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  • ChipSeg: an automatic tool to segment bacteria and mammalian cells cultured in microfluidic devices
    bioRxiv. Synth. Biol. Pub Date : 2020-08-03
    Irene de Cesare; Criseida G. Zamora-Chimal; Lorena Postiglione; Mahmoud Rabi Khazim; Elisa Pedone; Barbara Shannon; Gianfranco Fiore; Giansimone Perrino; Sara Napolitano; Diego di Bernardo; Nigel Savery; Claire Grierson; Mario di Bernardo; Lucia Marucci

    Extracting quantitative measurements from time-lapse images is necessary in external feedback control applications, where segmentation results are used to inform control algorithms. While such image segmentation applications have been previously reported, there is in the literature a lack of open-source and documented code for the community. We describe ChipSeg, a computational tool to segment bacterial

  • BioCRNpyler: Compiling Chemical Reaction Networks from Biomolecular Parts in Diverse Contexts
    bioRxiv. Synth. Biol. Pub Date : 2020-08-03
    William Poole; Ayush Pandey; Zoltan Tuza; Andrey Shur; Richard M Murray

    Biochemical interactions in systems and synthetic biology are often modeled with Chemical Reaction Networks (CRNs). CRNs provide a principled modeling environment capable of expressing a huge range of biochemical processes. In this paper, we present a software toolbox, written in python, that complies high-level design specifications to CRN representations. This compilation process offers three advantages

  • De novo design of picomolar SARS-CoV-2 miniprotein inhibitors
    bioRxiv. Synth. Biol. Pub Date : 2020-08-03
    David Baker; Longxing Cao; Inna Goreshnik; Brian Coventry; James Brett Case; Lauren Miller; Lisa Kozodoy; Rita E. Chen; Lauren Carter; Alexandra Walls; Young-Jun Park; Lance Stewart; Michael Diamond; David Veesler

    We used two approaches to design proteins with shape and chemical complementarity to the receptor binding domain (RBD) of SARS-CoV-2 Spike protein near the binding site for the human ACE2 receptor. Scaffolds were built around an ACE2 helix that interacts with the RBD, or de novo designed scaffolds were docked against the RBD to identify new binding modes. In both cases, designed sequences were optimized

  • CRISPR interference as low burden logic inverters in synthetic circuits: characterization and tuning
    bioRxiv. Synth. Biol. Pub Date : 2020-08-03
    Massimo Bellato; Angelica Frusteri Chiacchiera; Elia Salibi; Michela Casanova; Davide De Marchi; Maria Gabriella Cusella De Angelis; Lorenzo Pasotti; Paolo Magni

    The rational design of complex biological systems through the interconnection of single functional building blocks is hampered by many unpredictability sources; this is mainly due to the tangled context-dependency behavior of those parts once placed into an intrinsically complex living system. Among others, the finite amount of translational resources in prokaryotic cells leads to load effects in heterologous

  • Minicells as a potential chassis for engineering lineage-agnostic organisms
    bioRxiv. Synth. Biol. Pub Date : 2020-08-02
    Eric Wei; Anton Jackson-Smith; Drew Endy

    Background: Genomes encode for organisms and thus genome synthesis implies the possibility of organismal synthesis, including the synthesis of organisms without constraint to lineage. Current genome-scale engineering projects are focused on minimization, refactoring, or recoding within the context of existing natural lineages. Minicells arise naturally as anucleate cells that are devoid of heritable

  • Predator: A novel method for targeted protein degradation
    bioRxiv. Synth. Biol. Pub Date : 2020-08-02
    Chuanyang Liu; Jingyu Kuang; Xinyuan Qiu; Lu Min; Wenying Li; Jiaxin Ma; Lingyun Zhu

    Protein expression and degradation are fundamental to cell function and physiological status of organisms. Interfering with protein expression not only provides powerful strategies to analyze the function of proteins but also inspires effective treatment methods for diseases caused by protein dysfunction. Recently, harnessing the power of the ubiquitin-proteasome system for targeted protein degradation

  • Genetically-encoded fluorescent biosensor for rapid detection of protein expression
    bioRxiv. Synth. Biol. Pub Date : 2020-07-30
    Matthew G Eason; Antonia T Pandelieva; Marc M Mayer; Safwat T Khan; Hernan G Garcia; Roberto A Chica

    Fluorescent proteins are widely used as fusion tags to detect protein expression in vivo. To become fluorescent, these proteins must undergo chromophore maturation, a slow process with a half-time of 5 to >30 min, which causes delays in real-time detection of protein expression. Here, we engineer a genetically-encoded fluorescent biosensor to enable detection of protein expression within seconds in

  • Optimized gene expression from bacterial chromosome by high-throughput integration and screening
    bioRxiv. Synth. Biol. Pub Date : 2020-07-29
    Tatyana E Saleski; Meng Ting Chung; David N Carruthers; Azzaya Khasbaatar; Katsuo Kurabayashi; Xiaoxia (Nina) Lin

