21.  Gander, M.W., Vrana, J.D., Voje, W.E., Carothers, J.M., Klavins, E. Digital logic circuits in yeast with CRISPR-dCas9 NOR gates.  Nature Commun. Accepted. LINK

ABSTRACT: Natural genetic circuits enable cells to make sophisticated digital decisions. Building equally complex synthetic circuits in eukaryotes remains difficult, however, because commonly used genetic components leak transcriptionally, do not allow arbitrary interconnections, or do not have digital responses. Here, we designed a new dCas9-Mxi1 based NOR gate architecture in S. cerevisiae that allows arbitrary connectivity and large genetic circuits. Because we used the strong chromatin remodeler Mxi1, our system showed very little leak and exhibits a highly digital response. In particular, we built a combinatorial library of NOR gates that each directly convert guide RNA (gRNA) input signals into gRNA output signals, enabling NOR gates to be “wired” together. We constructed and characterized logic circuits with up to seven independent gRNAs, including repression cascades with up to seven layers. Modeling predicted that the NOR gates have Hill Coefficients of approximately 1.71±0.09, explaining the minimal signal degradation we observed in these deeply layered circuits. Our approach enables the construction of the largest, eukaryotic gene circuits to date and will form the basis for large, synthetic, decision making systems in living cells.

20.  Hwang, C., and Carothers, J.M.  Label-free selections of aptamers for metabolic engineering. Methods. 2016. LINK PDF

ABSTRACT: RNA aptamers can be assembled into genetic regulatory devices that sense and respond to levels of specific cellular metabolites and thus serve an integral part of designing dynamic control into engineered metabolic pathways. Here, we describe a practical method for generating specific and high affinity aptamers to enable the wider use of in vitro selection and a broader application of aptamers for metabolic engineering. Conventional selection methods involving either radioactive labeling of RNA or the use of label-free methods such as SPR to track aptamer enrichment require resources that are not widely accessible to research groups. We present a label-free selection method that uses small volume spectrophotometers to track RNA enrichment paired with previously characterized affinity chromatography methods. Borrowing techniques used in solid phase peptide synthesis, we present an approach for immobilizing a wide range of metabolites to an amino PEGA matrix. As an illustration, we detail laboratory techniques employed to generate aptamers that bind p-aminophenylalanine, a metabolic precursor for bio-based production of plastics and the pristinamycin family of antibiotics. We focused on the development of methods for ligand immobilization, selection via affinity chromatography, and nucleic acid quantification that can be performed with common laboratory equipment.

19.  Beck, D.A.C., Carothers, J.M. Subramanian, V., Pfaendtner, J. Data Science: Accelerating innovation and discovery in chemical engineering.  AIChE J. 2016. 62, 1402-1416. (Cover) LINK PDF

ABSTRACT: All of science and engineering, including chemical engineering, is being transformed by new sources of data from high-throughput experiments, observational studies, and simulation. In this new era of data-enabled science and engineering, discovery is no longer limited by the collection and processing of data but by data management, knowledge extraction, and the visualization of information. The termdata sciencehas become increasingly popular across industry, and academic disciplines to refer to the combination of strategies and tools for addressing the oncoming deluge of data. The term data scientist is a common descriptor of an engineer or scientist from any disciplinary background who is equipped to seamlessly process, analyze, and communicate in this data-intensive context. The core areas of data science are often identified as data management, statistical and machine learning, and visualization. In this Perspective, we present an overview of these core areas, discuss application areas from within chemical engineering research, and conclude with perspectives on how data science principles can be included in our training.

18.  Sparkman-Yager, D., Correa-Rojas, D., Carothers, J.M. Kinetic folding design of aptazyme-regulated expression devices as riboswitches for metabolic engineering. Methods in Enzymol. 2015. 550, 321-340.  LINK PDF

ABSTRACT: Recent developments in the fields of synthetic biology and metabolic engineering have opened the doors for the microbial production of biofuels and other valuable organic compounds. There remain, however, significant metabolic hurdles to the production of these compounds in cost-effective quantities. This is due, in part, to mismatches between the metabolic engineer's desire for high yields and the microbe's desire to survive. Many valuable compounds, or the intermediates necessary for their biosynthesis, prove deleterious at the desired production concentrations. One potential solution to these toxicity-related issues is the implementation of nonnative dynamic genetic control mechanisms that sense excessively high concentrations of metabolic intermediates and respond accordingly to alleviate their impact. One potential class of dynamic regulator is the riboswitch: cis-acting RNA elements that regulate the expression of downstream genes based on the presence of an effector molecule. Here, we present combined methods for constructing aptazyme-regulated expression devices (aREDs) through computational cotranscriptional kinetic folding design and experimental validation. These approaches can be used to engineer aREDs within novel genetic contexts for the predictable, dynamic regulation of gene expression in vivo.

