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bioRxiv2018

Marionette: E. coli containing 12 highly-optimized small molecule sensors

Cellular processes are carried out by many interacting genes and their study and optimization requires multiple levers by which they can be independently controlled. The most common method is via a genetically-encoded sensor that responds to a small molecule (an \"inducible system\"). However, these sensors are often suboptimal, exhibiting high background expression and low dynamic range. Further, using multiple sensors in one cell is limited by cross-talk and the taxing of cellular resources. Here, we have developed a directed evolution strategy to simultaneously select for less background, high dynamic range, increased sensitivity, and low crosstalk. Libraries of the regulatory protein and output promoter are built based on random and rationally-guided mutations. This is applied to generate a set of 12 high-performance sensors, which exhibit >100-fold induction with low background and cross-reactivity. These are combined to build a single \"sensor array\" and inserted into the genomes of E. coli MG1655 (wild-type), DH10B (cloning), and BL21 (protein expression). These \"Marionette\" strains allow for the independent control of gene expression using 2,4-diacetylphophloroglucinol (DAPG), cuminic acid (Cuma), 3-oxohexanoyl-homoserine lactone (OC6), vanillic acid (Van), isopropyl {beta}-D-1-thiogalactopyranoside (IPTG), anhydrotetracycline (aTc), L-arabinose (Ara), choline chloride (Cho), naringenin (Nar), 3,4-dihydroxybenzoic acid (DHBA), sodium salicylate (Sal), and 3-hydroxytetradecanoyl-homoserine lactone (OHC14).

synthetic biology↗

bioRxiv2018

Optical activation of TrkA signaling

Nerve growth factor/tropomyosin receptor kinase A (NGF/TrkA) signaling plays a key role in neuronal development, function, survival, and growth. The pathway is implicated in neurodegenerative disorders including Alzheimers disease, chronic pain, inflammation, and cancer. NGF binds the extracellular domain of TrkA, leading to the activation of the receptors intracellular kinase domain. TrkA signaling is highly dynamic, thus mechanistic studies would benefit from a tool with high spatial and temporal resolution. Here we present the design and evaluation of four strategies for light-inducible activation of TrkA in the absence of NGF. Our strategies involve the light-sensitive protein Arabidopsis cryptochrome 2 (CRY2) and its binding partner CIB1. We demonstrate successful recapitulation of native NGF/TrkA functions by optical induction of plasma membrane recruitment and homo-interaction of the intracellular domain of TrkA. This approach activates PI3K/AKT and Raf/ERK signaling pathways, promotes neurite growth in PC12 cells, and supports the survival of dorsal root ganglion neurons in the absence of NGF. This ability to activate TrkA using light bestows high spatial and temporal resolution for investigating NGF/TrkA signaling.

synthetic biology↗

bioRxiv2018

Astroplastic: A start-to-finish process for polyhydroxybutyrate production from solid human waste using genetically engineered bacteria to address the challenges for future manned Mars missions