    Chromosomal integration of recombinant genes is desirable compared to expression from plasmids due to increased stability, reduced cell-to-cell variability, and the elimination of antibiotics for plasmid maintenance. Here, we present a new approach for tuning pathway gene expression levels via random integrations followed by high-throughput screening. We demonstrate multiplexed pathway gene integration

  • Mapping the Functional Landscape of the Receptor Binding Domain of T7 Bacteriophage by Deep Mutational Scanning
    bioRxiv. Synth. Biol. Pub Date : 2020-07-29
    Phil Huss; Anthony Meger; Megan Leander; Kyle Nishikawa; Srivatsan Raman

    The interaction between a bacteriophage and its host is mediated by the phage's receptor binding protein (RBP). Despite its fundamental role in governing phage activity and host range, molecular rules of RBP function remain a mystery. Here, we systematically dissect the functional role of every residue in the tip domain of T7 phage RBP using a high-throughput, locus-specific, phage engineering method

  • Testing theoretical minimal genomes using whole-cell models
    bioRxiv. Synth. Biol. Pub Date : 2020-07-28
    Joshua Rees-Garbutt; Jake Rightmyer; Oliver Chalkley; Lucia Marucci; Claire Grierson

    Numerous authors have pondered what the minimal gene set for life might be and many hypothetical minimal gene sets have been proposed, including at least 10 for Mycoplasma genitalium (M. genitalium). None of these have been reported to be tested in-vivo or in-silico. In-vivo testing would be extremely difficult as M.genitalium is very difficult to grow in the laboratory and laborious to engineer. However

  • Engineering DNA templated nonribosomal peptide synthesis
    bioRxiv. Synth. Biol. Pub Date : 2020-07-27
    Hsin-Mei Huang; Philipp Stephan; Hajo Kries

    Nanocontainers or macromolecular scaffolds for artificial biocatalytic cascades facilitate sequential enzyme reactions but diffusive escape of intermediates limits rate enhancement. Nonribosomal peptide synthetases (NRPS) naturally form gigantic assembly lines and prevent escape by covalently tethering intermediates. Here, we have built DNA-templated NRPS (DT-NRPS) by adding zinc finger tags to split

  • The E. coli Cas1/2 endonuclease complex reduces CRISPR/Cascade guide array stability
    bioRxiv. Synth. Biol. Pub Date : 2020-07-27
    Zhixia Ye; Juliana Lebeau; Eirik Adim Moreb; Romel Menacho-Melgar; Michael D Lynch

    CRISPR based interference has become common in various applications from genetic circuits to dynamic metabolic control. In E. coli the native CRISPR Cascade system can be utilized for silencing by deletion of the cas3 nuclease along with expression of guide RNA arrays, where multiple genes can be silenced from a single transcript. We notice the loss of protospacer sequences from guide arrays utilized

  • De novo designed receptor transmembrane domains enhance CAR-T cell cytotoxicity and attenuate cytokine release
    bioRxiv. Synth. Biol. Pub Date : 2020-07-26
    Assaf Elazar; Nicholas J Chandler; Ashleigh S Davey; Jonathan Y Weinstein; Julie V Nguyen; Raphael Trenker; Misty R Jenkins; Melissa J Call; Matthew E Call; Sarel J Fleishman

    De novo designed receptor transmembrane domains (TMDs) present opportunities for precise control of cellular functions. We develop a strategy for generating programmed membrane proteins (proMPs): single-pass α-helical TMDs that form oligomeric complexes through computationally defined and crystallographically validated interfaces. To demonstrate their usefulness, we program specific oligomeric interactions

  • A modular two yeast species secretion system for the production and preparative application of fungal peroxygenases
    bioRxiv. Synth. Biol. Pub Date : 2020-07-22
    Pascal Puellmann; Anja Knorrscheidt; Judith Muench; Paul Robin Palme; Wolfgang Hoehenwarter; Sylvestre Marillonnet; Miguel Alcalde; Bernhard Westermann; Martin J Weissenborn

    Fungal unspecific peroxygenases (UPOs) are biocatalysts of outstanding interest. Providing access to novel UPOs using a modular secretion system was the central goal of this work. UPOs represent an enzyme class, catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and solely rely on

  • Systematic Quantification of Sequence and Structural Determinants Controlling mRNA stability in Bacterial Operons
    bioRxiv. Synth. Biol. Pub Date : 2020-07-22
    Daniel P Cetnar; Howard M Salis

    mRNA degradation is a central process that affects all gene expression levels, and yet the determinants that control mRNA decay rates remain poorly characterized. Here, we applied a synthetic biology, learn-by-design approach to elucidate the sequence and structural determinants that control mRNA stability in bacterial operons. We designed, constructed, and characterized 82 operons, systematically

