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  • Construction of a third NAD+ de novo biosynthesis pathway
    bioRxiv. Synth. Biol. Pub Date : 2020-10-18
    Yong Ding; Xinli Li; Geoff P. Horsman; Pengwei Li; Min Wang; Jine Li; Zhilong Zhang; Weifeng Liu; Bian Wu; Yong Tao; Yihua Chen

    Only two de novo biosynthetic routes to nicotinamide adenine dinucleotide (NAD+) have been described, both of which start from a proteinogenic amino acid and are tightly controlled. Here we establish a C3N pathway starting from chorismate in Escherichia coli as a third NAD+ de novo biosynthesis pathway. Significantly, the C3N pathway yielded extremely high cellular concentrations of NAD(H) in E. coli

  • Programmable Aggregation of Artificial Cells with DNA Signals
    bioRxiv. Synth. Biol. Pub Date : 2020-10-18
    Hengming Qiu; Feiran Li; Yancheng Du; Ruixin Li; Ji Yeon Hyun; Sei Young Lee; Jong Hyun Choi

    Cell aggregation is a complex behavior, which is closely related to the viability, differentiation, and migration of cells. An effort to create synthetic analogs could lead to considerable advances in cell physiology and biophysics. Rendering and modulating such a dynamic artificial cell system require mechanisms for receiving, transducing, and transmitting intercellular signals, yet effective tools

  • Multiomics data collection, visualization, and utilization for guiding metabolic engineering
    bioRxiv. Synth. Biol. Pub Date : 2020-10-16
    Somtirtha Roy; Tijana Radivojevic; Mark Forrer; Jose Manuel Marti; Vamshi Jonnalagadda; Tyler Backman; William Morrell; Hector Plahar; Joonhoon Kim; Nathan Hillson; Hector Garcia Martin

    Biology has changed radically in the past two decades, growing from a purely descriptive science into also a design science. The availability of tools that enable the precise modification of cells, as well as the ability to collect large amounts of multimodal data, open the possibility of sophisticated bioengineering to produce fuels, specialty and commodity chemicals, materials, and other renewable

  • Substitutional landscape of a split fluorescent protein fragment
    bioRxiv. Synth. Biol. Pub Date : 2020-10-16
    Oana Nicoleta Antonescu; Andreas Rasmussen; Nicole A.M. Damm; Ditte F. Heidemann; Roman Popov; Alexander Nesterov-Mueller; Kristoffer E. Johansson; Jakob Rahr Winther

    Split fluorescent proteins have wide applicability as biosensors for protein-protein interactions, genetically encoded tags for protein detection and localization, as well as fusion partners in super-resolution microscopy. In this work, we have established and validated a novel platform for functional analysis of leave-one-out split fluorescent proteins (LOO-FPs) in high throughput and with rapid turnover

  • Pilot production of SARS-CoV-2 related proteins in plants: a proof of concept for rapid repurposing of indoors farms into biomanufacturing facilities
    bioRxiv. Synth. Biol. Pub Date : 2020-10-13
    Borja Diego-Martin; Beatriz González; Marta Vazquez-Vilar; Sara Selma; Rubén Mateos-Fernández; Silvia Gianoglio; Asun Fernández-del-Carmen; Diego Orzaez

    The current CoVid-19 crisis is revealing the strengths and the weaknesses of the world's capacity to respond to a global health crisis. A critical weakness has resulted from the excessive centralization of the current biomanufacturing capacities, a matter of great concern, if not a source of nationalistic tensions. On the positive side, scientific data and information have been shared at an unprecedented

  • A synthetic RNA-based biosensor for fructose-1,6-bisphosphate that reports glycolytic flux
    bioRxiv. Synth. Biol. Pub Date : 2020-10-11
    Alvaro D Ortega; Vakil Takhaveev; Silke Bonsing-Vedelaar; Yi Long; Neus Mestre-Farras; Danny Incarnato; Franziska Ersoy; Lars Folke Olsen; Gunter Mayer; Matthias Heinemann

    Metabolic heterogeneity, the occurrence of different metabolic phenotypes among cells, represents a key challenge in health and biotechnology. To unravel its molecular basis, tools probing metabolism of single cells are needed. While RNA devices harbor huge potential for the development of such tools, until today, it is challenging to create in vivo-functional sensors for any given metabolite. Here

  • The Bioprocess TEA Calculator: An online techno-economic analysis tool to evaluate the commercial competitiveness of potential bioprocesses
    bioRxiv. Synth. Biol. Pub Date : 2020-10-08
    Michael D. Lynch