17.  Stevens, J.T., and Carothers, J.M. Designing RNA-based genetic control systems for efficient production from engineered metabolic pathways. ACS Synth. Biol. 2015. 4, 107-115. LINK PDF

ABSTRACT: Engineered metabolic pathways can be augmented with dynamic regulatory controllers to increase production titers by minimizing toxicity and helping cells maintain homeostasis. We investigated the potential for dynamic RNA-based genetic control systems to increase production through simulation analysis of an engineered p-aminostyrene (p-AS) pathway in E. coli. To map the entire design space, we formulated 729 unique mechanistic models corresponding to all of the possible control topologies and mechanistic implementations in the system under study. 2,000 sampled simulations were performed for each of the 729 system designs to relate the potential effects of dynamic control to increases in p-AS production (total of 3×106 simulations). Our analysis indicates that dynamic control strategies employing aptazyme-regulated expression devices (aREDs) can yield >10-fold improvements over static control. We uncovered generalizable trends in successful control architectures and found that highly performing RNA-based control systems are experimentally tractable. Analyzing the metabolic control state space to predict optimal genetic control strategies promises to enhance the design of metabolic pathways.

16.  Thimmaiah, T., Voje, Jr., W.E., and Carothers, J.M.  Computational design of RNA parts, devices, and transcripts with kinetic folding algorithms implemented on multiprocessor clusters. Computational Methods in Synthetic Biology, Methods in Mol. Biol., Marchisio (ed.). 2015, 1244. DOI: 10.1007/978-1-4939-1878-2_3LINK PDF 

ABSTRACT: With progress toward inexpensive, large-scale DNA assembly, the demand for simulation tools that allow the rapid construction of synthetic biological devices with predictable behaviors continues to increase. By combining engineered transcript components, such as ribosome binding sites, transcriptional terminators, ligand-binding aptamers, catalytic ribozymes, and aptamer-controlled ribozymes (aptazymes), gene expression in bacteria can be fine-tuned, with many corollaries and applications in yeast and mammalian cells. The successful design of genetic constructs that implement these kinds of RNA-based control mechanisms requires modeling and analyzing kinetically-determined co-transcriptional folding pathways. Transcript design methods using stochastic kinetic folding simulations to search spacer sequence libraries for motifs enabling the assembly of RNA component parts into static ribozyme- and dynamic aptazyme-regulated expression devices with quantitatively predictable functions (rREDs and aREDs, respectively) have been described (Science 2011, 334, 1716 1719). Here, we provide a detailed practical procedure for computational transcript design by illustrating a high throughput, multiprocessor approach for evaluating spacer sequences and generating functional rREDs. This chapter is written as a tutorial, complete with pseudo-code and step-by-step instructions for setting up a computational cluster with an Amazon, Inc. web server and performing the large numbers of kinefold-based stochastic kinetic co-transcriptional folding simulations needed to design functional rREDs and aREDs. The method described here should be broadly applicable for designing and analyzing a variety of synthetic RNA parts, devices and transcripts.

15. Stevens, J.T., and Carothers, J.M. Advanced Review: Programming gene expression by engineering transcript stability control and processing in bacteria. Wiley-Blackwell Biotechnology Series (Synthetic Biology). In Press.

ABSTRACT: Through control of messenger RNA stability, bacteria are able to process information, respond to changing conditions, and maintain homeostasis. Many of the naturally occurring mechanisms for Transcript Stability Control (TSC) have been elucidated, and a number of studies have leveraged this understanding to demonstrate that transcript stability can be engineered to control static and dynamic gene expression. Collectively, that body of work represents a foundation for developing new forward-engineering approaches that harness mechanistic understanding to build predictive computational models to guide the development of large-scale genetic devices based on TSC and other means. Further increasing our understanding of RNA degradation pathways and mechanisms will also improve the ability to anticipate how undesired variations in transcript stability may confound device output goals and frustrate engineering efforts. Here, we discuss the current state of the art and identify routes for using TSC to design increasingly large and complex synthetic biological systems.

