Source 1primary paper2014Proceedings of the National Academy of Sciences
Toolkit/phage display
phage display
Taxonomy: Technique Branch / Method. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
Phage display is an assay and selection method used during engineering workflows for light-responsive protein tools. In the cited context, it is applied alongside computational protein design and high-throughput binding assays in development of LOV2-based optogenetic systems such as improved light-induced dimers.
Usefulness & Problems
Why this is useful
This method is useful for identifying or enriching protein variants within photoswitch engineering pipelines. The evidence places it as part of a broader toolkit used to validate, improve, and apply newly designed optogenetic switches.
Source:
Cellular optogenetic switches, a novel class of biological tools, have improved our understanding of biological phenomena that were previously intractable.
Source:
We demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.
Problem solved
Phage display helps address the need to screen or select candidate binding variants during development of light-controlled protein interactions. The supplied evidence does not specify the exact library format, target, or selection stringency used in these studies.
Source:
Cellular optogenetic switches, a novel class of biological tools, have improved our understanding of biological phenomena that were previously intractable.
Source:
We demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.
Problem links
Phage display is a concrete selection method that could support rapid discovery of antibody-like binders against newly emerging microbial targets, which aligns with the gap's need to keep pace with evolving threats. Its selection/enrichment mechanism is also compatible with higher-throughput discovery workflows.
Taxonomy & Function
Primary hierarchy
Technique Branch
Method: A concrete measurement method used to characterize an engineered system.
Mechanisms
selection enrichmentTarget processes
No target processes tagged yet.
Input: Light
Implementation Constraints
cofactor dependency: cofactor requirement unknownencoding mode: genetically encodedimplementation constraint: context specific validationimplementation constraint: multi component delivery burdenimplementation constraint: spectral hardware requirementoperating role: sensorswitch architecture: multi componentswitch architecture: uncaging
The evidence indicates that protocols including phage display were provided together with fluorescent polarization and microscopy assays. No construct architecture, phage system, host strain, display valency, or illumination conditions are specified in the supplied text.
The supplied evidence does not report specific outcomes from phage display, such as enrichment factors, affinity improvements, false-positive rates, or library coverage. It also does not establish whether the method itself was independently replicated across multiple groups in the exact optogenetic application described.
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Source 2primary paper2020UNC Libraries
Ranked Claims
Cellular optogenetic switches have improved understanding of previously intractable biological phenomena.
Cellular optogenetic switches, a novel class of biological tools, have improved our understanding of biological phenomena that were previously intractable.
Optogenetic switches designed to date are based on borrowed elements from plant and bacterial photoreceptors.
they are all based on borrowed elements from plant and bacterial photoreceptors
Thorough biophysical characterization of the isolated LOV2 domain has created a strong foundation for engineering photoswitches.
its thorough biophysical characterization as an isolated domain has created a strong foundation for engineering of photoswitches
Optogenetic switches exploit endogenous light-induced photoreceptor conformational changes and repurpose their effects to different biological phenomena.
each of the optogenetic switches designed to date exploits the endogenous light induced change in photoreceptor conformation while repurposing its effect to target a different biological phenomena
The chapter provides protocols for fluorescent polarization, phage display, and microscopy optimized for validating, improving, and using newly designed photoswitches.
we provide protocols for assays including fluorescent polarization, phage display, and microscopy that are optimized for validating, improving, and using newly designed photoswitches
The chapter provides protocols for fluorescent polarization, phage display, and microscopy optimized for validating, improving, and using newly designed photoswitches.
we provide protocols for assays including fluorescent polarization, phage display, and microscopy that are optimized for validating, improving, and using newly designed photoswitches
The chapter provides protocols for fluorescent polarization, phage display, and microscopy optimized for validating, improving, and using newly designed photoswitches.
we provide protocols for assays including fluorescent polarization, phage display, and microscopy that are optimized for validating, improving, and using newly designed photoswitches
The chapter provides protocols for fluorescent polarization, phage display, and microscopy optimized for validating, improving, and using newly designed photoswitches.
