17
In another example, small molecule-dependent sensors were engineered based on ligand receptors that are proteolytically degraded,
but stabilized upon binding their cognate ligand [138]. The resulting ligand sensors can, in turn, be fused either to a fluorescent
protein or a transcription factor to regulate the activity of a reporter
gene. Yet, this strategy heavily relies on screening millions of
mutants in Saccharomyces cerevisae using a suitable high- throughput
screening procedure, while destabilizing mutations cannot be
identified computationally. In another example, a protease-based
screening strategy has been devised in Saccharomyces cerevisae
based on controlling the export of a reporter protein to the cell
surface by cleaving off a protein export tag [139]. This screen was
specifically applied to engineer the substrate specificity of TEV
protease using sequential positive and negative selection cycles and
could be readily adapted to screen the function of synthetic protein
switches in the presence and absence of a target analyte.
Beyond FACS-based screening procedures, μ-droplet-based
screening strategies have been developed for greater control of
protein expression and reaction conditions in microfluidic devices
[140–142]. While technically challenging and not widely available
in the majority of molecular and cell biology oriented labs,
μ-droplet screening strategies carry a number of advantages that
are directly applicable to the construction of synthetic protein
switches: Firstly, the function of a synthetic protein switch including catalytic functions can be assayed extracellularly, which facilitates the control of the reaction conditions. To this end, individual
proteins can be expressed in Escherichia coli [143, 144],
Saccharomyces cerevisiae [145, 146], or mammalian cells [147] and
displayed on the cell surface or released following cell lysis. Droplet
fusion technology can, in turn, be employed to deliver a substrate
or a target analyte that is used to analyze a distinct protein function. μ-droplet screening strategies are, however, technically challenging and limited to laboratories with the specialist expertise,
especially considering only few studies have successfully screened
protein function using integrated devices that can fuse droplets,
deliver reagents, incubate for defined time periods and assay protein function such as enzymatic activity [143–147].
Beyond spectroscopic readouts based on synthetic or genetically
encoded fluorescent, luminescent, and absorbant reporter molecules, selecting for the function of enzyme-based actuators can
also be directly linked to the survival of a microorganism by
means of growth-based selection assays. Both Escherichia coli
and Saccharomyces cerevisiae are suitable for this purpose providing
a range of antibiotic and auxotrophic markers. The most widely
used selectable marker in Escherichia coli is based on β-lactamase
that can also be assayed spectroscopically and has pioneered the
design of synthetic protein switches by means of domain insertion
4.5 Growth-Based
Genetic Selection
Assays
Engineering Synthetic Protein Switches
In another example, small molecule-dependent sensors were engineered based on ligand receptors that are proteolytically degraded,
but stabilized upon binding their cognate ligand [138]. The resulting ligand sensors can, in turn, be fused either to a fluorescent
protein or a transcription factor to regulate the activity of a reporter
gene. Yet, this strategy heavily relies on screening millions of
mutants in Saccharomyces cerevisae using a suitable high- throughput
screening procedure, while destabilizing mutations cannot be
identified computationally. In another example, a protease-based
screening strategy has been devised in Saccharomyces cerevisae
based on controlling the export of a reporter protein to the cell
surface by cleaving off a protein export tag [139]. This screen was
specifically applied to engineer the substrate specificity of TEV
protease using sequential positive and negative selection cycles and
could be readily adapted to screen the function of synthetic protein
switches in the presence and absence of a target analyte.
Beyond FACS-based screening procedures, μ-droplet-based
screening strategies have been developed for greater control of
protein expression and reaction conditions in microfluidic devices
[140–142]. While technically challenging and not widely available
in the majority of molecular and cell biology oriented labs,
μ-droplet screening strategies carry a number of advantages that
are directly applicable to the construction of synthetic protein
switches: Firstly, the function of a synthetic protein switch including catalytic functions can be assayed extracellularly, which facilitates the control of the reaction conditions. To this end, individual
proteins can be expressed in Escherichia coli [143, 144],
Saccharomyces cerevisiae [145, 146], or mammalian cells [147] and
displayed on the cell surface or released following cell lysis. Droplet
fusion technology can, in turn, be employed to deliver a substrate
or a target analyte that is used to analyze a distinct protein function. μ-droplet screening strategies are, however, technically challenging and limited to laboratories with the specialist expertise,
especially considering only few studies have successfully screened
protein function using integrated devices that can fuse droplets,
deliver reagents, incubate for defined time periods and assay protein function such as enzymatic activity [143–147].
Beyond spectroscopic readouts based on synthetic or genetically
encoded fluorescent, luminescent, and absorbant reporter molecules, selecting for the function of enzyme-based actuators can
also be directly linked to the survival of a microorganism by
means of growth-based selection assays. Both Escherichia coli
and Saccharomyces cerevisiae are suitable for this purpose providing
a range of antibiotic and auxotrophic markers. The most widely
used selectable marker in Escherichia coli is based on β-lactamase
that can also be assayed spectroscopically and has pioneered the
design of synthetic protein switches by means of domain insertion
4.5 Growth-Based
Genetic Selection
Assays
Engineering Synthetic Protein Switches