Christina Smolke

All Publications

• A quantitative framework for the forward design of synthetic miRNA circuits NATURE METHODS Bloom, R. J., Winkler, S. M., Smolke, C. D. 2014; 11 (11): 1147-1153

  View details for DOI 10.1038/NMETH.3100

  View details for Web of Science ID 000344580600022

• A microbial biomanufacturing platform for natural and semisynthetic opioids NATURE CHEMICAL BIOLOGY Thodey, K., Galanie, S., Smolke, C. D. 2014; 10 (10): 837-U78

  View details for DOI 10.1038/NCHEMBIO.1613

  View details for Web of Science ID 000342462100010

• Realizing the potential of synthetic biology NATURE REVIEWS MOLECULAR CELL BIOLOGY Church, G. M., Elowitz, M. B., Smolke, C. D., Voigt, C. A., Weiss, R. 2014; 15 (4): 289-294

  Abstract

  Synthetic biology, despite still being in its infancy, is increasingly providing valuable information for applications in the clinic, the biotechnology industry and in basic molecular research. Both its unique potential and the challenges it presents have brought together the expertise of an eclectic group of scientists, from cell biologists to engineers. In this Viewpoint article, five experts discuss their views on the future of synthetic biology, on its main achievements in basic and applied science, and on the bioethical issues that are associated with the design of new biological systems.

  View details for DOI 10.1038/nrm3767

  View details for Web of Science ID 000333586000012

  View details for PubMedID 24622617

• Kinetic and Equilibrium Binding Characterization of Aptamers to Small Molecules using a Label-Free, Sensitive, and Scalable Platform ANALYTICAL CHEMISTRY Chang, A. L., McKeague, M., Liang, J. C., Smoke, C. D. 2014; 86 (7): 3273-3278

  Abstract

  Nucleic acid aptamers function as versatile sensing and targeting agents for analytical, diagnostic, therapeutic, and gene-regulatory applications, but their limited characterization and functional validation have hindered their broader implementation. We report the development of a surface plasmon resonance-based platform for rapid characterization of kinetic and equilibrium binding properties of aptamers to small molecules. Our system is label-free and scalable and enables analysis of different aptamer-target pairs and binding conditions with the same platform. This method demonstrates improved sensitivity, flexibility, and stability compared to other aptamer characterization methods. We validated our assay against previously reported aptamer affinity and kinetic measurements and further characterized a diverse panel of 12 small molecule-binding RNA and DNA aptamers. We report the first kinetic characterization for six of these aptamers and affinity characterization of two others. This work is the first example of direct comparison of in vitro selected and natural aptamers using consistent characterization conditions, thus providing insight into the influence of environmental conditions on aptamer binding kinetics and affinities, indicating different possible regulatory strategies used by natural aptamers, and identifying potential in vitro selection strategies to improve resulting binding affinities.

  View details for DOI 10.1021/ac5001527

  View details for Web of Science ID 000333776600006

• Molecular tools for chemical biotechnology CURRENT OPINION IN BIOTECHNOLOGY Galanie, S., Siddiqui, M. S., Smolke, C. D. 2013; 24 (6): 1000-1009

  Abstract

  Biotechnological production of high value chemical products increasingly involves engineering in vivo multi-enzyme pathways and host metabolism. Recent approaches to these engineering objectives have made use of molecular tools to advance de novo pathway identification, tunable enzyme expression, and rapid pathway construction. Molecular tools also enable optimization of single enzymes and entire genomes through diversity generation and screening, whole cell analytics, and synthetic metabolic control networks. In this review, we focus on advanced molecular tools and their applications to engineered pathways in host organisms, highlighting the degree to which each tool is generalizable.

  View details for DOI 10.1016/j.cophio.2013.03.001

  View details for Web of Science ID 000328806200007

  View details for PubMedID 23528237

• Dynamically Reshaping Signaling Networks to Program Cell Fate via Genetic Controllers SCIENCE Galloway, K. E., Franco, E., Smolke, C. D. 2013; 341 (6152): 1358-?

