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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.


Formats for artificial covalent genotype–phenotype linkages based on droplet compartmentalization. The key initial step of both display methods is that a DNA library (coding for SNAP-tag-fused variants of the POI) is compartmentalized in water-in-oil emulsion droplets, so that each compartment contains no more than one DNA template (Poisson distribution). (a) In SNAP display [29–33], the POI is in vitro expressed from a single gene in fusion to the SNAP-tag (1). The SNAP-tag of the expressed fusion protein then reacts with its substrate, BG, that has been covalently linked to the DNA template. As a result, the SNAP-tag connects genotype and the displayed protein (responsible for the phenotype). (2) SNAP-tagged proteins are de-emulsified and challenged for binding against an antigen by affinity panning. (3) After non-binders are washed away, binders are eluted together with their encoding genes that can feed the next round of selection. (b) In BeSD display [23], the DNA is amplified by ePCR (using appropriate labelled primers), captured on the beads via a biotin–streptavidin linkage and the POI is in vitro expressed. After the emulsion is broken, beads are incubated with the labelled target and the affinity for the target is measured via fluorescence-activated sorting (FACS). The binding affinity of each recovered variant can be measured by subsequent FACS analysis on the bead display construct. The bead connects genotype and the megavalently displayed protein (responsible for the phenotype).
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RSFS20150035F3: Formats for artificial covalent genotype–phenotype linkages based on droplet compartmentalization. The key initial step of both display methods is that a DNA library (coding for SNAP-tag-fused variants of the POI) is compartmentalized in water-in-oil emulsion droplets, so that each compartment contains no more than one DNA template (Poisson distribution). (a) In SNAP display [29–33], the POI is in vitro expressed from a single gene in fusion to the SNAP-tag (1). The SNAP-tag of the expressed fusion protein then reacts with its substrate, BG, that has been covalently linked to the DNA template. As a result, the SNAP-tag connects genotype and the displayed protein (responsible for the phenotype). (2) SNAP-tagged proteins are de-emulsified and challenged for binding against an antigen by affinity panning. (3) After non-binders are washed away, binders are eluted together with their encoding genes that can feed the next round of selection. (b) In BeSD display [23], the DNA is amplified by ePCR (using appropriate labelled primers), captured on the beads via a biotin–streptavidin linkage and the POI is in vitro expressed. After the emulsion is broken, beads are incubated with the labelled target and the affinity for the target is measured via fluorescence-activated sorting (FACS). The binding affinity of each recovered variant can be measured by subsequent FACS analysis on the bead display construct. The bead connects genotype and the megavalently displayed protein (responsible for the phenotype).

Mentions: In the SNAP display [13,29,30], a display construct is assembled with the help of an in vitro compartment (figure 3a). A link between the POI and DNA is brought about by compartmentalizing a single DNA molecule in each water-in-oil emulsion microdroplet, expressing the POI in vitro and retaining both together by the microdroplet boundary. The corresponding protein is expressed as a fusion with a protein tag that reacts covalently with a label on its coding DNA (a benzylguanine (BG) [34] coupled to DNA) and the droplet compartment keeps gene and cognate protein together (assuring monoclonality). Inspired by the linkage of DNA and POI on a phage, the bare-bones SNAP-display is a reductionist model of the natural phage display system.Figure 3.


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

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

Formats for artificial covalent genotype–phenotype linkages based on droplet compartmentalization. The key initial step of both display methods is that a DNA library (coding for SNAP-tag-fused variants of the POI) is compartmentalized in water-in-oil emulsion droplets, so that each compartment contains no more than one DNA template (Poisson distribution). (a) In SNAP display [29–33], the POI is in vitro expressed from a single gene in fusion to the SNAP-tag (1). The SNAP-tag of the expressed fusion protein then reacts with its substrate, BG, that has been covalently linked to the DNA template. As a result, the SNAP-tag connects genotype and the displayed protein (responsible for the phenotype). (2) SNAP-tagged proteins are de-emulsified and challenged for binding against an antigen by affinity panning. (3) After non-binders are washed away, binders are eluted together with their encoding genes that can feed the next round of selection. (b) In BeSD display [23], the DNA is amplified by ePCR (using appropriate labelled primers), captured on the beads via a biotin–streptavidin linkage and the POI is in vitro expressed. After the emulsion is broken, beads are incubated with the labelled target and the affinity for the target is measured via fluorescence-activated sorting (FACS). The binding affinity of each recovered variant can be measured by subsequent FACS analysis on the bead display construct. The bead connects genotype and the megavalently displayed protein (responsible for the phenotype).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSFS20150035F3: Formats for artificial covalent genotype–phenotype linkages based on droplet compartmentalization. The key initial step of both display methods is that a DNA library (coding for SNAP-tag-fused variants of the POI) is compartmentalized in water-in-oil emulsion droplets, so that each compartment contains no more than one DNA template (Poisson distribution). (a) In SNAP display [29–33], the POI is in vitro expressed from a single gene in fusion to the SNAP-tag (1). The SNAP-tag of the expressed fusion protein then reacts with its substrate, BG, that has been covalently linked to the DNA template. As a result, the SNAP-tag connects genotype and the displayed protein (responsible for the phenotype). (2) SNAP-tagged proteins are de-emulsified and challenged for binding against an antigen by affinity panning. (3) After non-binders are washed away, binders are eluted together with their encoding genes that can feed the next round of selection. (b) In BeSD display [23], the DNA is amplified by ePCR (using appropriate labelled primers), captured on the beads via a biotin–streptavidin linkage and the POI is in vitro expressed. After the emulsion is broken, beads are incubated with the labelled target and the affinity for the target is measured via fluorescence-activated sorting (FACS). The binding affinity of each recovered variant can be measured by subsequent FACS analysis on the bead display construct. The bead connects genotype and the megavalently displayed protein (responsible for the phenotype).
Mentions: In the SNAP display [13,29,30], a display construct is assembled with the help of an in vitro compartment (figure 3a). A link between the POI and DNA is brought about by compartmentalizing a single DNA molecule in each water-in-oil emulsion microdroplet, expressing the POI in vitro and retaining both together by the microdroplet boundary. The corresponding protein is expressed as a fusion with a protein tag that reacts covalently with a label on its coding DNA (a benzylguanine (BG) [34] coupled to DNA) and the droplet compartment keeps gene and cognate protein together (assuring monoclonality). Inspired by the linkage of DNA and POI on a phage, the bare-bones SNAP-display is a reductionist model of the natural phage display system.Figure 3.

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.