-hybrid replication (C2HR).

Project description:Directed evolution methodologies benefit from read-outs quantitatively linking genotype to phenotype. We therefore devised a method that couples protein-peptide interactions to the dynamic read-out provided by an engineered DNA polymerase. Fusion of a processivity clamp protein to a thermostable nucleic acid polymerase enables polymerase activity and DNA amplification in otherwise prohibitive high-salt buffers. Here, we recapitulate this phenotype by indirectly coupling the Sso7d processivity clamp to Taq DNA polymerase via respective fusion to a high affinity and thermostable interacting protein-peptide pair. Escherichia coli cells co-expressing protein-peptide pairs can directly be used in polymerase chain reactions to determine relative interaction strengths by the measurement of amplicon yields. Conditional polymerase activity is further used to link genotype to phenotype of interacting protein-peptide pairs co-expressed in E. coli using the compartmentalized self-replication directed evolution platform. We validate this approach, termed compartmentalized two-hybrid replication, by selecting for high-affinity peptides that bind two model protein partners: SpyCatcher and the large fragment of NanoLuc luciferase. We further demonstrate directed co-evolution by randomizing both protein and peptide components of the SpyCatcher-SpyTag pair and co-selecting for functionally interacting variants.

| S-EPMC7736784 | biostudies-literature

Compartmentalized 3D Tissue Culture Arrays under Controlled Microfluidic Delivery.

Project description:We demonstrate an in vitro microfluidic cell culture platform that consists of periodic 3D hydrogel compartments with controllable shapes. The microchip is composed of approximately 500 discontinuous collagen gel compartments locally patterned in between PDMS pillars, separated by microfluidic channels. The typical volume of each compartment is 7.5 nanoliters. The compartmentalized design of the microchip and continuous fluid delivery enable long-term culturing of Caco-2 human intestine cells. We found that the cells started to spontaneously grow into 3D folds on day 3 of the culture. On day 8, Caco-2 cells were co-cultured for 36 hours under microfluidic perfusion with intestinal bacteria (E. coli) which did not overgrow in the system, and adhered to the Caco-2 cells without affecting cell viability. Continuous perfusion enabled the preliminary evaluation of drug effects by treating the co-culture of Caco-2 and E. coli with 34 µg ml-1 chloramphenicol during 36 hours, resulting in the death of the bacteria. Caco-2 cells were also cultured in different compartment geometries with large and small hydrogel interfaces, leading to differences in proliferation and cell spreading profile of Caco-2 cells. The presented approach of compartmentalized cell culture with facile microfluidic control can substantially increase the throughput of in vitro drug screening in the future.

| S-EPMC5469754 | biostudies-literature

3D-Printed Soft Lithography for Complex Compartmentalized Microfluidic Neural Devices.

Project description:Compartmentalized microfluidic platforms are an invaluable tool in neuroscience research. However, harnessing the full potential of this technology remains hindered by the lack of a simple fabrication approach for the creation of intricate device architectures with high-aspect ratio features. Here, a hybrid additive manufacturing approach is presented for the fabrication of open-well compartmentalized neural devices that provides larger freedom of device design, removes the need for manual postprocessing, and allows an increase in the biocompatibility of the system. Suitability of the method for multimaterial integration allows to tailor the device architecture for the long-term maintenance of healthy human stem-cell derived neurons and astrocytes, spanning at least 40 days. Leveraging fast-prototyping capabilities at both micro and macroscale, a proof-of-principle human in vitro model of the nigrostriatal pathway is created. By presenting a route for novel materials and unique architectures in microfluidic systems, the method provides new possibilities in biological research beyond neuroscience applications.

| S-EPMC7435242 | biostudies-literature

A Titratable Cell Lysis-on-Demand System for Droplet-Compartmentalized Ultrahigh-Throughput Screening in Functional Metagenomics and Directed Evolution.

Project description:Water-in-oil emulsion droplets are an attractive format for ultrahigh-throughput screening in functional metagenomics and directed evolution applications that allow libraries with more than 107 members to be characterized in a day. Single library members are compartmentalized in droplets that are generated in microfluidic devices and tested for the presence of target biocatalysts. The target proteins can be produced intracellularly, for example, in bacterial hosts in-droplet cell lysis is therefore necessary to allow the enzymes to encounter the substrate to initiate an activity assay. Here, we present a titratable lysis-on-demand (LoD) system enabling the control of the cell lysis rate in Escherichia coli. We demonstrate that the rate of cell lysis can be controlled by adjusting the externally added inducer concentration. This LoD system is evaluated both at the population level (by optical density measurements) and at the single-cell level (on single-cell arrays and in alginate microbeads). Additionally, we validate the LoD system by droplet screening of a phosphotriesterase expressed from E. coli, with cell lysis triggered by inducer concentrations in the μM range. The LoD system yields sufficient release of the intracellularly produced enzymes to bring about a detectable quantity of product (measured by fluorescence in flow cytometry of double emulsions), while leaving viable cells for the downstream recovery of the genetic material.

| S-EPMC8383311 | biostudies-literature

Directed evolution of excited state lifetime and brightness in FusionRed using a microfluidic sorter.