14. Goler, J.A., Carothers, J.M., and Keasling, J.D. Dual-selection for evolution of in vivo functional aptazymes as riboswitch parts. Methods Mol. Biol. 2014. 1111, 221-35.  LINK   PDF

ABSTRACT: Synthetic biology and metabolic engineering both are aided by the development of genetic control parts. One class of riboswitch parts that has great potential for sensing and regulation of protein levels is aptamer-coupled ribozymes (aptazymes). These devices are comprised of an aptamer domain selected to bind a particular ligand, a ribozyme domain, and a communication module that regulates the ribozyme activity based on the state of the aptamer. We describe a broadly-applicable method for coupling a novel, newly selected aptamer to a ribozyme to generate functional aptazymes via in vitro and in vivo selection. To illustrate this approach, we describe experimental procedures for selecting aptazymes assembled from aptamers that bind p -amino-phenylalanine and a hammerhead ribozyme. Because this method uses selection, it does not rely on sequence-specific design and thus should be generalizable for the generation of in vivo operational aptazymes that respond to any targeted molecules.

13. Carothers, J.M. Design-driven, multi-use research agendas to enable applied synthetic biology for global health. Syst. Synth. Biol. 2013.  7, 79-86. LINK   PDF

ABSTRACT: Many of the synthetic biological devices, pathways and systems that can be engineered aremulti-use, in the sense that they could be used both for commercially-important applications and to help meet global health needs. The on-going development of models and simulation tools for assembling component parts into functionally-complex devices and systems will enable successful engineering with much less trial-and-error experimentation and laboratory infrastructure. As illustrations, I draw upon recent examples from my own work and the broader Keasling research group at the University of California Berkeley and the Joint BioEnergy Institute, of which I was formerly a part. By combining multi-use synthetic biology research agendas with advanced computer-aided design tool creation, it may be possible to more rapidly engineer safe and effective synthetic biology technologies that help address a wide range of global health problems.

12. Cambray, G., Guimaraes J., Mutalik V., Lam C., May Q.A., Thimmaiah T., Carothers J.M., Arkin A.P., and Endy D. Quantification and prediction of intrinsic transcription termination efficiency. Nucl. Acids Res. 2013. 41, 5139-5148. LINK 

ABSTRACT: The reliable forward engineering of genetic systems remains limited by the ad hoc reuse of many types of basic genetic elements. Although a few intrinsic prokaryotic transcription terminators are used routinely, termination efficiencies have not been studied systematically. Here, we developed and validated a genetic architecture that enables reliable measurement of termination efficiencies. We then assembled a collection of 61 natural and synthetic terminators that collectively encode termination efficiencies across an ∼800-fold dynamic range withinEscherichia coli. We simulated co-transcriptional RNA folding dynamics to identify competing secondary structures that might interfere with terminator folding kinetics or impact termination activity. We found that structures extending beyond the core terminator stem are likely to increase terminator activity. By excluding terminators encoding such context-confounding elements, we were able to develop a linear sequence-function model that can be used to estimate termination efficiencies (r = 0.9, n = 31) better than models trained on all terminators (r = 0.67, n = 54). The resulting systematically measured collection of terminators should improve the engineering of synthetic genetic systems and also advance quantitative modeling of transcription termination.

11. Zhang, F., Carothers, J.M., and Keasling, J.D. Design of a dynamic sensor-regulator system for production of fatty acid-based chemicals and fuels. Nature Biotechnol. 2012.  30, 354-359LINK
ABSTRACT: Microbial production of chemicals is now an attractive alternative to chemical synthesis. Current efforts focus mainly on constructing pathways to produce different types of molecules. However, there are few strategies for engineering regulatory components to improve product titers and conversion yields of heterologous pathway. Here we developed a dynamic sensor-regulator system (DSRS) to produce fatty acid–based products in Escherichia coli, and demonstrated its use for biodiesel production.  The DSRS uses a transcription factor that senses a key intermediate and dynamically regulates the expression of genes involved in biodiesel production. This DSRS substantially improved the stability of biodiesel-producing strains and increased the titer to 1.5 g/l and the yield threefold to 28% of the theoretical maximum. Given the large number of natural sensors available, this DSRS strategy can be extended to many other biosynthetic pathways to balance metabolism, thereby increasing product titers and conversion yields and stabilizing production hosts.