  View details for DOI 10.1126/science.1235005

  View details for Web of Science ID 000324597200036

• A yeast-based rapid prototype platform for gene control elements in mammalian cells BIOTECHNOLOGY AND BIOENGINEERING Wei, K. Y., Chen, Y. Y., Smolke, C. D. 2013; 110 (4): 1201-1210

  Abstract

  Programming genetic circuits in mammalian cells requires flexible, tunable, and user-tailored gene-control systems. However, most existing control systems are either mechanistically specific for microbial organisms or must be laboriously re-engineered to function in mammalian cells. Here, we demonstrate a ribozyme-based device platform that can be directly transported from yeast to mammalian cells in a "plug-and-play" manner. Ribozyme switches previously prototyped in yeast are shown to regulate gene expression in a predictable, ligand-responsive manner in human HEK 293, HeLa, and U2OS cell lines without any change to device sequence nor further optimization. The ribozyme-based devices, which exhibit activation ratios comparable to the best RNA-based regulatory devices demonstrated in mammalian cells to-date, retain their prescribed functions (ON or OFF switch), tunability of regulatory stringency, and responsiveness to different small-molecule inputs in mammalian hosts. Furthermore, we observe strong correlations of device performance between yeast and all mammalian cell lines tested (R(2)  = 0.63-0.97). Our unique device architecture can therefore act as a rapid prototyping platform (RPP) based on a yeast chassis, providing a well-developed and genetically tractable system that supports rapid and high-throughput screens for generating gene-controllers with a broad range of functions in mammalian cells. This platform will accelerate development of mammalian gene-controllers for diverse applications, including cell-based therapeutics and cell-fate reprogramming.

  View details for DOI 10.1002/bit.24792

  View details for Web of Science ID 000315360100020

• A versatile cis-blocking and trans-activation strategy for ribozyme characterization NUCLEIC ACIDS RESEARCH Kennedy, A. B., Liang, J. C., Smolke, C. D. 2013; 41 (2)

  Abstract

  Synthetic RNA control devices that use ribozymes as gene-regulatory components have been applied to controlling cellular behaviors in response to environmental signals. Quantitative measurement of the in vitro cleavage rate constants associated with ribozyme-based devices is essential for advancing the molecular design and optimization of this class of gene-regulatory devices. One of the key challenges encountered in ribozyme characterization is the efficient generation of full-length RNA from in vitro transcription reactions, where conditions generally lead to significant ribozyme cleavage. Current methods for generating full-length ribozyme-encoding RNA rely on a trans-blocking strategy, which requires a laborious gel separation and extraction step. Here, we develop a simple two-step gel-free process including cis-blocking and trans-activation steps to support scalable generation of functional full-length ribozyme-encoding RNA. We demonstrate our strategy on various types of natural ribozymes and synthetic ribozyme devices, and the cleavage rate constants obtained for the RNA generated from our strategy are comparable with those generated through traditional methods. We further develop a rapid, label-free ribozyme cleavage assay based on surface plasmon resonance, which allows continuous, real-time monitoring of ribozyme cleavage. The surface plasmon resonance-based characterization assay will complement the versatile cis-blocking and trans-activation strategy to broadly advance our ability to characterize and engineer ribozyme-based devices.

  View details for DOI 10.1093/nar/gks1036

  View details for Web of Science ID 000314121100008

  View details for PubMedID 23155065

• Synthetic Biology: Advancing the Design of Diverse Genetic Systems ANNUAL REVIEW OF CHEMICAL AND BIOMOLECULAR ENGINEERING, VOL 4 Wang, Y., Wei, K. Y., Smolke, C. D. 2013; 4: 69-102

  Abstract

  A major objective of synthetic biology is to make the process of designing genetically encoded biological systems more systematic, predictable, robust, scalable, and efficient. Examples of genetic systems in the field vary widely in terms of operating hosts, compositional approaches, and network complexity, ranging from simple genetic switches to search-and-destroy systems. While significant advances in DNA synthesis capabilities support the construction of pathway- and genome-scale programs, several design challenges currently restrict the scale of systems that can be reasonably designed and implemented. Thus, while synthetic biology offers much promise in developing systems to address challenges faced in the fields of manufacturing, environment and sustainability, and health and medicine, the realization of this potential is currently limited by the diversity of available parts and effective design frameworks. As researchers make progress in bridging this design gap, advances in the field hint at ever more diverse applications for biological systems. Expected final online publication date for the Annual Review of Chemical and Biomolecular Engineering Volume 4 is June 07, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

  View details for DOI 10.1146/annurev-chembioeng-061312-103351

  View details for Web of Science ID 000321740100005

• Identification and treatment of heme depletion attributed to overexpression of a lineage of evolved P450 monooxygenases PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Michener, J. K., Nielsen, J., Smolke, C. D. 2012; 109 (47): 19504-19509