Space exploration has long been a source of inspiration, challenging scientists and engineers to find innovative solutions to various problems. One of the current focuses in space exploration is to send humans to Mars. However, the challenge of transporting materials to Mars and the need for waste management processes are two major obstacles for these long-duration missions.\n\nTo address these two challenges a process called Astroplastic was developed that produces polyhydroxybutyrate (PHB) from solid human waste, which can be used to 3D print useful items for astronauts. PHB granules are naturally produced by bacteria such as Ralstonia eutropha and Pseudomonas aeruginosa for carbon and energy storage. The phaJ, phaC, and phaCBA genes were cloned from these native PHB-producing bacteria into Escherichia coli. These genes code for enzymes that aid in PHB production by converting products of glycolysis and {beta}-oxidation pathways, such as acetyl-CoA and enoyl-CoA, into PHB. To ensure a continuous PHB production system and to eliminate the need for cell lysis to extract PHB, recombinant E. coli was engineered to use the genes in its natural type I secretion system to secrete PHB. The C-terminal of the HlyA secretion tag was fused to phasin (PhaP), a protein originally from R. eutropha. Phasin-HlyA electrostatically binds PHB granules and transports them outside of the cell.\n\nIn addition to genetically engineering bacteria, a concept for start-to-finish PHB production process was designed. Integrating expert feedback and experimental results, conditions for each step of the process including the collection and storage of waste, volatile fatty acid (VFA) fermentation, VFA extraction, PHB fermentation, and PHB extraction were optimized. The optimized system will provide a sustainable and continuous PHB production system, which will address the problems of transportation costs and waste management for future space missions.\n\nFinancial DisclosureMindfuel Science Alberta Foundation Genome Alberta GenScript Polyferm Canada GeekStarter Alberta Integrated DNA Technologies University of Calgary University of Calgary Cumming School of Medicine University of Calgary Bachelor of Sciences University of Calgary Schulich School of Engineering University of Calgary OBrien Centre for the Bachelor of Health Sciences City of Calgary Alberta Innovates The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\nCompeting InterestsThe authors have declared that no competing interests exist.\n\nEthics StatementN/A\n\nData AvailabilityAll data are freely available without restriction.

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bioRxiv2018

Discovery and Characterization of Novel Lignocellulose-Degrading Enzymes from the Porcupine Microbiome

Plant cell walls are comprised of cellulose, hemicellulose, and lignin, collectively known as lignocellulose. Microorganisms degrade these components to liberate sugars to meet metabolic demands. Using a metagenomic sequencing approach, we previously demonstrated that the microbiome of the North American porcupine (Erethizon dorsatum) is replete with novel lignocellulose-degrading enzymes. Here, we report the identification, synthesis and partial characterization of four genes from the porcupine microbiome encoding putative novel lignocellulose-degrading enzymes, including a {beta}-xylanase, endoxylanase, {beta}-glucosidase, and an -L-arabinofuranosidase. These genes were identified via conserved catalytic domains associated with cellulose and hemicellulose degradation. We cloned the putative {beta}-xylanase into the pET26b(+) plasmid, enabling inducible gene expression in Escherichia coli (E. coli) and periplasmic localization. We demonstrated IPTG-inducible accumulation of {beta}-xylanase protein but failed to detect xylobiose degrading activity in a reporter assay. Alternative assays may be required to measure activity of this putative {beta}-xylanase. In this report, we describe how a synthetic metagenomic pipeline can be used to identify novel microbial lignocellulose-degrading enzymes and take initial steps to introduce a hemicellulose-degradation pathway into E. coli to enable biofuel production from wood pulp feedstock.

synthetic biology↗

bioRxiv2018

Quantification of DNA samples by Ethidium Bromide Spot Technique

Accurate and quick determination of DNA concentration is critical for the assembly of synthetic constructs, as well as a multitude of other experiments. We sought to optimize an under-utilized and inexpensive approach for determining DNA concentration: a spotting technique that uses the intercalating dye Ethidium Bromide. This technique does not require specialized equipment such as a spectrophotometer, but instead relies on visualization of dye-DNA complex fluorescence when excited by UV light. We modelled and tested a range of parameters for dye concentration and spot size, finding that 15uL spots with 1.0ug/mL Ethidium Bromide produced the most reliable standard curve. More importantly, we hope that our approach can help other labs optimize this protocol for their own experimental setup. Adoption of this technique may help enable development of iGEM teams in resource limited environments and laboratories which do not or cannot employ a satisfactory method for determining DNA concentration.\n\nFinancial DisclosureWe obtained funding support from Canon Solutions America, and indirect support for this work through the CUNY Research Scholars Program. However, the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\nCompeting InterestsThe authors have declared that no competing interests exist.\n\nEthics StatementN/A\n\nData AvailabilityYes - all data are fully available without restriction. The data can be found in the associated supplementary materials.\n\nArticle TagsDNA, Ethidium Bromide, EtBr, Concentration, Method, UV, Gibson Assembly\n\nHeader StatementThis work was assessed during the iGEM/PLOS Realtime Peer Review Jamboree on 23rd February 2018 and has been revised in response to the reviewers. Responses to Reviewers, and supplementary information (including Fig. S1) are available as separate files.