HIGHLIGHTED IN: LBNL Press Release | Scientific American Online |Genetic Engineering & Biotechnology News (GEN)

10. Carothers, J.M., Goler, J.A., Juminaga, D, and Keasling, J.D. Model-driven engineering of RNA devices to quantitatively-program gene expression. Science. 2011. 334, 1716-1719. LINK

ABSTRACT:  The models and simulation tools available to design functionally complex synthetic biological devices are very limited. We formulated a design-driven approach that used mechanistic modeling and kinetic RNA folding simulations to engineer RNA-regulated genetic devices that control gene expression. Ribozyme and metabolite-controlled, aptazyme-regulated expression devices with quantitatively predictable functions were assembled from components characterized in vitro, in vivo, and in silico. The models and design strategy were verified by constructing 28 Escherichia coli expression devices that gave excellent quantitative agreement between the predicted and measured gene expression levels (r = 0.94). These technologies were applied to engineer RNA-regulated controls in metabolic pathways. More broadly, we provide a framework for studying RNA functions and illustrate the potential for the use of biochemical and biophysical modeling to develop biological design methods.

DOE Press Release (w/ quote from Secretary of Energy Steve Chu) | LBNL Press Release | Chemical & Engineering News (C&EN) | Scientific American Online | Nature Reviews Genetics|Genetic Engineering & Biotechnology News (GEN)

9. Carothers, J.M., Goler, J.A., Kapoor, R., Lara, L., and Keasling, J.D. Selecting aptamers for synthetic biology: investigating magnesium dependence and predicting binding affinity. Nucl. Acids Res. 2010. 38, 2736-2747. LINK 

ABSTRACT:  The ability to generate RNA aptamers for synthetic biology using in vitro selection depends on the informational complexity (IC) needed to specify functional structures that bind target ligands with desired affinities in physiological concentrations of magnesium. We investigate how selection for high-affinity aptamers is constrained by chemical properties of the ligand and the need to bind in low magnesium. We select two sets of RNA aptamers that bind planar ligands with dissociation constants (Kds) ranging from 65 nM to 100 μM in physiological buffer conditions. Aptamers selected to bind the non-proteinogenic amino acid, p-amino phenylalanine (pAF), are larger and more informationally complex (i.e., rarer in a pool of random sequences) than aptamers selected to bind a larger fluorescent dye, tetramethylrhodamine (TMR). Interestingly, tighter binding aptamers show less dependence on magnesium than weaker-binding aptamers. Thus, selection for high-affinity binding may automatically lead to structures that are functional in physiological conditions (1–2.5 mM Mg2+). We hypothesize that selection for high-affinity binding in physiological conditions is primarily constrained by ligand characteristics such as molecular weight (MW) and the number of rotatable bonds. We suggest that it may be possible to estimate aptamer–ligand affinities and predict whether a particular aptamer-based design goal is achievable before performing the selection.

HIGHLIGHTED IN:  BioTechniques

8. Carothers, J.M., Goler, J.A., and Keasling, J.D. Chemical synthesis using synthetic biology. Curr. Opin. Biotechnol. 2009. 20, 498-503. LINK

ABSTRACT:  An immense array of naturally occurring biological systems have evolved that convert simple substrates into the products that cells need for growth and persistence. Through the careful application of metabolic engineering and synthetic biology, this biotransformation potential can be harnessed to produce chemicals that address unmet clinical and industrial needs. Developing the capacity to utilize biology to perform chemistry is a matter of increasing control over both the function of synthetic biological systems and the engineering of those systems. Recent efforts have improved general techniques and yielded successes in the use of synthetic biology for the production of drugs, bulk chemicals, and fuels in microbial platform hosts. Synthetic promoter systems and novel RNA-based, or riboregulator, mechanisms give more control over gene expression. Improved methods for isolating, engineering, and evolving enzymes give more control over substrate and product specificity and better catalysis inside the cell. New computational tools and methods for high-throughput system assembly and analysis may lead to more rapid forward engineering. We highlight research that reduces reliance upon natural biological components and point to future work that may enable more rational design and assembly of synthetic biological systems for synthetic chemistry.