  Abstract

  Recent advances in metabolic engineering have demonstrated that microbial biosynthesis can provide a viable alternative to chemical synthesis for the production of bulk and fine chemicals. Introduction of a new biosynthetic pathway typically requires the expression of multiple heterologous enzymes in the production host, which can impose stress on the host cell and, thereby, limit performance of the pathway. Unfortunately, analysis and treatment of the host stress response can be difficult, because there are many sources of stress that may interact in complex ways. We use a systems biological approach to analyze the stress imposed by expressing different enzyme variants from a lineage of soluble P450 monooxygenases, previously evolved for heterologous activity in Saccharomyces cerevisiae. Our analysis identifies patterns of stress imposed on the host by heterologous enzyme overexpression that are consistent across the evolutionary lineage, ultimately implicating heme depletion as the major stress. We show that the monooxygenase evolution, starting from conditions of either high or low stress, caused the cellular stress to converge to a common level. Overexpression of rate-limiting enzymes in the endogenous heme biosynthetic pathway alleviates the stress imposed by expression of the P450 monooxygenases and increases the enzymatic activity of the final evolved P450 by an additional 2.3-fold. Heme overexpression also increases the total activity of an endogenous cytosolic heme-containing catalase but not a heterologous P450 that is membrane-associated. This work demonstrates the utility of combining systems and synthetic biology to analyze and optimize heterologous enzyme expression.

  View details for DOI 10.1073/pnas.1212287109

  View details for Web of Science ID 000311997200094

  View details for PubMedID 23129650

• A high-throughput, quantitative cell-based screen for efficient tailoring of RNA device activity NUCLEIC ACIDS RESEARCH Liang, J. C., Chang, A. L., Kennedy, A. B., Smolke, C. D. 2012; 40 (20)

  Abstract

  Recent advances have demonstrated the use of RNA-based control devices to program sophisticated cellular functions; however, the efficiency with which these devices can be quantitatively tailored has limited their broader implementation in cellular networks. Here, we developed a high-efficiency, high-throughput and quantitative two-color fluorescence-activated cell sorting-based screening strategy to support the rapid generation of ribozyme-based control devices with user-specified regulatory activities. The high-efficiency of this screening strategy enabled the isolation of a single functional sequence from a library of over 10(6) variants within two sorting cycles. We demonstrated the versatility of our approach by screening large libraries generated from randomizing individual components within the ribozyme device platform to efficiently isolate new device sequences that exhibit increased in vitro cleavage rates up to 10.5-fold and increased in vivo activation ratios up to 2-fold. We also identified a titratable window within which in vitro cleavage rates and in vivo gene-regulatory activities are correlated, supporting the importance of optimizing RNA device activity directly in the cellular environment. Our two-color fluorescence-activated cell sorting-based screen provides a generalizable strategy for quantitatively tailoring genetic control elements for broader integration within biological networks.

  View details for DOI 10.1093/nar/gks636

  View details for Web of Science ID 000310970700002

  View details for PubMedID 22810204

• Synthetic RNA switches as a tool for temporal and spatial control over gene expression CURRENT OPINION IN BIOTECHNOLOGY Chang, A. L., Wolf, J. J., Smolke, C. D. 2012; 23 (5): 679-688

  Abstract

  The engineering of biological systems offers significant promise for advances in areas including health and medicine, chemical synthesis, energy production, and environmental sustainability. Realizing this potential requires tools that enable design of sophisticated genetic systems. The functional diversity of RNA makes it an attractive and versatile substrate for programming sensing, information processing, computation, and control functions. Recent advances in the design of synthetic RNA switches capable of detecting and responding to molecular and environmental signals enable dynamic modulation of gene expression through diverse mechanisms, including transcription, splicing, stability, RNA interference, and translation. Furthermore, implementation of these switches in genetic circuits highlights the potential for building synthetic cell systems targeted to applications in environmental remediation and next-generation therapeutics and diagnostics.

  View details for DOI 10.1016/j.copbio.2012.01.005

  View details for Web of Science ID 000310406700007

  View details for PubMedID 22305712

• High-throughput enzyme evolution in Saccharomyces cerevisiae using a synthetic RNA switch METABOLIC ENGINEERING Michener, J. K., Smolke, C. D. 2012; 14 (4): 306-316

  Abstract

  Metabolic engineering can produce a wide range of bulk and fine chemicals using renewable resources. These approaches frequently require high levels of activity from multiple heterologous enzymes. Directed evolution techniques have been used to improve the activity of a wide range of enzymes but can be difficult to apply when the enzyme is used in whole cells. To address this limitation, we developed generalizable in vivo biosensors using engineered RNA switches to link metabolite concentrations and GFP expression levels in living cells. Using such a sensor, we quantitatively screened large enzyme libraries in high throughput based on fluorescence, either in clonal cultures or in single cells by fluorescence activated cell sorting (FACS). By iteratively screening libraries of a caffeine demethylase, we identified beneficial mutations that ultimately increased the enzyme activity in vivo by 33 fold and the product selectivity by 22 fold. As aptamer selection strategies allow RNA switches to be readily adapted to recognize new small molecules, these RNA-based screening techniques are applicable to a broad range of enzymes and metabolic pathways.