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bioRxiv2018

A thermo-responsive plasmid for biconditional protein expression

Here we develop a temperature sensitive expression vector that allows the selective production of distinct proteins over different temperature ranges with a single plasmid. We use the E. coli cold shock translational control system (the cspA 5Untranslated Transcribed Region - UTR) to drive the expression of a desired protein below a certain temperature threshold, and the lambda phage pL/cI857 transcriptional repressor system to drive the expression of a different protein above a certain temperature threshold. In this developmental work we use the chromogenic reporter proteins amilCP (blue chromoprotein) and monomeric red fluorescent protein (mRFP) to assess the function of the thermo-sensitive regulatory sequences over the desired temperature ranges. Our results show temperature dependent response of the cold shock regulatory sequence. However, our sequence design for the heat shock regulatory sequence did not give the intended result. The integration of these two temperature sensitive elements into a single plasmid awaits the re-design of the heat shock sequence.

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bioRxiv2018

Quantitative characterization of translational riboregulators using an in vitro transcription-translation system

Riboregulators are short RNA sequences that, upon binding to a ligand, change their secondary structure and influence the expression rate of a downstream gene. They constitute an attractive alternative to transcription factors for building synthetic gene regulatory networks because they can be engineered de novo and they have a fast turnover and a low metabolic burden. However, riboregulators are generally designed in silico and tested in vivo, which only provides a yes/no evaluation of their performances, thus hindering the improvement of design algorithms. Here we show that a cell-free transcription-translation (TX-TL) system provides valuable quantitative information about the performances of in silico designed riboregulators. In particular, we use the ribosome as an exquisite molecular machine that detects functional riboregulators, precisely measures their concentration and linearly amplifies the signal by generating a fluorescent protein. We apply this method to characterize two types of translational riboregulators composed of a cis-repressed (cr) and a trans-activating (ta) strand. At the DNA level we demonstrate that high concentrations of taDNA poisoned the activator until total shut off. At the RNA level, we show that this approach provides a fast and simple way to measure dissociation constants of functional riboregulators, in contrast to standard mobility-shift assays. Our method opens the route for using cell-free TX-TL systems for the quantitative characterization of functional riboregulators in order to improve their design in silico.

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bioRxiv2018

Towards a bioelectronic computer: A theoretical study of a multi-layer biomolecular computing system that can process electronic inputs

DNA molecular machines have great potential for use in computing systems. Since Adleman originally introduced the concept of DNA computing through his use of DNA strands to solve a Hamiltonian path problem, a range of DNA-based computing elements have been developed, including logic gates, neural networks, finite state machines (FSMs) and non-deterministic universal Turing machines. It has also been established that DNA molecular machines can be controlled using electrical signals and that the state of DNA nanodevices can be measured using an electrochemical readout. However, to the best of our knowledge there has as yet been no demonstration of a fully integrated biomolecular computing system that has multiple levels of information processing capacity, can accept electronic inputs and is capable of independent operation. In this paper we address the question of how such a system could work. We present simulation results showing that such an integrated hybrid system could convert electrical impulses into biomolecular signals, perform logical operations and take a decision, storing its history. We also illustrate theoretically how such a system could potentially be used to perform a task such as controlling an autonomous robot navigating through a maze.