  • Automated Classification of Bacterial Cell Sub-Populations with Convolutional Neural Networks.
    bioRxiv. Synth. Biol. Pub Date : 2020-07-22
    Denis Tamiev; Paige Elisabeth Furman; Nigel Forest Reuel

    Quantification of phenotypic heterogeneity present amongst bacterial cells can be a challenging task. Conventionally, classification and counting of bacteria sub-populations is achieved with manual microscopy, due to the lack of alternative, high-throughput, autonomous approaches. In this work, we apply classification-type convolutional neural networks (cCNN) to classify and enumerate bacterial cell

  • Modular cell-free expression plasmids to accelerate biological design in cells
    bioRxiv. Synth. Biol. Pub Date : 2020-07-22
    Ashty Karim; Eric Liew; Shivani Garg; Bastian Vögeli; Blake Rasor; Aislinn Gonnot; Marilene Pavan; Alex Juminaga; Sean D. Simpson; Michael Köpke; Michael C. Jewett

    Industrial biotechnology aims to produce high-value products from renewable resources. This can be challenging because model microorganisms--organisms that are easy to use like Escherichia coli--often lack the machinery required to utilize desired feedstocks like lignocellulosic biomass or syngas. Non-model organisms, such as Clostridium, are industrially proven and have the desired metabolic features

  • Increasing cell-free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA-binding proteins
    bioRxiv. Synth. Biol. Pub Date : 2020-07-22
    Bo Zhu; Rui Gan; Maria D Cabezas; Takaaki Kojima; Michael C Jewett; Hideo Nakano

    In crude extract-based cell-free protein synthesis (CFPS), DNA templates are transcribed and translated into functional proteins. Although linear expression templates (LETs) are less laborious and expensive to generate, plasmid templates are often desired over PCR-generated LETs due to increased stability and protection against exonucleases present in the extract of the reaction. Here we demonstrate

  • Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles
    bioRxiv. Synth. Biol. Pub Date : 2020-07-21
    Jasmine M. Hershewe; Katherine F. Warfel; Shaelyn M. Iyer; Justin A. Peruzzi; Claretta J. Sullivan; Eric W. Roth; Matthew P. DeLisa; Neha P. Kamat; Michael C. Jewett

    Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for accelerating the design of cellular function, on-demand biomanufacturing, portable diagnostics, and educational kits. Many essential biological processes that could endow CFE systems with desired functions, such as protein glycosylation, rely on the activity of membrane-bound components. However,

  • The variation of promoter strength in different gene contexts
    bioRxiv. Synth. Biol. Pub Date : 2020-07-21
    Tam T Tran; Trevor C Charles

    Background Promoter engineering has been employed as a strategy to enhance and optimize the production of bio-products. There have been many effortless studies searching the best promoter for biological application. However, whether promoter strengths stay unchanged in different gene contexts remains unknown. Results Six consecutive promoters at different strength levels were used to construct six

  • Precise and programmable C:G to G:C base editing in genomic DNA
    bioRxiv. Synth. Biol. Pub Date : 2020-07-21
    Liwei Chen; Jung Eun Park; Peter Paa; Priscilla Dorothy Rajakumar; Yi Ting Chew; Swathi N. Manivannan; Wei Leong Chew

    Many genetic diseases are caused by single-nucleotide polymorphisms (SNPs). Base editors can correct SNPs at single-nucleotide resolution, but until recently, only allowed for C:G to T:A and A:T to G:C transition edits, addressing four out of twelve possible DNA base substitutions. Here we developed a novel class of C:G to G:C Base Editors (CGBEs) to create single-base genomic transversions in human

  • De novo design of modular and tunable allosteric biosensors
    bioRxiv. Synth. Biol. Pub Date : 2020-07-20
    Alfredo Quijano-Rubio; Hsien-Wei Yeh; Jooyoung Park; Hansol Lee; Robert A. Langan; Scott E. Boyken; Marc J. Lajoie; Longxing Cao; Cameron M. Chow; Marcos C. Miranda; Jimin Wi; Hyo Jeong Hong; Lance Stewart; Byung-Ha Oh; David Baker

    Naturally occurring allosteric protein switches have been repurposed for developing novel biosensors and reporters for cellular and clinical applications, but the number of such switches is limited, and engineering them is often challenging as each is different. Here, we show that a very general class of allosteric protein-based biosensors can be created by inverting the flow of information through

  • Generation of ordered protein assemblies using rigid three-body fusion
    bioRxiv. Synth. Biol. Pub Date : 2020-07-19
    Ivan Vulovic; Qing Yao; Young-Jun Park; Alexis Courbet; Andrew Norris; Florian Busch; Aniruddha Sahasrabuddhe; Hannes Merten; Danny D Sahtoe; George Ueda; Jorge A Fallas; Yang Hsia; Sara J Weaver; Robert A Langan; Andreas Pluckthun; Vicki H Wysocki; David Veesler; Grant J Jensen; David Baker

    Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A longstanding design approach uses genetic fusion to join protein homo-oligomer subunits via α-helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method that performs

  • Single cell characterization of a synthetic bacterial clock with a hybrid feedback loop containing dCas9-sgRNA
    bioRxiv. Synth. Biol. Pub Date : 2020-07-16
    John Henningsen; Matthaeus Schwarz-Schilling; Andreas Leibl; Joaquin A.M. Guttierez; Sandra Sagredo; Friedrich C Simmel

    Genetic networks that generate oscillations in gene expression activity are found in a wide range of organisms throughout all kingdoms of life. Oscillatory dynamics facilitates the temporal orchestration of metabolic and growth processes inside cells and organisms, as well as the synchronization of such processes with periodically occurring changes in the environment. Synthetic oscillator gene circuits

  • Selected reaction monitoring for the quantification of Escherichia coli ribosomal proteins
    bioRxiv. Synth. Biol. Pub Date : 2020-07-16
    Yuishin Kosaka; Wataru Aoki; Megumi Mori; Shunsuke Aburaya; Yuta Ohtani; Hiroyoshi Minakuchi; Mitsuyoshi Ueda

    Ribosomes are the sophisticated machinery that is responsible for protein synthesis in a cell. Recently, quantitative mass spectrometry (qMS) based on data-dependent acquisition (DDA) have been widely used to understand the biogenesis and function of ribosomes. However, DDA-based qMS sometimes does not provide the reproducible and quantitatively reliable analysis that is needed for high-throughput

  • Cas9 fusions for precision in vivo editing
    bioRxiv. Synth. Biol. Pub Date : 2020-07-16
    Ryan R. Richardson; Marilyn Steyert; Jeffrey Inen; Saovleak Khim; Andrea J. Romanowski; Bekir Altas; Alexandros Poulopoulos

    Current Cas9 reagents can target genomic loci with high specificity. However, when used for knockin, on-target outcomes are inherently imprecise, often leading to unintended knockout rather than intended edits. This restricts applications of genome editing to ex vivo approaches, where clonal selection is possible. Here we describe a workflow using iterative high-throughput in vitro and high-yield in

  • Room Temperature Isothermal Colorimetric Padlock Probe Rolling Circle Amplification for Viral RNA Detection
    bioRxiv. Synth. Biol. Pub Date : 2020-07-15
    Wilson Huang; Hannah Hsu; Joyce Ting; Jimmy Su; Matthew Fang; Tsuyoshi Misaki; Derek Chan; Justin Yang; Ting-Yu Yeh; Kelly Yang; Vera Chien; Tiffany Huang; Andrew Chen; Claire Wei; Jonathan Hsu; Jude C. Clapper

    Seasonal flu and pandemics, which account for millions of infections and hundreds of thousands of deaths, require rapid and reliable detection mechanisms for preventive and therapeutic measures. Current methods of viral detection have limitations in speed, accuracy, accessibility, and usability. This project presents a novel, widely applicable viral diagnosis that uses a modified version of the traditional

  • Contextual dependencies expand the re-usability of genetic inverters
    bioRxiv. Synth. Biol. Pub Date : 2020-07-15
    Huseyin Tas; Lewis Grozinger; Ruud Stoof; Victor de Lorenzo; Angel Goni-Moreno

    The design and implementation of Boolean logic functions in living cells has become a very active field within synthetic biology. By controlling networks of regulatory proteins, novel genetic circuits are engineered to generate predefined output responses. Although many current implementations focus solely on the genetic components of the circuit, the host context in which the circuit performs is crucial

  • Systematic Engineering of Artificial Metalloenzymes for New-to-Nature Reactions
    bioRxiv. Synth. Biol. Pub Date : 2020-07-15
    Tobias Vornholt; Fadri Christoffel; Michela Pellizzoni; Sven Panke; Thomas R. Ward; Markus Jeschek

    Artificial metalloenzymes (ArMs) catalyze new-to-nature reactions under mild conditions and could therefore play an important role in the transition to a sustainable, circular economy. While ArMs have been created for a variety of reactions, their activity for most biorthogonal transformations has remained modest and attempts at optimizing them by means of enzyme engineering have been case-specific

  • In situ generation of RNA complexes for synthetic molecular strand displacement circuits in autonomous systems
    bioRxiv. Synth. Biol. Pub Date : 2020-07-15
    Wooli Bae; Guy-Bart V Stan; Thomas E Ouldridge