7. Hazen, R.M., Griffin, P.L., Carothers, J.M., and Szostak, J.W. Functional information and the emergence of biocomplexity. Proc. Natl. Acad. Sci. 2007. 104, 8574-8581. LINK

ABSTRACT:  Complex emergent systems of many interacting components, including complex biological systems, have the potential to perform quantifiable functions. Accordingly, we define “functional information,” I(Ex ), as a measure of system complexity. For a given system and function, x (e.g., a folded RNA sequence that binds to GTP), and degree of function, Ex (e.g., the RNA–GTP binding energy), I(Ex ) = −log2[F(E x)], where F(Ex ) is the fraction of all possible configurations of the system that possess a degree of function ≥ Ex . Functional information, which we illustrate with letter sequences, artificial life, and biopolymers, thus represents the probability that an arbitrary configuration of a system will achieve a specific function to a specified degree. In each case we observe evidence for several distinct solutions with different maximum degrees of function, features that lead to steps in plots of information versus degree of function.

6. Carothers, J.M., and Szostak, J.W. In vitro selection of functional oligonucleotides and the origins of biochemical activity. In The Aptamer Handbook Functional Oligonucleotides and Their Applications (ed. S. Klussmann). Springer-Verlag Press, Berlin: 2006, 3-28. LINK Available free under "Read Excerpt: Chapter (PDF)"

INTRODUCTION:  In vitro selection is an experimental method for searching oligonucleotide sequence spaces for synthetic structures and activities. Oligonucleotide sequence spaces are very large – they contain the ensemble of all possible sequences of a given length separated by point mutations. For example, the sequence space of an RNA the length of a small tRNA (74 nucleotides) encompasses 1043 different molecules. The largest libraries typically synthesized in the laboratory, approximately 1016 different sequences, represent only a minute fraction of the total number of possible sequences for any nucleic acid molecule of even modest size. How can such necessarily sparse samplings of sequence space produce so many different aptamers, ribozymes, and deoxyribozymes? In this chapter, we focus on the technology of in vitro selection and what its application teaches us about the quantity and quality of functional structures in nucleic acid sequence spaces.

5. Carothers, J.M., Oestreich, S.C., and Szostak, J.W. Aptamers selected for higher-affinity binding are not more specific for the target ligand. J. Am. Chem. Soc. 2006. 128, 7929-7937. LINK

ABSTRACT:  Previous study of eleven different in vitro-selected RNA aptamers that bind guanosine triphosphate (GTP) with Kds ranging from 8 μM to 9 nM showed that more information is required to specify the structures of the higher-affinity aptamers. We are interested in understanding how the more complex aptamers achieve higher affinities for the ligand. In vitro selection produces structural solutions to a functional problem that are are as simple as possible in terms of the information content needed to define them. It has long been assumed that the simplest way to improve the affinity of an aptamer is to increase the shape and functional group complementarity of the RNA binding pocket for the ligand. This argument underlies the hypothesis that selection for higher-affinity aptamers automatically leads to structures that bind more specifically to the target molecule. Here, we examined the binding specificities of the eleven GTP aptamers by carrying out competition binding studies with sixteen different chemical analogues of GTP. The aptamers have distinct patterns of specificity, implying that each RNA is a structurally unique solution to the problem of GTP binding. However, these experiments failed to provide evidence that higher-affinity aptamers bind more specifically to GTP. We suggest that the simplest way to improve aptamer Kds may be to increase the stability of the RNA tertiary structure with additional intramolecular RNA−RNA interactions; increasingly specific ligand binding may emerge only in response to direct selection for specificity.

