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Results for “Synthetic Biology”

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bioRxiv2017

Engineering post-translational proofreading to discriminate non-standard amino acids

Progress in genetic code expansion requires accurate, selective, and high-throughput detection of non-standard amino acid (NSAA) incorporation into proteins. Here, we discover how the N-end rule pathway of protein degradation applies to commonly used NSAAs. We show that several NSAAs are N-end stabilizing and demonstrate that other NSAAs can be made stabilizing by rationally engineering the N-end rule adaptor protein ClpS. We use these insights to engineer a synthetic quality control method, termed \"Post-Translational Proofreading\" (PTP). By implementing PTP, false positive proteins resulting from misincorporation of structurally similar standard amino acids or undesired NSAAs rapidly degrade, enabling high-accuracy discrimination of desired NSAA incorporation. We illustrate the utility of PTP during evolution of the biphenylalanine orthogonal translation system used for synthetic biocontainment. Our new OTS is more selective and confers lower escape frequencies and greater fitness in all tested biocontained strains. Our approach presents a new paradigm for molecular recognition of amino acids in target proteins.

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bioRxiv2017

Cell-free and in vivo characterization of Lux, Las, and Rpa quorum activation systems in E. coli

Synthetic biologists have turned towards quorum systems as a path for building sophisticated microbial consortia that exhibit group decision making. Currently, however, even the most complex consortium circuits rely on only one or two quorum sensing systems, greatly restricting the available design space. High-throughput characterization of available quorum sensing systems is useful for finding compatible sets of systems that are suitable for a defined circuit architecture. Recently, cell-free systems have gained popularity as a test-bed for rapid prototyping of genetic circuitry.\n\nWe take advantage of the transcription-translation cell-free system to characterize three commonly used Lux-type quorum activators, Lux, Las, and Rpa. We then compare the cell-free characterization to results obtained in vivo. We find significant genetic crosstalk in both the Las and Rpa systems and substantial signal crosstalk in Lux activation. We show that cell-free characterization predicts crosstalk observed in vivo.

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bioRxiv2017

Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks

The RNA-guided DNA endonuclease Cas9 has emerged as a powerful new tool for genome engineering. Cas9 creates targeted double-strand breaks (DSBs) in the genome. Knock-in of specific mutations (precision genome editing) requires homology-directed repair (HDR) of the DSB by synthetic donor DNAs containing the desired edits, but HDR has been reported to be variably efficient. Here, we report that linear DNAs (single and double-stranded) engage in a high-efficiency HDR mechanism that requires only [~]35 nucleotides of homology with the targeted locus to introduce edits ranging from 1 to 1000 nucleotides. We demonstrate the utility of linear donors by introducing fluorescent protein tags in human cells and mouse embryos using PCR fragments. We find that repair is local, polarity-sensitive, and prone to template switching, characteristics that are consistent with gene conversion by synthesis-dependent strand-annealing (SDSA). Our findings enable rational design of synthetic donor DNAs for efficient genome editing.\n\nSignificanceGenome editing, the introduction of precise changes in the genome, is revolutionizing our ability to decode the genome. Here we describe a simple method for genome editing that takes advantage of an efficient mechanism for DNA repair called synthesis-dependent strand annealing. We demonstrate that synthetic linear DNAs (ssODNs and PCR fragments) with [~]35bp homology arms function as efficient donors for SDSA repair of Cas9-induced double-strand breaks. Edits from 1 to 1000 base pairs can be introduced in the genome without cloning or selection.

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bioRxiv2017

Automated Parameterization of Predictive Kinetic Metabolic Models from Sparse Datasets for Efficient Optimization of Many-Enzyme Heterologous Pathways