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bioRxiv2018

Development of a GFP Fluorescent Bacterial Biosensor for the Detection and Quantification of Silver and Copper Ions

Ionic silver is known to be an effective antimicrobial agent widely used in the cleaning and medical industries, however, there are several concerns regarding the release of silver pollutants into the environment. Presented here are two engineered bacterial biosensors for the detection and quantification of silver. The biosensors contain a silver resistance operon and a GFP gene that is strictly regulated through silver activated regulatory regions that control expression of the sil operons. The two biosensors are responsive to a wide range of silver ion concentrations, and a correlation between silver and GFP signal is seen at select concentration ranges. The biosensors were shown to detect silver ions released from silver nanoparticles, and have the potential to become a method for monitoring ion release rates of different nanoparticles. Interestingly, the close homology of the silver resistance and copper resistance genes allowed for the biosensor to also be responsive to copper ions, implying that copper ions activate silver resistance. Further development of this biosensor could lead to commercial applications for environmental monitoring.\n\nImportanceIonic silver is known to have many harmful environmental effects. Silver pollutants have been found in various environmental settings such as natural waterways and tailings from mining operations, raising concern. In addition, persistent exposure to silver in medical and environmental settings has led to the development of silver resistant bacteria, many of which are also resistant to a wide range of antibiotics. Some of these have the potential to develop into human pathogens. It then becomes important to have standardized methods for detecting and monitoring silver concentrations in various environments so that appropriate measures can be taken to prevent further silver ion release. This research shows that bacterial biosensors engineered to detect and quantify silver ions can be developed as effective alternatives to traditional analytical techniques. Further development of such biosensors could result in a commercial system for short and long term environmental monitoring, which is important as products containing silver and other heavy metals become increasingly popular.

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bioRxiv2018

Systematically investigating the key features of the nuclease deactivated Cpf1 for tunable multiplex genetic regulation

With a unique crRNA processing capability, the CRISPR associated Cpf1 protein holds great potential for multiplex gene regulation. Unlike the well-studied Cas9 protein, however, conversion of Cpf1 to a transcription regulator and its related properties have not been systematically explored yet. In this study, we investigated the mutation schemes and crRNA requirements for the nuclease deactivated Cpf1 (dCpf1). By shortening the direct repeat sequence, we obtained genetically stable crRNA co-transcripts and improved gene repression with multiplex targeting. A screen of diversity-enriched PAM library was designed to investigate the PAM-dependency of gene regulation by dCpf1 from Francisella novicida and Lachnospiraceae bacterium. We found novel PAM patterns that elicited strong or medium gene repressions. Using a computational algorithm, we predicted regulatory outputs for all possible PAM sequences, which spanned a large dynamic range that could be leveraged for regulatory purposes. These newly identified features will facilitate the efficient design of CRISPR-dCpf1 based systems for tunable multiplex gene regulation.

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bioRxiv2018

Rational design of a compact CRISPR-Cas9 activator for AAV- mediated delivery

Akin to Zinc Finger and Transcription Activator Like Effector based transcriptional modulators, nuclease-null CRISPR-Cas9 provides a groundbreaking programmable DNA binding platform, begetting an arsenal of targetable regulators for transcriptional and epigenetic perturbation, by either directly tethering, or recruiting, transcription enhancing effectors to either component of the Cas9/guide RNA complex. Application of these programmable regulators is now gaining traction for the modulation of disease-causing genes or activation of therapeutic genes, in vivo. Adeno-Associated Virus (AAV) is an optimal delivery vehicle for in vivo delivery of such regulators to adult somatic tissue, due to the efficacy of viral delivery with minimal concerns about immunogenicity or integration. However, present Cas9 activator systems are notably beyond the packaging capacity of a single AAV delivery vector capsid. Here, we engineer a compact CRISPR-Cas9 activator for convenient AAV-mediated delivery. We validate efficacy of the CRISPR-Cas9 transcriptional activation using AAV delivery in several cell lines.