  View details for DOI 10.1016/j.ymben.2012.04.004

  View details for Web of Science ID 000305275600003

  View details for PubMedID 22554528

• Applications of genetically-encoded biosensors for the construction and control of biosynthetic pathways METABOLIC ENGINEERING Michener, J. K., Thodey, K., Liang, J. C., Smolke, C. D. 2012; 14 (3): 212-222

  Abstract

  Cells are filled with biosensors, molecular systems that measure the state of the cell and respond by regulating host processes. In much the same way that an engineer would monitor a chemical reactor, the cell uses these sensors to monitor changing intracellular environments and produce consistent behavior despite the variable environment. While natural systems derive a clear benefit from pathway regulation, past research efforts in engineering cellular metabolism have focused on introducing new pathways and removing existing pathway regulation. Synthetic biology is a rapidly growing field that focuses on the development of new tools that support the design, construction, and optimization of biological systems. Recent advances have been made in the design of genetically-encoded biosensors and the application of this class of molecular tools for optimizing and regulating heterologous pathways. Biosensors to cellular metabolites can be taken directly from natural systems, engineered from natural sensors, or constructed entirely in vitro. When linked to reporters, such as antibiotic resistance markers, these metabolite sensors can be used to report on pathway productivity, allowing high-throughput screening for pathway optimization. Future directions will focus on the application of biosensors to introduce feedback control into metabolic pathways, providing dynamic control strategies to increase the efficient use of cellular resources and pathway reliability.

  View details for DOI 10.1016/j.ymben.2011.09.004

  View details for Web of Science ID 000302876000005

  View details for PubMedID 21946159

• Synthetic biology: Emerging methodologies to catalyze the metabolic engineering design cycle METABOLIC ENGINEERING Smolke, C. D., Tyo, K. E. 2012; 14 (3): 187-188

  View details for DOI 10.1016/j.ymben.2012.03.009

  View details for Web of Science ID 000302876000001

  View details for PubMedID 22465683

• Metabolic engineering of Saccharomyces cerevisiae for alkaloid production Smolke, C. D. FEDERATION AMER SOC EXP BIOL. 2012

  View details for Web of Science ID 000310711300219

• Advancing secondary metabolite biosynthesis in yeast with synthetic biology tools FEMS YEAST RESEARCH Siddiqui, M. S., Thodey, K., Trenchard, I., Smolke, C. D. 2012; 12 (2): 144-170

  Abstract

  Secondary metabolites are an important source of high-value chemicals, many of which exhibit important pharmacological properties. These valuable natural products are often difficult to synthesize chemically and are commonly isolated through inefficient extractions from natural biological sources. As such, they are increasingly targeted for production by biosynthesis from engineered microorganisms. The budding yeast species Saccharomyces cerevisiae has proven to be a powerful microorganism for heterologous expression of biosynthetic pathways. S. cerevisiae's usefulness as a host organism is owed in large part to the wealth of knowledge accumulated over more than a century of intense scientific study. Yet many challenges are currently faced in engineering yeast strains for the biosynthesis of complex secondary metabolite production. However, synthetic biology is advancing the development of new tools for constructing, controlling, and optimizing complex metabolic pathways in yeast. Here, we review how the coupling between yeast biology and synthetic biology is advancing the use of S. cerevisiae as a microbial host for the construction of secondary metabolic pathways.

  View details for DOI 10.1111/j.1567-1364.2011.00774.x

  View details for Web of Science ID 000300500100005

  View details for PubMedID 22136110

• Synthetic biology: advancing biological frontiers by building synthetic systems GENOME BIOLOGY Chen, Y. Y., Galloway, K. E., Smolke, C. D. 2012; 13 (2)

  Abstract

  Advances in synthetic biology are contributing to diverse research areas, from basic biology to biomanufacturing and disease therapy. We discuss the theoretical foundation, applications, and potential of this emerging field.

  View details for DOI 10.1186/gb-2012-13-2-240

  View details for Web of Science ID 000305391700014

  View details for PubMedID 22348749

• From DNA to Targeted Therapeutics: Bringing Synthetic Biology to the Clinic SCIENCE TRANSLATIONAL MEDICINE Chen, Y. Y., Smolke, C. D. 2011; 3 (106)

  Abstract

  Synthetic biology aims to make biological engineering more scalable and predictable, lowering the cost and facilitating the translation of synthetic biological systems to practical applications. Increasingly sophisticated, rationally designed synthetic systems that are capable of complex functions pave the way to translational applications, including disease diagnostics and targeted therapeutics. Here, we provide an overview of recent developments in synthetic biology in the context of translational research and discuss challenges at the interface between synthetic biology and clinical medicine.