Engineered heterologous metabolic pathways can convert low-cost feedstock into high-value products, though it remains a significant challenge to reliably and efficiently maximize end-product biosynthesis, particularly when many enzymes must be co-expressed together. When current approaches are applied to many-enzyme pathways, the construction and characterization process is highly iterative and laborious, while generating high-dimensional datasets that remain difficult to analyze for forward engineering efforts. To overcome these challenges, we developed a new algorithm that determines the highly non-linear and high-dimensional relationship between a pathways enzyme expression levels and its end-product productivity from common characterization of a small number of heterologous pathway variants. We combined kinetic metabolic modeling, elementary mode analysis, model reduction, de-dimensionalization, and genetic algorithm optimization into an automated procedure that parameterizes accurate kinetic metabolic models from sparsely characterized pathway variant libraries with varied enzyme expression levels. The resulting Pathway Maps are used to determine rate-limiting steps, predict optimal expression levels, identify allosteric interactions, rank-order enzyme kinetics, and prioritize protein engineering efforts. We demonstrate the Pathway Map Calculator algorithm on two experimental datasets, a 3-enzyme carotenoid biosynthesis pathway and a 9-enzyme limonene biosynthesis pathway, as well as a series of in silico pathway examples to rigorously demonstrate the algorithms accuracy, linear scaling, and high tolerance to measurement noise. By greatly reducing experimental efforts and providing quantitative forward engineering predictions, the Pathway Map Calculator has the potential to dramatically accelerate the engineering of many-enzyme heterologous metabolic pathways.\n\nHighlightsO_LIWe developed an automated algorithm that uses a small number of characterized pathway variants to determine the pathways expression-productivity relationship.\nC_LIO_LIThe Pathway Map Calculator is accurate, scales linearly on many-enzyme pathways, distinguishes allosteric interactions, and tolerates substantial measurement noise.\nC_LIO_LIPathway Maps are used to predict optimal enzyme expression levels, identify rate-limiting steps, and prioritize protein engineering efforts\nC_LI

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bioRxiv2017

Reducing Pyrrolysine tRNA Copy Number Improves the Performance of Genetic Code Expansion in Live Cell Imaging of Bioorthogonally Labeled Proteins

Genetic code expansion enables the incorporation of non-canonical amino acids (ncAAs) into expressed proteins. ncAAs are usually encoded by a stop codon that is decoded by an exogenous orthogonal aminoacyl tRNA synthetase and its cognate suppressor tRNA, such as the pyrrolysine synthetase/[Formula] pair. In such systems, stop codon suppression is dependent on the intracellular levels of the exogenous tRNA. Therefore, multiple copies of the tRNAPyl gene (PylT) are encoded to improve ncAA incorporation. However, certain applications in mammalian cells, such as live-cell imaging applications, where labelled tRNA contributes to background fluorescence, can benefit from the use of less invasive minimal expression systems. Accordingly, we studied the effect of tRNAPyl on live-cell fluorescence imaging of bioorthogonally-labelled intracellular proteins. We found that in COS7 cells, a decrease in PylT copy numbers had no measurable effect on protein expression levels. Importantly, reducing PylT copy numbers improved the quality of live-cells images by enhancing the signal-to-noise ratio and reducing an immobile tRNAPyl population. This enabled us to improve live cell imaging of bioorthogonally labelled intracellular proteins, and to simultaneously label two different proteins in a cell. Our results indicate that the number of introduced PylT genes can be minimized according to the transfected cell line, incorporated ncAA, and application.

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bioRxiv2017

Engineered CRISPR-Cas9 system enables noiseless, fine-tuned and multiplexed repression of bacterial genes

Over the past few years, tools that make use of the Cas9 nuclease have led to many breakthroughs, including in the control of gene expression. The catalytically dead variant of Cas9 known as dCas9 can be guided by small RNAs to block transcription of target genes, in a strategy also known as CRISPRi. Here, we reveal that the level of complementarity between the guide RNA and the target controls the rate at which dCas9 successfully blocks the RNA polymerase. We use this mechanism to precisely and robustly reduce gene expression by defined relative amounts. We demonstrate broad applicability of this method to the study of genetic regulation and cellular physiology. First, we characterize feedback strength of a model auto-repressor. Second, we study the impact of copy-number variations of cell-wall synthesizing enzymes on cell morphology. Finally, we demonstrate that this system can be multiplexed to obtain any combination of fractional repression of two genes.