    Synthetic molecular circuits implementing DNA or RNA strand-displacement reactions can be used to build complex systems such as molecular computers and feedback control systems. Despite recent advances, application of nucleic acid-based circuits in vivo remains challenging due to a lack of efficient methods to produce their essential components - multi-stranded complexes known as 'gates' − in situ

  • A standardized broad host range inverter package for genetic circuitry design in Gram-negative bacteria
    bioRxiv. Synth. Biol. Pub Date : 2020-07-15
    Huseyin Tas; Angel Goni-Moreno; Victor de Lorenzo

    Genetically encoded logic gates, especially inverters NOT gates are the building blocks for designing circuits, engineering biosensors or decision-making devices in synthetic biology. However, the repertoire of inverters readily available for different species is rather limited. In this work, a large whole of NOT gates that was shown to function previously in a specific strain of Escherichia coli,

  • LoxTnSeq: Transposon mutagenesis coupled with ultra-sequencing to study large random genome reductions
    bioRxiv. Synth. Biol. Pub Date : 2020-07-14
    Daniel Shaw; Samuel Miravet-Verde; Carlos Pinero-Lambea; Luis Serrano; Maria Lluch-Senar

    Rational engineering in synthetic biology requires preliminary knowledge of which genomic regions are dispensable. Typically, these efforts are guided by transposon mutagenesis studies, coupled to ultra-sequencing (TnSeq) which determine single gene essentiality. However, epistatic interactions can rapidly alter these profiles post deletion, leading to the redundancy of these maps. Here, we present

  • Photochemical NOT Gate for DNA Computing
    bioRxiv. Synth. Biol. Pub Date : 2020-07-14
    Cole Emanuelson; Anirban Bardhan; Alexander Deiters

    DNA-based Boolean logic gates (AND, OR and NOT) can be assembled into complex computational circuits that generate an output signal in response to specific patterns of oligonucleotide inputs. However, the fundamental nature of NOT gates, which convert the absence of an input into an output, makes their implementation within DNA-based circuits difficult. Premature execution of a NOT gate before completion

  • Modelling co-translational dimerisation for programmable nonlinearity in synthetic biology
    bioRxiv. Synth. Biol. Pub Date : 2020-07-11
    Ruud Stoof; Angel Goni-Moreno

    Nonlinearity plays a fundamental role in the performance of both natural and synthetic biological networks. Key functional motifs in living microbial systems, such as the emergence of bistability or oscillations, rely on nonlinear molecular dynamics. Despite its core importance, the rational design of nonlinearity remains an unmet challenge. This is largely due to a lack of mathematical modelling that

  • The genotype-phenotype landscape of an allosteric protein
    bioRxiv. Synth. Biol. Pub Date : 2020-07-11
    Drew S Tack; Peter D Tonner; Abe Pressman; Nathanael D Olson; Sasha F Levy; Eugenia F Romantseva; Nina Alperovich; Olga Vasilyeva; David Ross

    Allostery is a fundamental biophysical mechanism that underlies cellular sensing, signaling, and metabolism. Quantitative methods to characterize the genotype-phenotype relationships for allosteric proteins would provide data needed to improve engineering of biological systems, to uncover the role of allosteric mis-regulation in disease, and to develop allosterically targeted drugs1. Here we report

  • Creating Red Light-Controlled Protein Dimerization Systems as Genetically Encoded Actuators with High Specificity
    bioRxiv. Synth. Biol. Pub Date : 2020-07-11
    Zhimin Huang; Zengpeng Li; Xiao Zhang; Runze Dong; Shoukai Kang; Li Sun; Xiaonan Fu; David Vaisar; Kurumi Watanabe; Liangcai Gu

    Protein dimerization systems that can be controlled by red light with increased tissue penetration depth are a highly needed optogenetic tool for clinical applications such as cell and gene therapies. However, existing red light-induced dimerization systems are all based on phytochrome photoreceptors and naturally occurring binding partners with complex structures and suboptimal in vivo performance

  • A Modular Computational Framework for the Design of Multicellular Genetic Circuits of Morphogenesis
    bioRxiv. Synth. Biol. Pub Date : 2020-07-11
    Calvin Lam; Leonardo Morsut

    Synthetic development is a nascent field of research that uses the tools of synthetic biology to design genetic programs directing cellular patterning and morphogenesis in higher eukaryotic cells, such as mammalian cells. Synthetic genetic networks comprising cell-cell communications and morphogenesis effectors (e.g. adhesion) are generated and integrated into a cellular genome. Current design methods

  • Genetic manipulation of the optical refractive index in living cells
    bioRxiv. Synth. Biol. Pub Date : 2020-07-09
    Junko Ogawa; Yoko Iwata; Nina U Tonnu; Chitra Gopinath; Ling Huang; Sachihiko Itoh; Ryoko Ando; Atsushi Miyawaki; Inder M Verma; Gerald M Pao