    Techno-economic analysis connects R&D, engineering, and business. By linking process parameters to financial metrics, it allows researchers to understand the factors controlling the potential success of their technologies. In particular, metabolic and bioprocess engineering, as disciplines, are aimed at engineering cells to synthesize products with an ultimate goal of commercial deployment. As a result

  • Genetic requirements for cell division in a genomically minimal cell
    bioRxiv. Synth. Biol. Pub Date : 2020-10-07
    James F. Pelletier; Lijie Sun; Kim S. Wise; Nacyra Assad-Garcia; Bogumil J. Karas; Thomas J. Deerinck; Mark H. Ellisman; Andreas Mershin; Neil Gershenfeld; Ray-Yuan Chuang; John I. Glass; Elizabeth A. Strychalski

    Genomically minimal cells, such as JCVI-syn3.0, offer a platform to clarify genes underlying core physiological processes. While this minimal cell includes genes essential for population growth, the physiology of its single cells remained uncharacterized. To investigate striking morphological variation in JCVI-syn3.0 cells, we present an approach to characterize cell propagation and determine genes

  • Functional Characterization of an Electromagnetic Perceptive Protein
    bioRxiv. Synth. Biol. Pub Date : 2020-10-07
    Sunayana Mitra; Carlo Barnaba; Jens Christopher Schmidt; Galit Pelled; Assaf A Gilad

    Magnetoreception, the response to geomagnetic fields is a well described phenomenon in nature. However, it is likely that convergent evolution led to different mechanisms in different organisms. One intriguing example is the unique Electromagnetic Perceptive Gene (EPG) from the glass catfish Kryptopterus vitreolus, that can remotely control cellular function, upon magnetic stimulation in in-vitro and

  • The NK Landscape as a Versatile Benchmark for Machine Learning Driven Protein Engineering
    bioRxiv. Synth. Biol. Pub Date : 2020-10-06
    Adam C Mater; Mahakaran Sandhu; Colin J Jackson

    Machine learning (ML) has the potential to revolutionize protein engineering. However, the field currently lacks standardized and rigorous evaluation benchmarks for sequence-fitness prediction, which makes accurate evaluation of the performance of different architectures difficult. Here we propose a unifying framework for ML-driven sequence-fitness prediction. Using simulated (the NK model) and empirical

  • Discovery of tREP-18, a novel class of tRNA encoded peptide with potent leishmanicidal activity
    bioRxiv. Synth. Biol. Pub Date : 2020-10-04
    Amrita Chakrabarti; Monika Kaushik; Juveria Khan; Deepanshu Soota; P Kalairasan; Sunil Saini; Siddharth Manvati; Jhalak Singhal; Anand Ranganathan; Soumya Pati; Pawan Dhar; Shailja Singh

    In this study we have identified a novel class of membrane binding microbial peptide that was generated in silico from non-translatable tRNA element of E. coli genome and chemically synthesized, which demonstrated exceptional anti-leishmanial property. Briefly, E.coli tRNAs were translated in silico into peptide sequence equivalents and a virtual set of full length hypothetical tRNA encoded peptides

  • Testing theoretical minimal genomes using whole-cell models
    bioRxiv. Synth. Biol. Pub Date : 2020-10-02
    Joshua Rees-Garbutt; Jake Rightmyer; Oliver Chalkley; Lucia Marucci; Claire Grierson

    The minimal gene set for life has often been theorised, with at least ten produced for Mycoplasma genitalium (M. genitalium). Due to the difficulty of using M. genitalium in the lab, combined with its long replication time of 12 - 15 hours, none of these theoretical minimal genomes have been tested, even with modern techniques. The publication of the M. genitalium whole-cell model provided the first

  • Model-guided design of mammalian genetic programs
    bioRxiv. Synth. Biol. Pub Date : 2020-10-02
    Joseph J Muldoon; Viswajit Kandula; Mihe Hong; Patrick S Donahue; Jonathan Boucher; Neda Bagheri; Joshua Nathaniel Leonard

    Genetically engineering cells to perform customizable functions is an emerging frontier with numerous technological and translational applications. However, it remains challenging to systematically engineer mammalian cells to execute complex functions. To address this need, we developed a method enabling accurate genetic program design using high-performing genetic parts and predictive computational

  • Engineering supramolecular organizing centers for optogenetic control of innate immune responses
    bioRxiv. Synth. Biol. Pub Date : 2020-10-01
    Peng Tan; Lian He; Yubin Zhou

    The spatiotemporal organization of oligomeric protein complexes and translocons, such as the supramolecular organizing centers (SMOC) made of MyDDosome and MAVSome, are essential for transcriptional activation of host inflammatory responses and immune metabolisms. Light-inducible assembly of MyDDosome and MAVSome are presented herein to induce activation of nuclear factor-kB (NF-κB) and type-I interferons