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bioRxiv2017

Screening and directed evolution of transporters to improve xylodextrin utilization in the yeast Saccharomyces cerevisiae

The economic production of cellulosic biofuel requires efficient and full utilization of all abundant carbohydrates naturally released from plant biomass by enzyme cocktails. Recently, we reconstituted the Neurospora crassa xylodextrin transport and consumption system in Saccharomyces cerevisiae, enabling growth of yeast on xylodextrins aerobically. However, the consumption rate of xylodextrin requires improvement for industrial applications, including consumption in anaerobic conditions. As a first step in this improvement, we report analysis of orthologues of the N. crassa transporters CDT-1 and CDT-2. Transporter ST16 from Trichoderma virens enables faster aerobic growth of S. cerevisiae on xylodextrins compared to CDT-2. ST16 is a xylodextrin-specific transporter, and the xylobiose transport activity of ST16 is not inhibited by cellobiose. Other transporters identified in the screen also enable growth on xylodextrins including xylotriose. Taken together, these results indicate that multiple transporters might prove useful to improve xylodextrin utilization in S. cerevisiae. Efforts to use directed evolution to improve ST16 from a chromosomally-integrated copy were not successful, due to background growth of yeast on other carbon sources present in the selection medium. Future experiments will require increasing the baseline growth rate of the yeast population on xylodextrins, to ensure that the selective pressure exerted on xylodextrin transport can lead to isolation of improved xylodextrin transporters.

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bioRxiv2017

An in vivo binding assay for RNA-binding proteins based on repression of a reporter gene

We employ a reporter assay and Selective 2'-hydroxyl acylation analysed by primer extension sequencing (SHAPE-seq) to study translational regulation by RNA-binding proteins, in bacteria. We designed 82 constructs, each with a single hairpin based on the binding sites of the RNA-binding coat proteins of phages MS2, PP7, GA, and Q{beta}, at various positions within the N-terminus of a reporter gene. In the absence of RNA-binding proteins, the translation level depends on hairpin location, and exhibits a three-nucleotide periodicity. For hairpin positions within the initiation region, we observe strong translational repression in the presence of its cognate RNA-binding protein. In vivo SHAPE-seq results for a representative construct indicate that the repression phenomenon correlates with a wide-swath of protection, including the hairpin and extending past the ribosome binding site. Consequently, our data suggest that the protection provided by the RBP-hairpin complex inhibits ribosomal initiation. Finally, utilizing the repression phenomenon for quantifying protein-RNA binding affinity in vivo, we both observe partially contrasting results to previous in vitro and in situ studies, and additionally, show that this method can be used in a high-throughput assay for a quantitative study of protein-RNA binding in vivo.

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bioRxiv2017

Rapid and scalable characterization of CRISPR technologies using an E. coli cell-free transcription-translation system

CRISPR-Cas systems have offered versatile technologies for genome engineering, yet their implementation has been outpaced by the ongoing discovery of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation systems (TXTL) to vastly improve the speed and scalability of CRISPR characterization and validation. Unlike prior approaches that require protein purification or live cells, TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression. To demonstrate the applicability of TXTL, we rapidly measure guide RNA-dependent DNA cleavage and gene repression for single- and multi-effector CRISPR-Cas systems, accurately predict the strength of gene repression in E. coli, quantify the inhibitory activity of anti-CRISPR proteins, and develop a fast and scalable high-throughput screen for protospacer-adjacent motifs. These examples underscore the potential of TXTL to facilitate the characterization and application of CRISPR technologies across their many uses.

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bioRxiv2017

A Prototype for Modular Cell Engineering

When aiming to produce a target chemical at high yield, titer, and productivity, various combinations of genetic parts available to build the target pathway can generate a large number of strains for characterization. This engineering approach will become increasingly laborious and expensive when seeking to develop desirable strains for optimal production of a large space of biochemicals due to extensive screening. Our recent theoretical development of modular cell (MODCELL) design principles can offer a promising solution for rapid generation of optimal strains by coupling a modular cell and exchangeable production modules in a plug-and-play fashion. In this study, we experimentally validated some designed properties of MODCELL by demonstrating: i) a modular (chassis) cell is required to couple with a production module, a heterologous ethanol pathway, as a testbed, ii) degree of coupling between the modular cell and production modules can be modulated to enhance growth and product synthesis, iii) a modular cell can be used as a host to select an optimal pyruvate decarboxylase (PDC) of the ethanol production module and to help identify a hypothetical PDC protein, and iv) adaptive laboratory evolution based on growth selection of the modular cell can enhance growth and product synthesis rates. We envision that the MODCELL design provides a powerful prototype for modular cell engineering to rapidly create optimal strains for synthesis of a large space of biochemicals.