we provide protocols for assays including fluorescent polarization, phage display, and microscopy that are optimized for validating, improving, and using newly designed photoswitches
The chapter provides protocols for fluorescent polarization, phage display, and microscopy optimized for validating, improving, and using newly designed photoswitches.
we provide protocols for assays including fluorescent polarization, phage display, and microscopy that are optimized for validating, improving, and using newly designed photoswitches
The chapter provides protocols for fluorescent polarization, phage display, and microscopy optimized for validating, improving, and using newly designed photoswitches.
we provide protocols for assays including fluorescent polarization, phage display, and microscopy that are optimized for validating, improving, and using newly designed photoswitches
The chapter provides protocols for fluorescent polarization, phage display, and microscopy optimized for validating, improving, and using newly designed photoswitches.
we provide protocols for assays including fluorescent polarization, phage display, and microscopy that are optimized for validating, improving, and using newly designed photoswitches
The switch was functionally demonstrated through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.
We demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.
The switch was functionally demonstrated through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.
We demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.
The switch was functionally demonstrated through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.
We demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.
The switch was functionally demonstrated through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.
We demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.
The switch was functionally demonstrated through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.
We demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.
The switch was functionally demonstrated through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.
We demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.
The switch was functionally demonstrated through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.
We demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.
The switch was created by embedding the bacterial SsrA peptide in the C-terminal helix of the Avena sativa LOV2 domain.
To create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa.
The switch was created by embedding the bacterial SsrA peptide in the C-terminal helix of the Avena sativa LOV2 domain.
To create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa.
The switch was created by embedding the bacterial SsrA peptide in the C-terminal helix of the Avena sativa LOV2 domain.
To create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa.
The switch was created by embedding the bacterial SsrA peptide in the C-terminal helix of the Avena sativa LOV2 domain.
To create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa.
The switch was created by embedding the bacterial SsrA peptide in the C-terminal helix of the Avena sativa LOV2 domain.
To create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa.
The switch was created by embedding the bacterial SsrA peptide in the C-terminal helix of the Avena sativa LOV2 domain.
To create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa.
The switch was created by embedding the bacterial SsrA peptide in the C-terminal helix of the Avena sativa LOV2 domain.
To create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa.
In the dark, the SsrA peptide is sterically blocked from binding SspB, and blue-light activation allows binding by undocking the LOV2 C-terminal helix.
In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB.
In the dark, the SsrA peptide is sterically blocked from binding SspB, and blue-light activation allows binding by undocking the LOV2 C-terminal helix.
In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB.
In the dark, the SsrA peptide is sterically blocked from binding SspB, and blue-light activation allows binding by undocking the LOV2 C-terminal helix.
In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB.
In the dark, the SsrA peptide is sterically blocked from binding SspB, and blue-light activation allows binding by undocking the LOV2 C-terminal helix.
In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB.
In the dark, the SsrA peptide is sterically blocked from binding SspB, and blue-light activation allows binding by undocking the LOV2 C-terminal helix.
In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB.
In the dark, the SsrA peptide is sterically blocked from binding SspB, and blue-light activation allows binding by undocking the LOV2 C-terminal helix.
In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB.
In the dark, the SsrA peptide is sterically blocked from binding SspB, and blue-light activation allows binding by undocking the LOV2 C-terminal helix.
In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB.
Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation.
Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation.
change in binding affinity for SspB with light stimulation twofold
Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation.
Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation.
change in binding affinity for SspB with light stimulation twofold
Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation.
Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation.
change in binding affinity for SspB with light stimulation twofold
Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation.
Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation.
change in binding affinity for SspB with light stimulation twofold
Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation.
Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation.
change in binding affinity for SspB with light stimulation twofold
Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation.
Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation.
change in binding affinity for SspB with light stimulation twofold
Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation.
Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation.
change in binding affinity for SspB with light stimulation twofold
The improved light inducible dimer iLID changes its affinity for SspB by over 50-fold with light stimulation.
create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation
change in affinity for SspB with light stimulation 50 fold
The improved light inducible dimer iLID changes its affinity for SspB by over 50-fold with light stimulation.
create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation
change in affinity for SspB with light stimulation 50 fold
The improved light inducible dimer iLID changes its affinity for SspB by over 50-fold with light stimulation.
create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation
change in affinity for SspB with light stimulation 50 fold
The improved light inducible dimer iLID changes its affinity for SspB by over 50-fold with light stimulation.
create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation
change in affinity for SspB with light stimulation 50 fold
The improved light inducible dimer iLID changes its affinity for SspB by over 50-fold with light stimulation.
create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation
change in affinity for SspB with light stimulation 50 fold
The improved light inducible dimer iLID changes its affinity for SspB by over 50-fold with light stimulation.
create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation
change in affinity for SspB with light stimulation 50 fold
The improved light inducible dimer iLID changes its affinity for SspB by over 50-fold with light stimulation.
create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation
change in affinity for SspB with light stimulation 50 fold
A crystal structure of iLID shows a critical interaction between the LOV2 surface and an engineered phenylalanine that more tightly pins the SsrA peptide against LOV2 in the dark.
A crystal structure of iLID shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark.
A crystal structure of iLID shows a critical interaction between the LOV2 surface and an engineered phenylalanine that more tightly pins the SsrA peptide against LOV2 in the dark.
A crystal structure of iLID shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark.
A crystal structure of iLID shows a critical interaction between the LOV2 surface and an engineered phenylalanine that more tightly pins the SsrA peptide against LOV2 in the dark.
A crystal structure of iLID shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark.
A crystal structure of iLID shows a critical interaction between the LOV2 surface and an engineered phenylalanine that more tightly pins the SsrA peptide against LOV2 in the dark.
A crystal structure of iLID shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark.
A crystal structure of iLID shows a critical interaction between the LOV2 surface and an engineered phenylalanine that more tightly pins the SsrA peptide against LOV2 in the dark.
A crystal structure of iLID shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark.
A crystal structure of iLID shows a critical interaction between the LOV2 surface and an engineered phenylalanine that more tightly pins the SsrA peptide against LOV2 in the dark.
A crystal structure of iLID shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark.
A crystal structure of iLID shows a critical interaction between the LOV2 surface and an engineered phenylalanine that more tightly pins the SsrA peptide against LOV2 in the dark.
A crystal structure of iLID shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark.
Approval Evidence
2 sources1 linked approval claimfirst-pass slug phage-display
we provide protocols for assays including fluorescent polarization, phage display, and microscopy
Source:
we describe the use of computational protein design, phage display, and high-throughput binding assays
Source:
protocol availabilitysupports
The chapter provides protocols for fluorescent polarization, phage display, and microscopy optimized for validating, improving, and using newly designed photoswitches.
we provide protocols for assays including fluorescent polarization, phage display, and microscopy that are optimized for validating, improving, and using newly designed photoswitches
Source:
Comparisons
Source-backed strengths
A clear strength is its integration with computational protein design and high-throughput binding assays, indicating compatibility with iterative protein engineering workflows. The cited literature also situates it within successful engineering efforts for optogenetic switches, although no quantitative performance metrics are provided here.
Source:
its thorough biophysical characterization as an isolated domain has created a strong foundation for engineering of photoswitches
Source:
To create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa.
Source:
Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation.
Source:
create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation
Compared with CLARITY technology
phage display and CLARITY technology address a similar problem space.
Shared frame: same top-level item type; same primary input modality: light
Strengths here: appears more independently replicated; looks easier to implement in practice.
Compared with Langendorff perfused heart electrical recordings
phage display and Langendorff perfused heart electrical recordings address a similar problem space.
Shared frame: same top-level item type; same primary input modality: light
Strengths here: appears more independently replicated; looks easier to implement in practice.
Compared with native green gel system
phage display and native green gel system address a similar problem space.
Shared frame: same top-level item type; same primary input modality: light
Strengths here: appears more independently replicated; looks easier to implement in practice.
Ranked Citations
- 1.
- 2.