  • Reprogramming Epiblast Stem Cells into Pre-Implantation Blastocyst Cell-like Cells
    bioRxiv. Synth. Biol. Pub Date : 2020-10-01
    Kiichiro Tomoda; Haiming Hu; Yoshiki Sahara; Hashimita Sanyal; Minoru Takasato; Cody Kime

    Recently, a new wave of synthetic embryo systems (SESs) have been established from cultured cells toward efficient and ethical embryonic development research. We recently reported our epiblast stem cell (EPISC) reprogramming SES that generates numerous blastocyst (BC)-like hemispheres (BCLH) with pluripotent and extraembryonic cell features detected microscopically. Here, we further explored the system

  • An Empirical Comparison of Preservation Methods for Synthetic DNA Data Storage
    bioRxiv. Synth. Biol. Pub Date : 2020-09-30
    Lee Wohlen Organick; Bichlien H. Nguyen; Rachel McAmis; Weida D. Chen; A. Xavier Kohll; Siena Dumas Ang; Robert N. Grass; Luis H Ceze; Karin Strauss

    Synthetic DNA has recently risen as a viable alternative for long-term digital data storage. To ensure that information is safely recovered after storage, it is essential to appropriately preserve the physical DNA molecules encoding the data. While preservation of biological DNA has been studied previously, synthetic DNA differs in that it is typically much shorter in length, it has different sequence

  • Synthetic auxotrophy remains stable after continuous evolution and in co-culture with mammalian cells
    bioRxiv. Synth. Biol. Pub Date : 2020-09-28
    Aditya M Kunjapur; Michael G Napolitano; Eriona Hysolli; Karen Noguera; Evan M. Appleton; Max G. Schubert; Michaela A. Jones; Siddharth Iyer; Daniel J. Mandell; George M. Church

    Understanding the evolutionary stability and possible context-dependence of biological containment techniques is critical as engineered microbes are increasingly under consideration for applications beyond biomanufacturing. While batch cultures of synthetic auxotrophic Escherichia coli previously exhibited undetectable escape throughout 14 days of monitoring, the long-term effectiveness of synthetic

  • Mapping the Functional Landscape of the Receptor Binding Domain of T7 Bacteriophage by Deep Mutational Scanning
    bioRxiv. Synth. Biol. Pub Date : 2020-09-28
    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, the 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 novel phage genome engineering method called ORACLE

  • Robust Self-Regeneratable Stiff Living Materials
    bioRxiv. Synth. Biol. Pub Date : 2020-09-25
    Avinash Manjula-Basavanna; Anna Duraj-Thatte; Neel S Joshi

    Living systems have not only the exemplary capability to fabricate materials (e.g. wood, bone) under ambient conditions but they also consist of living cells that imbue them with properties like growth and self-regeneration. Like a seed that can grow into a sturdy living wood, we wondered: can living cells alone serve as the primary building block to fabricate stiff materials? Here we report the fabrication

  • Loxd in translation: Contradictions in the nomenclature surrounding common lox site mutants and their implications in experiments
    bioRxiv. Synth. Biol. Pub Date : 2020-09-25
    Daniel Shaw; Luis Serrano; Maria Lluch-Senar

    The Cre-Lox system is a highly versatile and powerful DNA recombinase mechanism, mainly used in genetic engineering to insert or remove desired DNA sequences. It is widely utilised across multiple fields of biology, with applications ranging from plants, to mammals, to microbes. A key feature of this system is its ability to allow recombination between mutant lox sites, traditionally named lox66 and

  • Precision design of single and multi-heme de novo proteins
    bioRxiv. Synth. Biol. Pub Date : 2020-09-24
    George H. Hutchins; Claire E. H. Noble; Hector Blackburn; Ben Hardy; Charles Landau; Alice E. Parnell; Sathish Yadav; Christopher Williams; Paul R. Race; Sofia Oliveira; Matthew P. Crump; Christiane Berger-Schaffitzel; Adrian J. Mulholland; J. L. Ross Anderson

    The de novo design of simplified porphyrin-binding helical bundles is a versatile approach for the construction of valuable biomolecular tools to both understand and enhance protein functions such as electron transfer, oxygen binding and catalysis. However, the methods utilised to design such proteins by packing hydrophobic side chains into a buried binding pocket for ligands such as heme have typically

  • Design of Biologically Active Binary Protein 2D Materials
    bioRxiv. Synth. Biol. Pub Date : 2020-09-19
    Ariel J Ben-Sasson; Joseph Watson; William Sheffler; Matthew C Johnson; Alice Bittleston; Logeshwaran Somasundaram; Justin Decarreau; Fang Jiao; Jiajun Chen; Andrew A Drabek; Sanchez M Jarrett; Justin M Kollman; Stephen C Blacklow; James J De Yoreo; Hannele Ruohola-Baker; Emmanuel Derivery; David Baker

