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Bioinspired genotype-phenotype linkages: mimicking cellular compartmentalization for the engineering of functional proteins.

van Vliet LD, Colin PY, Hollfelder F - Interface Focus (2015)

Bottom Line: The idea of compartmentalization of genotype and phenotype in cells is key for enabling Darwinian evolution.This contribution describes bioinspired systems that use in vitro compartments-water-in-oil droplets and gel-shell beads-for the directed evolution of functional proteins.Technologies based on these principles promise to provide easier access to protein-based therapeutics, reagents for processes involving enzyme catalysis, parts for synthetic biology and materials with biological components.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry , University of Cambridge , 80 Tennis Court Road, Cambridge CB2 1GA , UK.

ABSTRACT
The idea of compartmentalization of genotype and phenotype in cells is key for enabling Darwinian evolution. This contribution describes bioinspired systems that use in vitro compartments-water-in-oil droplets and gel-shell beads-for the directed evolution of functional proteins. Technologies based on these principles promise to provide easier access to protein-based therapeutics, reagents for processes involving enzyme catalysis, parts for synthetic biology and materials with biological components.

No MeSH data available.


Related in: MedlinePlus

A workflow for directed evolution of a hydrolase by lysate screening in droplets [42]. (a) Unit operations from the ‘toolbox’ (figure 5) are assembled to miniaturize the steps necessary for single-cell assays of library members for directed evolution. (b) Workflow: (1) the protein of interest (POI), in this case an enzyme, is expressed in E. coli; (2) single cells are compartmentalized, together with substrate and cell lysis agents; (3) droplets are incubated to generate fluorescent product and (4) re-injected into a sorting device, where hits are detected by laser-induced fluorescence and steered into the upper channel by a variable electric field; (5) plasmid DNA from selected droplets is electroporated into E. coli. Repetition of such cycles increases the stringency of selection and enriches hits gradually to identify improved enzyme variants.
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RSFS20150035F4: A workflow for directed evolution of a hydrolase by lysate screening in droplets [42]. (a) Unit operations from the ‘toolbox’ (figure 5) are assembled to miniaturize the steps necessary for single-cell assays of library members for directed evolution. (b) Workflow: (1) the protein of interest (POI), in this case an enzyme, is expressed in E. coli; (2) single cells are compartmentalized, together with substrate and cell lysis agents; (3) droplets are incubated to generate fluorescent product and (4) re-injected into a sorting device, where hits are detected by laser-induced fluorescence and steered into the upper channel by a variable electric field; (5) plasmid DNA from selected droplets is electroporated into E. coli. Repetition of such cycles increases the stringency of selection and enriches hits gradually to identify improved enzyme variants.

Mentions: Instead of providing a template for the genotype–phenotype linkage that is later used for selection, the droplet compartment can also be maintained until selection, which makes it eminently suitable for selections of enzyme catalysts. Figure 4 shows how a substrate is co-compartmentalized with the protein catalyst in a droplet, multiple turnovers occur: now selections can be carried out based on product detection. To make product detection as precise as possible, microdroplets are prepared in monodisperse form in microfluidic devices (made, for example, conveniently by soft lithography from polydimethylsiloxane [15,58,59]) and interfaced with analytical systems. Figure 5 shows building blocks of integrated microfluidic devices that have recently been built. Many steps that are normally carried out in manual laboratory routines by pipetting are now automated in ‘lab-on-a-chip’ devices that process the bioinspired cell-like droplets on-chip on an assembly line at ultra-high throughput. In addition to droplet formation, the microfluidic format allows a number of other unit operations that are summarized in figure 4. Droplets are formed at rates well above 1 kHz [52,60] and can then be divided [44], fused [45–50], incubated [48,51], analysed [52–55], sorted [14,56,57] and broken up. An attractive feature of the microfluidic droplet platform is its modularity, where individual elements of a workflow correspond to experimental steps that are represented as jigsaw pieces [43]. Each piece of the jigsaw represents a unit operation and their integration translates a macroscopic workflow to the miniaturized scale within a microfluidic device. Integration of these steps with control over timing can potentially create a versatile system for directed evolution in which complex selection schemes can be realized.Figure 4.