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bioRxiv2017

A synthetic integral feedback controller for robust tunable regulation in bacteria

We report on the first engineered integral feedback control system in a living cell. The controller is based on the recently published antithetic integral feedback motif [1] which has been analytically shown to have favorable regulation properties. It is implemented along with test circuitry in Escherichia coli using seven genes and three small-molecule inducers. The closed-loop system is highly tunable, allowing a regulated protein of interest to be driven to a desired level and maintained there with precision. Realized using a sigma/anti-sigma protein pair, the integral controller ensures that regulation is maintained in the face of perturbations that lead to the regulated proteins degradation, thus serving as a proof-of-concept prototype of integral feedback implementation in living cells. When suitably optimized, this integral controller may be utilized as a general-purpose robust regulator for genetic circuits with unknown or partially-known topologies and parameters.

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bioRxiv2017

Cell-free prediction of protein expression costs for growing cells

Translating heterologous proteins places significant burden on host cells, consuming expression resources leading to slower cell growth and productivity. Yet predicting the cost of protein production for any gene is a major challenge, as multiple processes and factors determine translation efficiency. Here, to enable prediction of the cost of gene expression in bacteria, we describe a standard cell-free lysate assay that determines the relationship between in vivo and cell-free measurements and {gamma}, a relative measure of the resource consumption when a given protein is expressed. When combined with a computational model of translation, this enables prediction of the in vivo burden placed on growing E. coli cells for a variety of proteins of different functions and lengths. Using this approach, we can predict the burden of expressing multigene operons of different designs and differentiate between the fraction of burden related to gene expression compared to action of a metabolic pathway.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC=\"FIGDIR/small/172627_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (26K):\norg.highwire.dtl.DTLVardef@19afa63org.highwire.dtl.DTLVardef@1acafbforg.highwire.dtl.DTLVardef@46443borg.highwire.dtl.DTLVardef@12987e3_HPS_FORMAT_FIGEXP M_FIG C_FIG

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bioRxiv2017

Gossypol biosynthesis in cotton revealed through organ culture, plant grafting and gene expression profiling

Gossypol plays an important role in defense mechanism of Gossypium species and the presence of gossypol also limits the utilization of cottonseeds. However, little is known about the metabolism of gossypol in cotton plant. Here, Detection on the dynamic tendency of gossypol content illustrated that at the germination stage, the main source of gossypol was cotyledon, and at the later stages, gossypol mainly came from root system. Plant grafting between cottons and sunflower proved that gossypol was mainly synthesized in the root systems of cotton plants and both of the glanded and glandless cottons had the ability of gossypol biosynthesis. Besides, the pigment glands expression was uncoupled with gossypol biosynthesis. Root tip and rootless seedling organ culture in vitro further revealed other parts of the seedlings also got the ability to synthesize gossypol except root system. Moreover, root system produced the racemic gossypol and plant synthesized the optically active gossypol. The expression profiling of key genes in the gossypol biosynthetic pathway suggested that downstream key genes had relatively high expression levels in root systems which confirmed that gossypol was mainly synthesized in the root systems. Taken together, our results helped to clarify the complex mechanism of gossypol metabolism.

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bioRxiv2017

Labeling RNAs in live cells using malachite green aptamer scaffolds as fluorescent probes

RNAs mediate many different processes that are central to cellular function. The ability to quantify or image RNAs in live cells is very useful in elucidating such functions of RNA. RNA aptamerfluorogen systems have been increasingly used in labeling RNAs in live cells. Here, we use the malachite green aptamer (MGA), an RNA aptamer that can specifically bind to malachite green (MG) dye and induces it to emit far-red fluorescence signals. Previous studies on MGA showed a potential for the use of MGA for genetically tagging other RNA molecules in live cells. However, these studies also exhibited low fluorescence signals and high background noise. Here we constructed and tested RNA scaffolds containing multiple tandem repeats of MGA as a strategy to increase the brightness of the MGA aptamer-fluorogen system as well as to make the system fluoresce when tagging various RNA molecules, in live cells. We demonstrate that our MGA scaffolds can induce fluorescence signals by up to ~20 fold compared to the basal level as a genetic tag for other RNA molecules. We also show that our scaffolds function reliably as genetically-encoded fluorescent tags for mRNAs of fluorescent proteins and other RNA aptamers.