Project description:Green fluorescent proteins (GFP) and their blue, cyan and red counterparts offer unprecedented advantages as biological markers owing to their genetic encodability and straightforward expression in different organisms. Although significant advancements have been made towards engineering the key photo-physical properties of red fluorescent proteins (RFPs), they continue to perform sub-optimally relative to GFP variants. Advanced engineering strategies are needed for further evolution of RFPs in the pursuit of improving their photo-physics. In this report, a microfluidic sorter that discriminates members of a cell-based library based on their excited state lifetime and fluorescence intensity is used for the directed evolution of the photo-physical properties of FusionRed. In-flow measurements of the fluorescence lifetime are performed in a frequency-domain approach with sub-millisecond sampling times. Promising clones are sorted by optical force trapping with an infrared laser. Using this microfluidic sorter, mutants are generated with longer lifetimes than their precursor, FusionRed. This improvement in the excited state lifetime of the mutants leads to an increase in their fluorescence quantum yield up to 1.8-fold. In the course of evolution, we also identified one key mutation (L177M), which generated a mutant (FusionRed-M) that displayed ?2-fold higher brightness than its precursor upon expression in mammalian (HeLa) cells. Photo-physical and mutational analyses of clones isolated at the different stages of mutagenesis reveal the photo-physical evolution towards higher in vivo brightness.

| S-EPMC6141309 | biostudies-literature

Modeling Neurodegenerative Diseases Using <i>In Vitro</i> Compartmentalized Microfluidic Devices.

Project description:The human brain is a complex organ composed of many different types of cells interconnected to create an organized system able to efficiently process information. Dysregulation of this delicately balanced system can lead to the development of neurological disorders, such as neurodegenerative diseases (NDD). To investigate the functionality of human brain physiology and pathophysiology, the scientific community has been generated various research models, from genetically modified animals to two- and three-dimensional cell culture for several decades. These models have, however, certain limitations that impede the precise study of pathophysiological features of neurodegeneration, thus hindering therapeutical research and drug development. Compartmentalized microfluidic devices provide in vitro minimalistic environments to accurately reproduce neural circuits allowing the characterization of the human central nervous system. Brain-on-chip (BoC) is allowing our capability to improve neurodegeneration models on the molecular and cellular mechanism aspects behind the progression of these troubles. This review aims to summarize and discuss the latest advancements of microfluidic models for the investigations of common neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis.

| S-EPMC9263267 | biostudies-literature

A compartmentalized microfluidic neuromuscular co-culture system reveals spatial aspects of GDNF functions.

Project description:Bidirectional molecular communication between the motoneuron and the muscle is vital for neuromuscular junction (NMJ) formation and maintenance. The molecular mechanisms underlying such communication are of keen interest and could provide new targets for intervention in motoneuron disease. Here, we developed a microfluidic platform with motoneuron cell bodies on one side and muscle cells on the other, connected by motor axons extending through microgrooves to form functional NMJs. Using this system, we were able to differentiate between the proximal and distal effects of oxidative stress and glial-derived neurotrophic factor (GDNF), demonstrating a dying-back degeneration and retrograde transmission of pro-survival signaling, respectively. Furthermore, we show that GDNF acts differently on motoneuron axons versus soma, promoting axonal growth and innervation only when applied locally to axons. Finally, we track for the first time the retrograde transport of secreted GDNF from muscle to neuron. Thus, our data suggests spatially distinct effects of GDNF--facilitating growth and muscle innervation at axon terminals and survival pathways in the soma.

| S-EPMC4359927 | biostudies-literature

Microfluidic device for the formation of optically excitable, three-dimensional, compartmentalized motor units.

Project description:Motor units are the fundamental elements responsible for muscle movement. They are formed by lower motor neurons and their muscle targets, synapsed via neuromuscular junctions (NMJs). The loss of NMJs in neurodegenerative disorders (such as amyotrophic lateral sclerosis or spinal muscle atrophy) or as a result of traumatic injuries affects millions of lives each year. Developing in vitro assays that closely recapitulate the physiology of neuromuscular tissues is crucial to understand the formation and maturation of NMJs, as well as to help unravel the mechanisms leading to their degeneration and repair. We present a microfluidic platform designed to coculture myoblast-derived muscle strips and motor neurons differentiated from mouse embryonic stem cells (ESCs) within a three-dimensional (3D) hydrogel. The device geometry mimics the spinal cord-limb physical separation by compartmentalizing the two cell types, which also facilitates the observation of 3D neurite outgrowth and remote muscle innervation. Moreover, the use of compliant pillars as anchors for muscle strips provides a quantitative functional readout of force generation. Finally, photosensitizing the ESC provides a pool of source cells that can be differentiated into optically excitable motor neurons, allowing for spatiodynamic, versatile, and noninvasive in vitro control of the motor units.

| S-EPMC4972469 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data