  View details for DOI 10.1126/scitranslmed.3002944

  View details for Web of Science ID 000296586100002

  View details for PubMedID 22030748

• Synthetic RNA modules for fine-tuning gene expression levels in yeast by modulating RNase III activity NUCLEIC ACIDS RESEARCH Babiskin, A. H., Smolke, C. D. 2011; 39 (19): 8651-8664

  Abstract

  The design of synthetic gene networks requires an extensive genetic toolbox to control the activities and levels of protein components to achieve desired cellular functions. Recently, a novel class of RNA-based control modules, which act through post-transcriptional processing of transcripts by directed RNase III (Rnt1p) cleavage, were shown to provide predictable control over gene expression and unique properties for manipulating biological networks. Here, we increase the regulatory range of the Rnt1p control elements, by modifying a critical region for enzyme binding to its hairpin substrates, the binding stability box (BSB). We used a high throughput, cell-based selection strategy to screen a BSB library for sequences that exhibit low fluorescence and thus high Rnt1p processing efficiencies. Sixteen unique BSBs were identified that cover a range of protein expression levels, due to the ability of the sequences to affect the hairpin cleavage rate and to form active cleavable complexes with Rnt1p. We further demonstrated that the activity of synthetic Rnt1p hairpins can be rationally programmed by combining the synthetic BSBs with a set of sequences located within a different region of the hairpin that directly modulate cleavage rates, providing a modular assembly strategy for this class of RNA-based control elements.

  View details for DOI 10.1093/nar/gkr445

  View details for Web of Science ID 000296341100036

  View details for PubMedID 21737428

• Engineering Biological Systems with Synthetic RNA Molecules MOLECULAR CELL Liang, J. C., Bloom, R. J., Smolke, C. D. 2011; 43 (6): 915-926

  Abstract

  RNA molecules play diverse functional roles in natural biological systems. There has been growing interest in designing synthetic RNA counterparts for programming biological function. The design of synthetic RNA molecules that exhibit diverse activities, including sensing, regulatory, information processing, and scaffolding activities, has highlighted the advantages of RNA as a programmable design substrate. Recent advances in implementing these engineered RNA molecules as key control elements in synthetic genetic networks are highlighting the functional relevance of this class of synthetic elements in programming cellular behaviors.

  View details for DOI 10.1016/j.molcel.2011.08.023

  View details for Web of Science ID 000295309800007

  View details for PubMedID 21925380

• Cell biology. Bringing it together with RNA. Science Thodey, K., Smolke, C. D. 2011; 333 (6041): 412-413

  View details for DOI 10.1126/science.1209685

  View details for Web of Science ID 000292959600031

  View details for PubMedID 21778388

• Engineering ligand-responsive RNA controllers in yeast through the assembly of RNase III tuning modules NUCLEIC ACIDS RESEARCH Babiskin, A. H., Smolke, C. D. 2011; 39 (12): 5299-5311

  Abstract

  The programming of cellular networks to achieve new biological functions depends on the development of genetic tools that link the presence of a molecular signal to gene-regulatory activity. Recently, a set of engineered RNA controllers was described that enabled predictable tuning of gene expression in the yeast Saccharomyces cerevisiae through directed cleavage of transcripts by an RNase III enzyme, Rnt1p. Here, we describe a strategy for building a new class of RNA sensing-actuation devices based on direct integration of RNA aptamers into a region of the Rnt1p hairpin that modulates Rnt1p cleavage rates. We demonstrate that ligand binding to the integrated aptamer domain is associated with a structural change sufficient to inhibit Rnt1p processing. Three tuning strategies based on the incorporation of different functional modules into the Rnt1p switch platform were demonstrated to optimize switch dynamics and ligand responsiveness. We further demonstrated that these tuning modules can be implemented combinatorially in a predictable manner to further improve the regulatory response properties of the switch. The modularity and tunability of the Rnt1p switch platform will allow for rapid optimization and tailoring of this gene control device, thus providing a useful tool for the design of complex genetic networks in yeast.