    The optical refractive index of cellular components is generally not a property considered amenable to manipulation in microscopy as this is an intrinsic physical property of materials. Here we show that by targeting cephalopod reflectin protein nanoparticles one can manipulate the optical refractive index of mammalian cellular compartments. We further demonstrate that refractive index alteration based

  • Synthetic promoter based azide biosensor toolkit to advance chemical-biology
    bioRxiv. Synth. Biol. Pub Date : 2020-07-09
    Chandra Kanth Bandi; Kyle S. Skalenko; Ayushi Agrawal; Neelan Sivaneri; Margaux Thiry; Shishir P.S. Chundawat

    Real-time azide or azido-functionalized molecular detection inside living cells using bioorthogonal chemistry-based approaches has been revolutionary to advancing chemical-biology. These methods have enabled diverse applications ranging from understanding the role of cellular glycosylation pathways, identifying diseased cells, and targeting delivery of azido-based therapeutic drugs. However, while

  • AL-PHA beads: bioplastic-based protease biosensors for global health applications
    bioRxiv. Synth. Biol. Pub Date : 2020-07-09
    Richard J. R. Kelwick; Alexander J. Webb; Yizhou Wang; Amelie Heliot; Fiona Allan; Aidan M. Emery; Michael R. Templeton; Paul S. Freemont

    Proteases are multi-functional proteolytic enzymes that have complex roles in human health and disease. Therefore, the development of protease biosensors can be beneficial to global health applications. To this end, we developed Advanced proteoLytic detector PolyHydroxyAlkanoates (AL-PHA) beads, a library of over 20 low-cost, biodegradable, bioplastic-based protease biosensors. Broadly, these biosensors

  • Production of benzylglucosinolate by engineering and optimizing the biosynthetic pathway in Saccharomyces cerevisiae
    bioRxiv. Synth. Biol. Pub Date : 2020-07-09
    Cuiwei Wang; Christoph Crocoll; Christina Spuur Nodvig; Uffe Hasbro Mortensen; Sidsel Ettrup Clemmensen; Barbara Ann Halkier

    Glucosinolates are amino acid-derived defense compounds characteristic of the Brassicales order. Benzylglucosinolate (BGLS) derived from phenylalanine is associated with health-promoting effects, which has primed a desire to produce BGLS in microorganisms for a stable and rich source. In this study, we engineered the BGLS production in Saccharomyces cerevisiae by either stably integrating the biosynthetic

  • Robust bidirectional communication between electronics and an engineered multi-functional microbial community
    bioRxiv. Synth. Biol. Pub Date : 2020-07-08
    Jessica Terrell; Tanya Tschirhart; Justin Jahnke; Kristina Stephens; Yi Liu; Hong Dong; Margaret Hurley; Maria Pozo; Ryan McKay; Chen-Yu Tsao; Hsuan-Chen Wu; Gary Vora; Gregory Payne; Dimitra N Stratis-Cullum; William E Bentley

    We developed a bidirectional bioelectronic communication system that is enabled by a redox signal transduction modality to exchange information between a living cell-embedded bioelectronics interface and an engineered microbial network. A naturally communicating three-member microbial network is 'plugged into' an external electronic system that interrogates and controls biological function in real

  • Single strain control of microbial consortia
    bioRxiv. Synth. Biol. Pub Date : 2020-07-08
    Alex J H Fedorec; Behzad D Karkaria; Michael Sulu; Chris P Barnes

    The scale of the biological systems we can engineer is limited by the burden that host cells can bear. Division-of-labour can spread that burden across a community of cells but competitive exclusion inevitably leads to the removal of less fit community members over time. Here, we leverage amensalism and competitive exclusion to stabilise multi-species communities by engineering a strain of Escherichia

  • Development of a growth coupled dynamic regulation network balancing malonyl-CoA node to enhance (2S)-naringenin synthesis in E. coli
    bioRxiv. Synth. Biol. Pub Date : 2020-07-07
    Shenghu Zhou; Shuo-Fu Yuan; Priya H Nair; Hal S Alper; Yu Deng; Jingwen Zhou

    Generally, high- and low-performance nongenetic variants and young and aged cells co-existed in culture at all growth phases. In this regard, individually and dynamically regulating the metabolic flux of single cells based on their cellular state is highly useful for improving the performance of populations. However, balancing the trade-offs between biomass formation and compound over-production requires

  • Robust frequency-encoded dynamics in a minimal synthetic phytohormone crosstalk
    bioRxiv. Synth. Biol. Pub Date : 2020-07-06
    Sara Perez Garcia; Mario Garcia Navarrete; Diego Ruiz Sanchis; Cristina Prieto Navarro; Merisa Avdovic; Ornella Pucciariello; Krzysztof Wabnik

