In eukaryotes, links in gene regulatory networks are often maintained through cooperative self-as... more In eukaryotes, links in gene regulatory networks are often maintained through cooperative self-assembly between transcriptional regulators (TRs) and DNA cis-regulatory motifs, a strategy widely thought to enable highly specific regulatory connections to be formed between otherwise weakly-interacting, low-specificity molecular components. Here, we directly test whether this regulatory strategy can be used to engineer regulatory specificity in synthetic gene circuits constructed in yeast. We show that circuits composed of artificial zinc-finger TRs can be effectively insulated from aberrant misregulation of the host cell genome by using cooperative multivalent TR assemblies to program circuit connections. As we demonstrate in experiments and mathematical models, assembly-mediated regulatory connections enable mitigation of circuit-driven fitness defects, resulting in genetic and functional stability of circuits in long-term continuous culture. Our naturallyinspired approach offers a simple, generalizable means for building evolutionarily robust gene circuits that can be scaled to a wide range of host organisms and applications.
Cooperativity in synthetic gene circuits Synthetic biologists would like to be able to make gene ... more Cooperativity in synthetic gene circuits Synthetic biologists would like to be able to make gene regulatory circuits that mimic key properties of eukaryotic gene regulation. Taking a cue from multimeric transcription factor complexes, Bashor et al. developed synthetic transcriptional circuits that produce nonlinear behavior from cooperativity (see the Perspective by Ng and El-Samad). Their system uses clamp proteins with multiple protein-interaction domains. Circuit behavior can be tuned by altering the number or affinities of the interactions according to a mathematical model. The authors created synthetic circuits with desired functions common in biology, for example, switch-like behavior or Boolean decision functions. Science , this issue p. 593 ; see also p. 531
The ability to rationally engineer microorganisms has been a long-envisioned goal dating back mor... more The ability to rationally engineer microorganisms has been a long-envisioned goal dating back more than a half-century. With the genomics revolution and rise of systems biology in the 1990s came the development of a rigorous engineering discipline to create, control and programme cellular behaviour. The resulting field, known as synthetic biology, has undergone dramatic growth throughout the past decade and is poised to transform biotechnology and medicine. This Timeline article charts the technological and cultural lifetime of synthetic biology, with an emphasis on key breakthroughs and future challenges.
The transcription of genomic information in eukaryotes is regulated in large part by chromatin. H... more The transcription of genomic information in eukaryotes is regulated in large part by chromatin. How a diverse array of chromatin regulator (CR) proteins with different functions and genomic localization patterns coordinates chromatin activity to control transcription remains unclear. Here, we take a synthetic biology approach to decipher the complexity of chromatin regulation by studying emergent transcriptional behaviors from engineered combinatorial, spatial, and temporal patterns of individual CRs. We fuse 223 yeast CRs to programmable zinc finger proteins. Site-specific and combinatorial recruitment of CRs to distinct intralocus locations reveals a range of transcriptional logic and behaviors, including synergistic activation, long-range and spatial regulation, and gene expression memory. Comparing these transcriptional behaviors with annotated CR complex and function terms provides design principles for the engineering of transcriptional regulation. This work presents a bottom-...
The living cell is an incredibly complex entity, and the goal of predictively and quantitatively ... more The living cell is an incredibly complex entity, and the goal of predictively and quantitatively understanding its function is one of the next great challenges in biology. Much of what we know about the cell concerns its constituent parts, but to a great extent we have yet to decode how these parts are organized to yield complex physiological function. Classically, we have learned about the organization of cellular networks by disrupting them through genetic or chemical means. The emerging discipline of synthetic biology offers an additional, powerful approach to study systems. By rearranging the parts that comprise existing networks, we can gain valuable insight into the hierarchical logic of the networks and identify the modular building blocks that evolution uses to generate innovative function. In addition, by building minimal toy networks, one can systematically explore the relationship between network structure and function. Here, we outline recent work that uses synthetic bio...
Eukaryotic transcription factors (TFs) perform complex and combinatorial functions within transcr... more Eukaryotic transcription factors (TFs) perform complex and combinatorial functions within transcriptional networks. Here, we present a synthetic framework for systematically constructing eukaryotic transcription functions using artificial zinc fingers, modular DNA-binding domains found within many eukaryotic TFs. Utilizing this platform, we construct a library of orthogonal synthetic transcription factors (sTFs) and use these to wire synthetic transcriptional circuits in yeast. We engineer complex functions, such as tunable output strength and transcriptional cooperativity, by rationally adjusting a decomposed set of key component properties, e.g., DNA specificity, affinity, promoter design, protein-protein interactions. We show that subtle perturbations to these properties can transform an individual sTF between distinct roles (activator, cooperative factor, inhibitory factor) within a transcriptional complex, thus drastically altering the signal processing behavior of multi-input systems. This platform provides new genetic components for synthetic biology and enables bottom-up approaches to understanding the design principles of eukaryotic transcriptional complexes and networks.