  View details for DOI 10.1093/nar/gkr090

  View details for Web of Science ID 000292564900041

  View details for PubMedID 21355039

• Design of small molecule-responsive microRNAs based on structural requirements for Drosha processing NUCLEIC ACIDS RESEARCH Beisel, C. L., Chen, Y. Y., Culler, S. J., Hoff, K. G., Smolke, C. D. 2011; 39 (7): 2981-2994

  Abstract

  MicroRNAs (miRNAs) are prevalent regulatory RNAs that mediate gene silencing and play key roles in diverse cellular processes. While synthetic RNA-based regulatory systems that integrate regulatory and sensing functions have been demonstrated, the lack of detail on miRNA structure-function relationships has limited the development of integrated control systems based on miRNA silencing. Using an elucidated relationship between Drosha processing and the single-stranded nature of the miRNA basal segments, we developed a strategy for designing ligand-responsive miRNAs. We demonstrate that ligand binding to an aptamer integrated into the miRNA basal segments inhibits Drosha processing, resulting in titratable control over gene silencing. The generality of this control strategy was shown for three aptamer-small molecule ligand pairs. The platform can be extended to the design of synthetic miRNAs clusters, cis-acting miRNAs and self-targeting miRNAs that act both in cis and trans, enabling fine-tuning of the regulatory strength and dynamics. The ability of our ligand-responsive miRNA platform to respond to user-defined inputs, undergo regulatory performance tuning and display scalable combinatorial control schemes will help advance applications in biological research and applied medicine.

  View details for DOI 10.1093/nar/gkq954

  View details for Web of Science ID 000289628400050

  View details for PubMedID 21149259

• Informing Biological Design by Integration of Systems and Synthetic Biology CELL Smolke, C. D., Silver, P. A. 2011; 144 (6): 855-859

  Abstract

  Synthetic biology aims to make the engineering of biology faster and more predictable. In contrast, systems biology focuses on the interaction of myriad components and how these give rise to the dynamic and complex behavior of biological systems. Here, we examine the synergies between these two fields.

  View details for DOI 10.1016/j.cell.2011.02.020

  View details for Web of Science ID 000288543500004

  View details for PubMedID 21414477

• A synthetic library of RNA control modules for predictable tuning of gene expression in yeast MOLECULAR SYSTEMS BIOLOGY Babiskin, A. H., Smolke, C. D. 2011; 7

  Abstract

  Advances in synthetic biology have resulted in the development of genetic tools that support the design of complex biological systems encoding desired functions. The majority of efforts have focused on the development of regulatory tools in bacteria, whereas fewer tools exist for the tuning of expression levels in eukaryotic organisms. Here, we describe a novel class of RNA-based control modules that provide predictable tuning of expression levels in the yeast Saccharomyces cerevisiae. A library of synthetic control modules that act through posttranscriptional RNase cleavage mechanisms was generated through an in vivo screen, in which structural engineering methods were applied to enhance the insulation and modularity of the resulting components. This new class of control elements can be combined with any promoter to support titration of regulatory strategies encoded in transcriptional regulators and thus more sophisticated control schemes. We applied these synthetic controllers to the systematic titration of flux through the ergosterol biosynthesis pathway, providing insight into endogenous control strategies and highlighting the utility of this control module library for manipulating and probing biological systems.

  View details for DOI 10.1038/msb.2011.4

  View details for Web of Science ID 000289205600006

  View details for PubMedID 21364573

• Reprogramming Cellular Behavior with RNA Controllers Responsive to Endogenous Proteins SCIENCE Culler, S. J., Hoff, K. G., Smolke, C. D. 2010; 330 (6008): 1251-1255

  Abstract

  Synthetic genetic devices that interface with native cellular pathways can be used to change natural networks to implement new forms of control and behavior. The engineering of gene networks has been limited by an inability to interface with native components. We describe a class of RNA control devices that overcome these limitations by coupling increased abundance of particular proteins to targeted gene expression events through the regulation of alternative RNA splicing. We engineered RNA devices that detect signaling through the nuclear factor ?B and Wnt signaling pathways in human cells and rewire these pathways to produce new behaviors, thereby linking disease markers to noninvasive sensing and reprogrammed cellular fates. Our work provides a genetic platform that can build programmable sensing-actuation devices enabling autonomous control over cellular behavior.