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bioRxiv2018

Robust Perfect Adaptation in Biomolecular Reaction Networks

For control in biomolecular systems, the most basic objective of maintaining a small error in a target variable, say the expression level of some protein, is often difficult due to the presence of both large uncertainty of every type and intrinsic limitations on the controllers implementation. This paper explores the limits of biochemically plausible controller design for the problem of robust perfect adaptation (RPA), biologists term for robust steady state tracking. It is well-known that for a large class of nonlinear systems, a system has RPA iff it has integral feedback control (IFC), which has been used extensively in real control systems to achieve RPA. However, we show that due to intrinsic physical limitations on the dynamics of chemical reaction networks (CRNs), cells cannot implement IFC directly in the concentration of a chemical species. This contrasts with electronic implementations, particularly digital, where it is trivial to implement IFC directly in a single state. Therefore, biomolecular systems have to achieve RPA by encoding the integral control variable into the network architecture of a CRN. We describe a general framework to implement RPA in CRNs and show that well-known network motifs that achieve RPA, such as (negative) integral feedback (IFB) and incoherent feedforward (IFF), are examples of such implementations. We also develop methods to solve the problem of designing integral feedback variables for unknown plants. This standard control notion is surprisingly nontrivial and relatively unstudied in biomolecular control. The methods developed here connect different existing fields and approaches on the problem of biomolecular control, and hold promise for systematic chemical reaction controller synthesis as well as analysis.

synthetic biology↗

bioRxiv2018

Analysing disturbance response of cell-to-cell communication systems: a case study for activator-repressor-diffuser motif

In biomolecular communication networks, bacterial cells communicate with each other using a cell-to-cell communication mechanism mediated by diffusible signaling molecules. The dynamics of molecular concentrations in such systems are approximately modeled by reaction-diffusion equations. In this paper, we analyse the ability of cell-to-cell communication systems to attenuate impulsive disturbances with various spatial frequency profiles by computing the integrated squared concentration of molecules. In particular, we perform in-depth study of disturbance responses for an activator-repressor-diffuser biocircuit in the spatial frequency domain to characterize its spatial frequency gain.

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bioRxiv2018

Synthetic methanogenic communities reveal differential impact of ecological perturbations on aceto- and hydrogeno-trophic methanogens

Synthetic microbial communities provide reduced microbial ecologies that can be studied under defined conditions. Here, we use this approach to study the interactions underpinning anaerobic digestion communities and involving the key microbial populations of a sulfate reducer (Desulfovibrio vulgaris), and aceto-(Methanosarcina barkeri) and hydrogenotrophic (Methanococcus maripaludis) methanogens. We create all possible mixed culture combinations of these species and analyse the stability and productivity of each system over multiple sub-culturings and under different sulfate levels, mimicking ecological perturbation in the form of strong electron acceptor availability. We find that all three species can co-exist in the absence of sulfate, and that system productivity (in form of methane production from lactate) increases by almost two-fold compared to co-cultures. With increasing sulfate availability, co-existence is perturbed and both methanogenic populations display a diminishing trend. Interestingly, we find that, despite the continued presence of acetate in the system, the acetotrophic methanogens are more readily disrupted by sulfate perturbation. We show that this is due to a shift in M. barkeri metabolism towards increased co-utilisation of hydrogen with acetate, which we verified through experiments on mono cultures and mass balance calculations in co-cultures. We conclude that hydrogen is a key factor for both hydrogeno- and aceto-trophic methanogenesis and can influence these populations differentially under the common ecological perturbation of strong electron acceptor availability. These findings will help engineering of larger synthetic communities for specific applications in biodegradation and understanding complex anaerobic digestion communities found in animal guts, sediments, and bioreactors.