Bioinspired genotype-phenotype linkages: mimicking cellular compartmentalization for the engineering of functional proteins.

van Vliet LD, Colin PY, Hollfelder F - Interface Focus (2015)

A workflow for directed evolution of a hydrolase by lysate screening in droplets [42]. (a) Unit operations from the ‘toolbox’ (figure 5) are assembled to miniaturize the steps necessary for single-cell assays of library members for directed evolution. (b) Workflow: (1) the protein of interest (POI), in this case an enzyme, is expressed in E. coli; (2) single cells are compartmentalized, together with substrate and cell lysis agents; (3) droplets are incubated to generate fluorescent product and (4) re-injected into a sorting device, where hits are detected by laser-induced fluorescence and steered into the upper channel by a variable electric field; (5) plasmid DNA from selected droplets is electroporated into E. coli. Repetition of such cycles increases the stringency of selection and enriches hits gradually to identify improved enzyme variants.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4590426&req=5

RSFS20150035F4: A workflow for directed evolution of a hydrolase by lysate screening in droplets [42]. (a) Unit operations from the ‘toolbox’ (figure 5) are assembled to miniaturize the steps necessary for single-cell assays of library members for directed evolution. (b) Workflow: (1) the protein of interest (POI), in this case an enzyme, is expressed in E. coli; (2) single cells are compartmentalized, together with substrate and cell lysis agents; (3) droplets are incubated to generate fluorescent product and (4) re-injected into a sorting device, where hits are detected by laser-induced fluorescence and steered into the upper channel by a variable electric field; (5) plasmid DNA from selected droplets is electroporated into E. coli. Repetition of such cycles increases the stringency of selection and enriches hits gradually to identify improved enzyme variants.
Mentions: Instead of providing a template for the genotype–phenotype linkage that is later used for selection, the droplet compartment can also be maintained until selection, which makes it eminently suitable for selections of enzyme catalysts. Figure 4 shows how a substrate is co-compartmentalized with the protein catalyst in a droplet, multiple turnovers occur: now selections can be carried out based on product detection. To make product detection as precise as possible, microdroplets are prepared in monodisperse form in microfluidic devices (made, for example, conveniently by soft lithography from polydimethylsiloxane [15,58,59]) and interfaced with analytical systems. Figure 5 shows building blocks of integrated microfluidic devices that have recently been built. Many steps that are normally carried out in manual laboratory routines by pipetting are now automated in ‘lab-on-a-chip’ devices that process the bioinspired cell-like droplets on-chip on an assembly line at ultra-high throughput. In addition to droplet formation, the microfluidic format allows a number of other unit operations that are summarized in figure 4. Droplets are formed at rates well above 1 kHz [52,60] and can then be divided [44], fused [45–50], incubated [48,51], analysed [52–55], sorted [14,56,57] and broken up. An attractive feature of the microfluidic droplet platform is its modularity, where individual elements of a workflow correspond to experimental steps that are represented as jigsaw pieces [43]. Each piece of the jigsaw represents a unit operation and their integration translates a macroscopic workflow to the miniaturized scale within a microfluidic device. Integration of these steps with control over timing can potentially create a versatile system for directed evolution in which complex selection schemes can be realized.Figure 4.

Bottom Line: The idea of compartmentalization of genotype and phenotype in cells is key for enabling Darwinian evolution.This contribution describes bioinspired systems that use in vitro compartments-water-in-oil droplets and gel-shell beads-for the directed evolution of functional proteins.Technologies based on these principles promise to provide easier access to protein-based therapeutics, reagents for processes involving enzyme catalysis, parts for synthetic biology and materials with biological components.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry , University of Cambridge , 80 Tennis Court Road, Cambridge CB2 1GA , UK.

ABSTRACT
The idea of compartmentalization of genotype and phenotype in cells is key for enabling Darwinian evolution. This contribution describes bioinspired systems that use in vitro compartments-water-in-oil droplets and gel-shell beads-for the directed evolution of functional proteins. Technologies based on these principles promise to provide easier access to protein-based therapeutics, reagents for processes involving enzyme catalysis, parts for synthetic biology and materials with biological components.

No MeSH data available.


Related in: MedlinePlus