    How do dynamic hormone inputs translate into speed, and precision of response is one of the most challenging questions of science. To approach this question, we constructed minimal synthetic gene circuits capable of responding to plant hormones auxin and salicylic acid (SA). These circuits integrate bacterial multi antibiotic resistance (Mar) repressors that directly detect phytohormones through a

  • A designed high-affinity peptide that hijacks microtubule-based transport
    bioRxiv. Synth. Biol. Pub Date : 2020-07-06
    Jessica A Cross; Magda S Chegkazi; Roberto A Steiner; Derek N Woolfson; Mark Dodding

    Technologies that manipulate and augment the transport of vesicles and organelles by motor proteins along microtubules offer new routes to understanding its mechanistic basis, and could lead to therapeutics. Many cargoes for the kinesin-1 family of microtubule motors utilize adaptor proteins that harbor linear peptide motifs that are recognized by the tetratricopeptide repeats of kinesin light chains

  • Harnessing the central dogma for stringent multi-level control of gene expression
    bioRxiv. Synth. Biol. Pub Date : 2020-07-05
    F. Veronica Greco; Claire S Grierson; Thomas E Gorochowski

    Strictly controlled inducible gene expression is crucial when engineering biological systems where even tiny amounts of a protein have a large impact on function or host cell viability. In these cases, leaky protein production must be avoided at all costs. Here, we demonstrate how the central dogma offers a simple way to effectively address this challenge. By simultaneously regulating both transcriptional

  • Optogenetics in Sinorhizobium meliloti enables spatial control of exopolysaccharide production and biofilm structure
    bioRxiv. Synth. Biol. Pub Date : 2020-07-04
    Azady Pirhanov; Charles M Bridges; Reed A Goodwin; Yi-Syuan Guo; Jessica Furrer; Leslie M Shor; Daniel J Gage; Yong Ku Cho

    Microorganisms play a vital role in shaping the soil environment and enhancing plant growth by interacting with plant root systems. Due to the vast diversity of cell types involved, combined with dynamic and spatial heterogeneity, identifying the causal contribution of a defined factor, such as a microbial exopolysaccharide (EPS), remains elusive. Synthetic approaches that enable orthogonal control

  • A self-regenerating synthetic cell model
    bioRxiv. Synth. Biol. Pub Date : 2020-07-04
    Barbora Lavickova; Nadanai Laohakunakorn; Sebastian Josef Maerkl

    Self-regeneration is a fundamental function of all living systems. Here we demonstrate molecular self-regeneration in a synthetic cell model. By implementing a minimal transcription- translation system within microfluidic reactors, the system was able to regenerate essential protein components from DNA templates and sustained synthesis activity for over a day. By mapping genotype-phenotype landscapes

  • Spatial engineering of E. coli with addressable phase-separated RNAs
    bioRxiv. Synth. Biol. Pub Date : 2020-07-03
    Haotian Guo; Joseph C Ryan; Adeline Mallet; Xiaohu Song; Victor Pabst; Antoine Decrulle; Ariel B. Lindner

    Biochemical processes often require spatial regulation and specific microenvironments. The general lack of organelles in bacteria limits the potential of bioengineering complex intracellular reactions. Here we demonstrate Transcriptionally Engineered Addressable RNA Solvent droplets (TEARS) as synthetic microdomains within the Escherichia coli. TEARS are assembled from RNA-binding protein recruitment

  • “Scentsor”: A Whole-Cell Yeast Biosensor with an Olfactory Reporter for Low-Cost and Equipment-Free Detection of Pharmaceuticals
    bioRxiv. Synth. Biol. Pub Date : 2020-07-03
    Rachel A. Miller; Seryeong Lee; Ethan J. Fridmanski; Elsa Barron; Julia Pence; Marya Lieberman; Holly V. Goodson

    Portable and inexpensive analytical tools are required to monitor pharmaceutical quality in technology limited settings including low- and middle-income countries (LMICs). Whole cell yeast biosensors have the potential to help meet this need. However, most of the read-outs for yeast biosensors require expensive equipment or reagents. To overcome this challenge, we have designed a yeast biosensor that

  • Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation
    bioRxiv. Synth. Biol. Pub Date : 2020-07-03
    Elisa Pesenti; Mikhail Liskovykh; Koei Okazaki; Alessio Mallozzi; Caitlin Reid; Maria Alba Abad; A. Arockia Jeyaprakash; Natalay Kouprina; Vladimir Larionov; William C. Earnshaw

    Human Artificial Chromosomes (HACs) are important tools for epigenetic engineering, for measuring chromosome instability (CIN) and possible gene therapy. However, their use in the latter is potentially limited because the input HAC-seeding DNA can undergo an unpredictable series of rearrangements during HAC formation. As a result, after transfection and HAC formation, each cell clone contains a HAC