  View details for DOI 10.1126/science.1192128

  View details for Web of Science ID 000284613700044

  View details for PubMedID 21109673

• Functional selection and systematic analysis of intronic splicing elements identify active sequence motifs and associated splicing factors NUCLEIC ACIDS RESEARCH Culler, S. J., Hoff, K. G., Voelker, R. B., Berglund, J. A., Smolke, C. D. 2010; 38 (15): 5152-5165

  Abstract

  Despite the critical role of pre-mRNA splicing in generating proteomic diversity and regulating gene expression, the sequence composition and function of intronic splicing regulatory elements (ISREs) have not been well elucidated. Here, we employed a high-throughput in vivo Screening PLatform for Intronic Control Elements (SPLICE) to identify 125 unique ISRE sequences from a random nucleotide library in human cells. Bioinformatic analyses reveal consensus motifs that resemble splicing regulatory elements and binding sites for characterized splicing factors and that are enriched in the introns of naturally occurring spliced genes, supporting their biological relevance. In vivo characterization, including an RNAi silencing study, demonstrate that ISRE sequences can exhibit combinatorial regulatory activity and that multiple trans-acting factors are involved in the regulatory effect of a single ISRE. Our work provides an initial examination into the sequence characteristics and function of ISREs, providing an important contribution to the splicing code.

  View details for DOI 10.1093/nar/gkq248

  View details for Web of Science ID 000281345900029

  View details for PubMedID 20385591

• Engineering RNA controllers for programming cellular behavior Smolke, C. WILEY-BLACKWELL. 2010: 30-30

  View details for Web of Science ID 000278565100118

• Genetic control of mammalian T-cell proliferation with synthetic RNA regulatory systems PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chen, Y. Y., Jensen, M. C., Smolke, C. D. 2010; 107 (19): 8531-8536

  Abstract

  RNA molecules perform diverse regulatory functions in natural biological systems, and numerous synthetic RNA-based control devices that integrate sensing and gene-regulatory functions have been demonstrated, predominantly in bacteria and yeast. Despite potential advantages of RNA-based genetic control strategies in clinical applications, there has been limited success in extending engineered RNA devices to mammalian gene-expression control and no example of their application to functional response regulation in mammalian systems. Here we describe a synthetic RNA-based regulatory system and its application in advancing cellular therapies by linking rationally designed, drug-responsive, ribozyme-based regulatory devices to growth cytokine targets to control mouse and primary human T-cell proliferation. We further demonstrate the ability of our synthetic controllers to effectively modulate T-cell growth rate in response to drug input in vivo. Our RNA-based regulatory system exhibits unique properties critical for translation to therapeutic applications, including adaptability to diverse ligand inputs and regulatory targets, tunable regulatory stringency, and rapid response to input availability. By providing tight gene-expression control with customizable ligand inputs, RNA-based regulatory systems can greatly improve cellular therapies and advance broad applications in health and medicine.

  View details for DOI 10.1073/pnas.1001721107

  View details for Web of Science ID 000277591200010

  View details for PubMedID 20421500

• In Vivo Fluorescent Detection of Fe-S Clusters Coordinated by Human GRX2 CHEMISTRY & BIOLOGY Hoff, K. G., Culler, S. J., Nguyen, P. Q., McGuire, R. M., Silberg, J. J., Smolke, C. D. 2009; 16 (12): 1299-1308

  Abstract

  A major challenge to studying Fe-S cluster biosynthesis in higher eukaryotes is the lack of simple tools for imaging metallocluster binding to proteins. We describe the first fluorescent approach for in vivo detection of 2Fe2S clusters that is based upon the complementation of Venus fluorescent protein fragments via human glutaredoxin 2 (GRX2) coordination of a 2Fe2S cluster. We show that Escherichia coli and mammalian cells expressing Venus fragments fused to GRX2 exhibit greater fluorescence than cells expressing fragments fused to a C37A mutant that cannot coordinate a metallocluster. In addition, we find that maximal fluorescence in the cytosol of mammalian cells requires the iron-sulfur cluster assembly proteins ISCU and NFS1. These findings provide evidence that glutaredoxins can dimerize within mammalian cells through coordination of a 2Fe2S cluster as observed with purified recombinant proteins.

  View details for DOI 10.1016/j.chembiol.2009.11.011

  View details for Web of Science ID 000273410700015

  View details for PubMedID 20064440

• Building outside of the box: iGEM and the BioBricks Foundation NATURE BIOTECHNOLOGY Smolke, C. D. 2009; 27 (12): 1099-1102

  View details for DOI 10.1038/nbt1209-1099

  View details for Web of Science ID 000272708300020

  View details for PubMedID 20010584

• Cell biology. It's the DNA that counts. Science Smolke, C. D. 2009; 324 (5931): 1156-1157

  View details for DOI 10.1126/science.1174843

  View details for PubMedID 19478174

• Design Principles for Riboswitch Function PLOS COMPUTATIONAL BIOLOGY Beisel, C. L., Smolke, C. D. 2009; 5 (4)