Adenine phosphoribosyltransferase (APRT, EC 2.4.2.7) catalyzes the reversible phosphoribosylation... more Adenine phosphoribosyltransferase (APRT, EC 2.4.2.7) catalyzes the reversible phosphoribosylation of adenine from R-D-5-phosphoribosyl-1-pyrophosphate (PRPP) to form AMP and PP i. Threedimensional structures of the dimeric APRT enzyme from Leishmania donoVani (LdAPRT) bear many similarities to other members of the type 1 phosphoribosyltransferase family but do not reveal the structural basis for catalysis (
In eukaryotes, links in gene regulatory networks are often maintained through cooperative self-as... more In eukaryotes, links in gene regulatory networks are often maintained through cooperative self-assembly between transcriptional regulators (TRs) and DNA cis-regulatory motifs, a strategy widely thought to enable highly specific regulatory connections to be formed between otherwise weakly-interacting, low-specificity molecular components. Here, we directly test whether this regulatory strategy can be used to engineer regulatory specificity in synthetic gene circuits constructed in yeast. We show that circuits composed of artificial zinc-finger TRs can be effectively insulated from aberrant misregulation of the host cell genome by using cooperative multivalent TR assemblies to program circuit connections. As we demonstrate in experiments and mathematical models, assembly-mediated regulatory connections enable mitigation of circuit-driven fitness defects, resulting in genetic and functional stability of circuits in long-term continuous culture. Our naturallyinspired approach offers a simple, generalizable means for building evolutionarily robust gene circuits that can be scaled to a wide range of host organisms and applications.
Cooperativity in synthetic gene circuits Synthetic biologists would like to be able to make gene ... more Cooperativity in synthetic gene circuits Synthetic biologists would like to be able to make gene regulatory circuits that mimic key properties of eukaryotic gene regulation. Taking a cue from multimeric transcription factor complexes, Bashor et al. developed synthetic transcriptional circuits that produce nonlinear behavior from cooperativity (see the Perspective by Ng and El-Samad). Their system uses clamp proteins with multiple protein-interaction domains. Circuit behavior can be tuned by altering the number or affinities of the interactions according to a mathematical model. The authors created synthetic circuits with desired functions common in biology, for example, switch-like behavior or Boolean decision functions. Science , this issue p. 593 ; see also p. 531
The ability to rationally engineer microorganisms has been a long-envisioned goal dating back mor... more The ability to rationally engineer microorganisms has been a long-envisioned goal dating back more than a half-century. With the genomics revolution and rise of systems biology in the 1990s came the development of a rigorous engineering discipline to create, control and programme cellular behaviour. The resulting field, known as synthetic biology, has undergone dramatic growth throughout the past decade and is poised to transform biotechnology and medicine. This Timeline article charts the technological and cultural lifetime of synthetic biology, with an emphasis on key breakthroughs and future challenges.
The transcription of genomic information in eukaryotes is regulated in large part by chromatin. H... more The transcription of genomic information in eukaryotes is regulated in large part by chromatin. How a diverse array of chromatin regulator (CR) proteins with different functions and genomic localization patterns coordinates chromatin activity to control transcription remains unclear. Here, we take a synthetic biology approach to decipher the complexity of chromatin regulation by studying emergent transcriptional behaviors from engineered combinatorial, spatial, and temporal patterns of individual CRs. We fuse 223 yeast CRs to programmable zinc finger proteins. Site-specific and combinatorial recruitment of CRs to distinct intralocus locations reveals a range of transcriptional logic and behaviors, including synergistic activation, long-range and spatial regulation, and gene expression memory. Comparing these transcriptional behaviors with annotated CR complex and function terms provides design principles for the engineering of transcriptional regulation. This work presents a bottom-...
The living cell is an incredibly complex entity, and the goal of predictively and quantitatively ... more The living cell is an incredibly complex entity, and the goal of predictively and quantitatively understanding its function is one of the next great challenges in biology. Much of what we know about the cell concerns its constituent parts, but to a great extent we have yet to decode how these parts are organized to yield complex physiological function. Classically, we have learned about the organization of cellular networks by disrupting them through genetic or chemical means. The emerging discipline of synthetic biology offers an additional, powerful approach to study systems. By rearranging the parts that comprise existing networks, we can gain valuable insight into the hierarchical logic of the networks and identify the modular building blocks that evolution uses to generate innovative function. In addition, by building minimal toy networks, one can systematically explore the relationship between network structure and function. Here, we outline recent work that uses synthetic bio...
Eukaryotic transcription factors (TFs) perform complex and combinatorial functions within transcr... more Eukaryotic transcription factors (TFs) perform complex and combinatorial functions within transcriptional networks. Here, we present a synthetic framework for systematically constructing eukaryotic transcription functions using artificial zinc fingers, modular DNA-binding domains found within many eukaryotic TFs. Utilizing this platform, we construct a library of orthogonal synthetic transcription factors (sTFs) and use these to wire synthetic transcriptional circuits in yeast. We engineer complex functions, such as tunable output strength and transcriptional cooperativity, by rationally adjusting a decomposed set of key component properties, e.g., DNA specificity, affinity, promoter design, protein-protein interactions. We show that subtle perturbations to these properties can transform an individual sTF between distinct roles (activator, cooperative factor, inhibitory factor) within a transcriptional complex, thus drastically altering the signal processing behavior of multi-input systems. This platform provides new genetic components for synthetic biology and enables bottom-up approaches to understanding the design principles of eukaryotic transcriptional complexes and networks.
Adenine phosphoribosyltransferase (APRT, EC 2.4.2.7) catalyzes the reversible phosphoribosylation... more Adenine phosphoribosyltransferase (APRT, EC 2.4.2.7) catalyzes the reversible phosphoribosylation of adenine from R-D-5-phosphoribosyl-1-pyrophosphate (PRPP) to form AMP and PP i. Threedimensional structures of the dimeric APRT enzyme from Leishmania donoVani (LdAPRT) bear many similarities to other members of the type 1 phosphoribosyltransferase family but do not reveal the structural basis for catalysis (
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Papers by Caleb Bashor