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bioRxiv2018

Genome-wide CRISPR-Cas9 screen in E. coli identifies design rules for efficient targeting

The main outcome of efficient CRISPR-Cas9 cleavage in the chromosome of bacteria is cell death. This can be conveniently used to eliminate specific genotypes from a mixed population of bacteria, which can be achieved both in vitro, e.g. to select mutants, or in vivo as an antimicrobial strategy. The efficiency with which Cas9 kills bacteria has been observed to be quite variable depending on the specific target sequence, but little is known about the sequence determinants and mechanisms involved. Here we performed a genome-wide screen of Cas9 cleavage in the chromosome of E. coli to determine the efficiency with which each guide RNA kills the cell. Surprisingly we observed a large-scale pattern where guides targeting some regions of the chromosome are more rapidly depleted than others. Unexpectedly, this pattern arises from the influence of degrading specific chromosomal regions on the copy number of the plasmid carrying the guide RNA library. After taking this effect into account, it is possible to train a neural network to predict Cas9 efficiency based on the target sequence. We show that our model learns different features than previous models trained on Eukaryotic CRISPR-Cas9 knockout libraries. Our results highlight the need for specific models to design efficient CRISPR-Cas9 tools in bacteria.

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bioRxiv2018

A synthetic bacterial information transfer system functions in the mammalian gut

The gut microbiome is intricately involved with establishing and maintaining the health of the host. Engineering of gut microbes aims to add new functions and expand the scope of control over the gut microbiome. To create systems that can perform increasingly complex tasks in the gut with multiple engineered strains it is necessary to program communication among these bacteria in the gut. Towards this goal, we engineered an information transfer system for inter-cellular communication, using native gut Escherichia coli and attenuated Salmonella enterica serovar Typhimurium. Specifically, we have taken two genetic circuits-one for signaling from the quorum sensing system and the other for memory from the bacteriophage genetic switch-and integrated them into a robust system that can report on successful communication in the mammalian gut. Our system provides a basis for the construction of a programmable gut consortia as well as a basis for further understanding of bacterial interactions in an otherwise hard-to-study environment.

synthetic biology↗

bioRxiv2018

One-step assembly of large CRISPR arrays enables multi-functional targeting and reveals constraints on array design

CRISPR-Cas systems inherently multiplex through their CRISPR arrays--whether to confer immunity against multiple invaders or by mediating multi-target editing, regulation, imaging, and sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report an efficient, one-step scheme called CRATES to construct large CRISPR arrays through defined assembly junctions within the trimmed portion of array spacers. We show that the constructed arrays function with the single-effector nucleases Cas9, Cas12a, and Cas13a for multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. We also applied CRATES to assemble composite arrays utilized by multiple Cas nucleases, where these arrays enhanced DNA targeting specificity by blocking off-target sites. Finally, array characterization revealed context-dependent loss of spacer activity and processing of unintended guide RNAs derived from Cas12a terminal repeats. CRATES thus can facilitate diverse applications requiring CRISPR multiplexing and help elucidate critical factors influencing array function.

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bioRxiv2018

Scalable continuous evolution of genes at mutation rates above genomic error thresholds

Directed evolution is a powerful approach for engineering biomolecules and understanding adaptation1-3. However, experimental strategies for directed evolution are notoriously low-throughput, limiting access to demanding functions, multiple functions in parallel, and the study of molecular evolution in replicate. Here, we report OrthoRep, a yeast orthogonal DNA polymerase-plasmid pair that stably mutates ~100,000-fold faster than the host genome in vivo, exceeding error thresholds of genomic replication that lead to single-generation extinction4. User-defined genes in OrthoRep continuously and rapidly evolve through serial passaging, a highly scalable process. Using OrthoRep, we evolved drug resistant malarial DHFRs 90 times and uncovered a more complex fitness landscape than previously realized5-9. We find rare fitness peaks that resist the maximum soluble concentration of the antimalarial pyrimethamine - these resistant variants support growth at pyrimethamine concentrations >40,000-fold higher than the wild-type enzyme can tolerate - and also find that epistatic interactions direct adaptive trajectories to convergent outcomes. OrthoRep enables a new paradigm of routine, high-throughput evolution of biomolecular and cellular function.

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