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bioRxiv2017

Synthetic protein-sensing riboswitches

We study translational regulation by a 5 UTR sequence encoding the binding site of an RNA-binding protein (RBP) in bacteria, using a reporter assay and Selective 2-hydroxyl acylation analysed by primer extension sequencing (SHAPE-Seq). We tested constructs containing a single hairpin, based on the binding sites of the coat RBPs of bacteriophages GA, MS2, PP7, and Q{beta}, positioned in the 5 UTR of a reporter gene. With specifically-bound RBP present, either weak repression or up-regulation is observed, depending on the binding site and its flanking sequence. SHAPE-Seq data for a representative construct exhibiting up-regulation, indicates a partially-folded hairpin and non-reactive upstream and downstream flanking region, which we attribute to intermediate structures that apparently blocks translation. RBP binding stabilizes the fully-folded hairpin state and thus facilitates translation, suggesting that the up-regulating constructs are RBP-sensing riboswitches. This finding is further supported by lengthening the binding-site stem, which in turn destabilizes the translationally-inactive state, and abolishes the up-regulating behavior. Finally, we found that the combination of two binding sites, positioned in the 5 UTR and gene-header of the same transcript, can yield a cooperative regulatory response. Together, we show that the interaction of an RBP with its RNA target facilitates structural changes in the RNA, which is reflected by a controllable range of binding affinities and dose response behaviors. Thus, demonstrating that RNA-RBP interactions can provide a platform for constructing gene regulatory networks that are based on translational, rather than transcriptional, regulation.

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bioRxiv2017

Burden-driven feedback control of gene expression

Cells use feedback regulation to ensure robust growth despite fluctuating demands on resources and different environmental conditions. Yet the expression of foreign proteins from engineered constructs is an unnatural burden on resources that cells are not adapted for. Here we combined multiplex RNAseq with an in vivo assay to reveal the major transcriptional changes in two E. coli strains when a set of inducible synthetic constructs are expressed. We identified that native promoters related to the heat-shock response activate expression rapidly in response to synthetic expression, regardless of the construct. Using these promoters, we built a CRISPR/dCas9-based feedback regulation system that automatically adjusts synthetic construct expression in response to burden. Cells equipped with this general-use controller maintain capacity for native gene expression to ensure robust growth and as such outperform unregulated cells at protein yields in batch production. This engineered feedback is the first example of a universal, burden-based biomolecular control system and is modular, tuneable and portable.

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bioRxiv2017

Control of lineage-specific gene expression by functionalized gRNA barcodes

Lineage tracking delivers essential quantitative insight into dynamic, probabilistic cellular processes, such as somatic tumor evolution and differentiation. Methods for high diversity lineage quantitation rely on sequencing the population of DNA barcodes. However, molecular analysis of specific individual lineages is not possible with this approach. To address this challenge, we developed a functionalized lineage tracing tool that enables high diversity lineage tracing and lineage-specific manipulation of gene expression. This modular platform utilizes expressed barcode gRNAs to both track cell lineages and direct lineage specific gene expression.

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bioRxiv2017

Functional effects of heating and cooling gene networks

Everyday existence and survival of most organisms requires coping with temperature changes, which involves gene regulatory networks both as subjects and agents of cellular protection. Yet, how temperature affects gene network function remains unclear, partly because natural gene networks are complex and incompletely characterized. Here, we study how heating and cooling affect the function of single genes and well-characterized synthetic gene circuits in Saccharomyces cerevisiae. We found nontrivial, nonmonotone temperature-dependent gene expression trends at non-growth-optimal temperatures. In addition, heating caused unusual bimodality in the negative-feedback gene circuit expression and shifts upward the bimodal regime for the positive feedback gene circuit. Mathematical models incorporating temperature-dependent growth rates and Arrhenius scaling of reaction rates captured the effects of cooling, but not those of heating. Molecular dynamics simulations revealed that heating alters the conformational dynamics and allows DNA-binding of the TetR transcriptional repressor, fully explaining the experimental results for the negative-feedback gene circuit. Overall, we uncover how temperature shifts may corrupt gene networks, which may aid future designs of temperature-robust synthetic gene circuits.

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