  • RanDeL-seq: A high-throughput method to map viral cis- and trans-acting elements
    bioRxiv. Synth. Biol. Pub Date : 2020-07-02
    Timothy Notton; Joshua J Glazier; Victoria R Saykally; Cassandra E Thompson; Leor Weinberger

    It has long been known that noncoding genomic regions can be obligate cis elements acted upon in trans by gene products. In viruses, cis elements regulate gene expression, encapsidation, and other maturation processes but mapping these elements relies on targeted iterative deletion or laborious prospecting for rare, spontaneously occurring mutants. Here, we introduce a method to comprehensively map

  • Machine learning-based promoter strength prediction derived from a fine-tuned synthetic promoter library in Escherichia coli
    bioRxiv. Synth. Biol. Pub Date : 2020-07-01
    Mei Zhao; Shenghu Zhou; Longtao Wu; Yu Deng

    Promoters are one of the most critical regulatory elements controlling metabolic pathways. However, in recent years, researchers have simply perfected promoter strength, but ignored the relationship between the internal sequences and promoter strength. In this context, we constructed and characterized a mutant promoter library of Ptrc through dozens of mutation-construction-screening-characterization

  • Codon optimisation for maximising gene expression in multiple species and microbial consortia
    bioRxiv. Synth. Biol. Pub Date : 2020-07-01
    David James Skelton; Lucy E Eland; Martin Sim; Michael A White; Russell J Davenport; Anil Wipat

    Motivation: Codon optimisation, the process of adapting the codon composition of a coding sequence, is often used in synthetic biology to increase expression of a heterologous protein. Recently, a number of synthetic biology approaches that allow synthetic constructs to be deployed in multiple organisms have been published. However, so far, design tools for codon optimisation have not been updated

  • Catalytically Enhanced Cas9 through Directed Protein Evolution
    bioRxiv. Synth. Biol. Pub Date : 2020-07-01
    Travis H Hand; Mitchell O Roth; Chardasia L Smith; Emily Shiel; Kyle Klein; David M Gilbert; Hong Li

    The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 system has found widespread applications in genome manipulations due to its simplicity and effectiveness. Significant efforts in enzyme engineering have been made to improve the CRISPR-Cas9 systems beyond their natural power with additional functionalities such as DNA modification, transcriptional regulation, and high target

  • Automated design of synthetic microbial communities
    bioRxiv. Synth. Biol. Pub Date : 2020-07-01
    Behzad D Karkaria; Alex J H Fedorec; Chris P Barnes

    In naturally occurring microbial systems, species rarely exist in isolation. There is strong ecological evidence for a positive relationship between species diversity and the functional output of communities. The pervasiveness of these communities in nature highlights that there may be advantages for engineered strains to exist in cocultures as well. Building synthetic microbial communities allows

  • Reconstitution of contractile actomyosin rings in vesicles
    bioRxiv. Synth. Biol. Pub Date : 2020-07-01
    Thomas Litschel; Charlotte F Kelley; Danielle Holz; Maral Adeli Koudehi; Sven Kenjiro Vogel; Laura Burbaum; Naoko Mizuno; Dimitrios Vavylonis; Petra Schwille

    One of the grand challenges of bottom-up synthetic biology is the development of minimal machineries for cell division. The mechanical transformation of large-scale compartments, such as Giant Unilamellar Vesicles (GUVs), requires the geometry-specific coordination of active elements, several orders of magnitude larger than the molecular scale. Of all cytoskeletal structures, large-scale actomyosin

  • High-resolution mapping of glycoprotein structure-activity relationships by shotgun scanning glycomutagenesis
    bioRxiv. Synth. Biol. Pub Date : 2020-06-29
    Mingji Li; Xiaolu Zheng; Sudhanshu Shanker; Thapakorn Jaroentomeechai; Ilkay Kocer; Josef Byrne; Emily C. Cox; Jason W. Labonte; Jeffrey J. Gray; Matthew P DeLisa

    N-linked glycosylation serves to diversify the proteome and is crucial for the folding and activity of numerous cellular proteins. Consequently, there is great interest in uncovering the rules that govern how glycosylation modulates protein properties so that the effects of site-specific glycosylation might eventually be predicted. Towards this goal, we describe a combinatorial strategy termed shotgun

  • The SAGE genetic toolkit enables highly efficient, iterative site-specific genome engineering in bacteria
    bioRxiv. Synth. Biol. Pub Date : 2020-06-28
    Joshua R Elmore; Gara N Dexter; Ryan Francis; Lauren Riley; Jay Huenemann; Henri Baldino; Adam M Guss; Robert Egbert

    Sustainable enhancements to crop productivity and increased resilience to adverse conditions are critical for modern agriculture, and application of plant growth promoting rhizobacteria (PGPR) is a promising method to achieve these goals. However, many desirable PGPR traits are highly regulated in their native microbe, limited to certain plant rhizospheres, or insufficiently active for agricultural

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