  Abstract

  Scientific and technological advances that enable the tuning of integrated regulatory components to match network and system requirements are critical to reliably control the function of biological systems. RNA provides a promising building block for the construction of tunable regulatory components based on its rich regulatory capacity and our current understanding of the sequence-function relationship. One prominent example of RNA-based regulatory components is riboswitches, genetic elements that mediate ligand control of gene expression through diverse regulatory mechanisms. While characterization of natural and synthetic riboswitches has revealed that riboswitch function can be modulated through sequence alteration, no quantitative frameworks exist to investigate or guide riboswitch tuning. Here, we combined mathematical modeling and experimental approaches to investigate the relationship between riboswitch function and performance. Model results demonstrated that the competition between reversible and irreversible rate constants dictates performance for different regulatory mechanisms. We also found that practical system restrictions, such as an upper limit on ligand concentration, can significantly alter the requirements for riboswitch performance, necessitating alternative tuning strategies. Previous experimental data for natural and synthetic riboswitches as well as experiments conducted in this work support model predictions. From our results, we developed a set of general design principles for synthetic riboswitches. Our results also provide a foundation from which to investigate how natural riboswitches are tuned to meet systems-level regulatory demands.

  View details for DOI 10.1371/journal.pcbi.1000363

  View details for Web of Science ID 000266214200028

  View details for PubMedID 19381267

• Frameworks for Programming Biological Function through RNA Parts and Devices CHEMISTRY & BIOLOGY Win, M. N., Liang, J. C., Smolke, C. D. 2009; 16 (3): 298-310

  Abstract

  One of the long-term goals of synthetic biology is to reliably engineer biological systems that perform human-defined functions. Currently, researchers face several scientific and technical challenges in designing and building biological systems, one of which is associated with our limited ability to access, transmit, and control molecular information through the design of functional biomolecules exhibiting novel properties. The fields of RNA biology and nucleic acid engineering, along with the tremendous interdisciplinary growth of synthetic biology, are fueling advances in the emerging field of RNA programming in living systems. Researchers are designing functional RNA molecules that exhibit increasingly complex functions and integrating these molecules into cellular circuits to program higher-level biological functions. The continued integration and growth of RNA design and synthetic biology presents exciting potential to transform how we interact with and program biology.

  View details for DOI 10.1016/j.chembiol.2009.02.011

  View details for Web of Science ID 000264859200009

  View details for PubMedID 19318211

• Synthetic control of a fitness tradeoff in yeast nitrogen metabolism. Journal of biological engineering Bayer, T. S., Hoff, K. G., Beisel, C. L., Lee, J. J., Smolke, C. D. 2009; 3: 1-?

  Abstract

  Microbial communities are involved in many processes relevant to industrial and medical biotechnology, such as the formation of biofilms, lignocellulosic degradation, and hydrogen production. The manipulation of synthetic and natural microbial communities and their underlying ecological parameters, such as fitness, evolvability, and variation, is an increasingly important area of research for synthetic biology.Here, we explored how synthetic control of an endogenous circuit can be used to regulate a tradeoff between fitness in resource abundant and resource limited environments in a population of Saccharomyces cerevisiae. We found that noise in the expression of a key enzyme in ammonia assimilation, Gdh1p, mediated a tradeoff between growth in low nitrogen environments and stress resistance in high ammonia environments. We implemented synthetic control of an endogenous Gdh1p regulatory network to construct an engineered strain in which the fitness of the population was tunable in response to an exogenously-added small molecule across a range of ammonia environments.The ability to tune fitness and biological tradeoffs will be important components of future efforts to engineer microbial communities.

  View details for DOI 10.1186/1754-1611-3-1

  View details for PubMedID 19118500

• Model-guided design of ligand-regulated RNAi for programmable control of gene expression MOLECULAR SYSTEMS BIOLOGY Beisel, C. L., Bayer, T. S., Hoff, K. G., Smolke, C. D. 2008; 4

  Abstract

  Progress in constructing biological networks will rely on the development of more advanced components that can be predictably modified to yield optimal system performance. We have engineered an RNA-based platform, which we call an shRNA switch, that provides for integrated ligand control of RNA interference (RNAi) by modular coupling of an aptamer, competing strand, and small hairpin (sh)RNA stem into a single component that links ligand concentration and target gene expression levels. A combined experimental and mathematical modelling approach identified multiple tuning strategies and moves towards a predictable framework for the forward design of shRNA switches. The utility of our platform is highlighted by the demonstration of fine-tuning, multi-input control, and model-guided design of shRNA switches with an optimized dynamic range. Thus, shRNA switches can serve as an advanced component for the construction of complex biological systems and offer a controlled means of activating RNAi in disease therapeutics.

  View details for DOI 10.1038/msb.2008.62

  View details for Web of Science ID 000260722900006

  View details for PubMedID 18956013