4. Carothers, J.M., Davis, J.H., Chou, J.J., and Szostak, J.W. Solution structure of an informationally- complex high-affinity RNA aptamer to GTP. RNA. 2006. 12, 567-579. LINK

ABSTRACT:  Higher-affinity RNA aptamers to GTP are more informationally complex than lower-affinity aptamers. Analog binding studies have shown that the additional information needed to improve affinity does not specify more interactions with the ligand. In light of those observations, we would like to understand the structural characteristics that enable complex aptamers to bind their ligands with higher affinity. Here we present the solution structure of the 41-nt Class I GTP aptamer (Kd = 75 nM) as determined by NMR. The backbone of the aptamer forms a reverse-S that shapes the binding pocket. The ligand nucleobase stacks between purine platforms and makes hydrogen bonds with the edge of another base. Interestingly, the local modes of interaction for the Class I aptamer and an RNA aptamer that binds ATP with a Kdof 6 μM are very much alike. The aptamers exhibit nearly identical levels of binding specificity and fraction of ligand sequestered from the solvent (81%–85%). However, the GTP aptamer is more informationally complex (~45 vs. 35 bits) and has a larger recognition bulge (15 vs. 12 nucleotides) with many more stabilizing base–base interactions. Because the aptamers have similar modes of ligand binding, we conclude that the stabilizing structural elements in the Class I aptamer are responsible for much of the difference in Kd. These results are consistent with the hypothesis that increasing the number of intra-RNA interactions, rather than adding specific contacts to the ligand, is the simplest way to improve binding affinity.

3. Plummer, K.A., Carothers, J.M., Yoshimura, M. Szostak, J.W., and Verdine, G.L. In vitro selection of RNA aptamers against a composite small molecule-protein surface. Nucl. Acids Res. 2005, 33, 5602- 5610. LINK

ABSTRACT:  A particularly challenging problem in chemical biology entails developing systems for modulating the activity of RNA using small molecules. One promising new approach towards this problem exploits the phenomenon of ‘surface borrowing,’ in which the small molecule is presented to the RNA in complex with a protein, thereby expanding the overall surface area available for interaction with RNA. To extend the utility of surface borrowing to include potential applications in synthetic biology, we set out to create an ‘orthogonal’ RNA-targeting system, one in which all components are foreign to the cell. Here we report the identification of small RNA modules selected in vitro to bind a surface-engineered protein, but only when the two macromolecules are bound to a synthetic bifunctional small molecule.

2. Carothers, J.M., Oestreich, S.C., Davis, J.H., and Szostak, J.W. Informational complexity and functional activity of RNA structures. J. Am. Chem. Soc. 2004, 12: 5130-5137. LINK

ABSTRACT:  Very little is known about the distribution of functional DNA, RNA, and protein molecules in sequence space. The question of how the number and complexity of distinct solutions to a particular biochemical problem varies with activity is an important aspect of this general problem. Here we present a comparison of the structures and activities of eleven distinct GTP-binding RNAs (aptamers). By experimentally measuring the amount of information required to specify each optimal binding structure, we show that defining a structure capable of 10-fold tighter binding requires approximately 10 additional bits of information. This increase in information content is equivalent to specifying the identity of five additional nucleotide positions and corresponds to an 1000-fold decrease in abundance in a sample of random sequences. We observe a similar relationship between structural complexity and activity in a comparison of two catalytic RNAs (ribozyme ligases), raising the possibility of a general relationship between the complexity of RNA structures and their functional activity. Describing how information varies with activity in other heteropolymers, both biological and synthetic, may lead to an objective means of comparing their functional properties. This approach could be useful in predicting the functional utility of novel heteropolymers.

1. Kopp, E., Medzhitov, R., Carothers, J., Xiao, C., Douglas, I., Janeway, C.A., and Ghosh, S. ECSIT is an evolutionarily conserved intermediate in the Toll/IL-1 signal transduction pathway. Genes Develop. 1999, 13: 2059-2071.  LINK

ABSTRACT:  Activation of NF-κB as a consequence of signaling through the Toll and IL-1 receptors is a major element of innate immune responses. We report the identification and characterization of a novel intermediate in these signaling pathways that bridges TRAF6 to MEKK-1. This adapter protein, which we have named ECSIT (evolutionarilyconserved signaling intermediate inToll pathways), is specific for the Toll/IL-1 pathways and is a regulator of MEKK-1 processing. Expression of wild-type ECSIT accelerates processing of MEKK-1, whereas a dominant-negative fragment of ECSIT blocks MEKK-1 processing and activation of NF-κB. These results indicate an important role for ECSIT in signaling to NF-κB and suggest that processing of MEKK-1 is required for its function in the Toll/IL-1 pathway.