    Proteins that assemble into ordered two-dimensional arrays such as S-layers and designed analogues have intrigued bioengineers, but with the exception of a single lattice formed through non-rigid template streptavidin linkers, they are constituted from just one protein component. For modulating assembly dynamics and incorporating more complex functionality, materials composed of two components would

  • Genetic design automation for autonomous formation of multicellular shapes from a single cell progenitor
    bioRxiv. Synth. Biol. Pub Date : 2020-09-18
    Evan Appleton; Noushin Mehdipour; Tristan Daifuku; Demarcus Briers; Iman Haghighi; Michael Moret; George Chao; Timothy Wannier; Anush Chiappino-Pepe; Jeremy Huang; Calin Belta; George M Church

    Multi-cellular organisms originate from a single cell, ultimately giving rise to mature organisms of heterogeneous cell type composition in complex structures. Recent work in the areas of stem cell biology and tissue engineering have laid major groundwork in the ability to convert certain types of cells into other types, but there has been limited progress in the ability to control the morphology of

  • Frequency Spectra and the Color of Cellular Noise
    bioRxiv. Synth. Biol. Pub Date : 2020-09-17
    Ankit Gupta; Mustafa Khammash

    The invention of the Fourier integral in the 19th century laid the foundation for today's modern spectral analysis methods. By decomposing a (time) signal into its essential frequency components, these methods uncovered deep insights into the signal and its generating process, precipitating tremendous inventions and discoveries in many fields of engineering, technology, and physical science. In systems

  • Does co-expression of Yarrowia lipolytica genes encoding Yas1p, Yas2p and Yas3p make a potential alkane-responsive biosensor in Saccharomyces cerevisiae?
    bioRxiv. Synth. Biol. Pub Date : 2020-09-16
    Yasaman Dabirian; Christos Skrekas; Florian David; Verena Siewers

    Alkane-based biofuels are desirable to produce at a commercial scale as these have properties similar to our current petroleum-derived transportation fuels. Rationally engineering microorganisms to produce a desirable compound, such as alkanes, is, however, challenging. Metabolic engineers are therefore increasingly implementing evolutionary engineering approaches combined with high-throughput screening

  • Discovery of a Novel Polymer for Xeno-free, Long-term Culture of Human Pluripotent Stem Cell Expansion
    bioRxiv. Synth. Biol. Pub Date : 2020-09-16
    Jordan Thorpe; Aishah Nasir; Laurence Burroughs; Joris Meurs; Sara Pijuan-Galito; Derek J Irvine; Morgan R Alexander; Chris Denning

    Human pluripotent stem cells (hPSCs) can be expanded and differentiated in vitro into almost any adult tissue cell type, and thus have great potential as a source for cell therapies with biomedical application. In this study, a fully-defined polymer synthetic substrate is identified for hPSC culture in completely defined, xeno-free conditions. This system can overcome the cost, scalability and reproducibility

  • Engineering sensitivity and specificity of AraC-based biosensors responsive to triacetic acid lactone and orsellinic acid
    bioRxiv. Synth. Biol. Pub Date : 2020-09-16
    Zhiqing Wang; Aarti Doshi; Ratul Chowdhury; Yixi Wang; Costas D Maranas; Patrick C Cirino

    We previously described the design of triacetic acid lactone (TAL) biosensor 'AraC-TAL1', based on the AraC regulatory protein. While useful as a tool to screen for enhanced TAL biosynthesis, this variant shows elevated background (leaky) expression, poor sensitivity, and relaxed inducer specificity, including responsiveness to orsellinic acid (OA). More sensitive biosensors specific to either TAL

  • Engineering a culturable Serratia symbiotica strain for aphid paratransgenesis
    bioRxiv. Synth. Biol. Pub Date : 2020-09-16
    Katherine M Elston; Julie Perreau; Gerald P Maeda; Nancy A Moran; Jeffrey E Barrick

    Aphids are global agricultural pests and important models for bacterial symbiosis. To date, none of the native symbionts of aphids have been genetically manipulated, which limits our understanding of how they interact with their hosts. Serratia symbiotica CWBI-2.3T is a culturable, gut-associated bacterium isolated from the black bean aphid. Closely related Serratia symbiotica strains are facultative

  • Semiconducting bacterial biofilm based on graphene-MoS2 template and component dependent gating behavior
    bioRxiv. Synth. Biol. Pub Date : 2020-09-13
    Sanhita Ray; Arpita Das; Anjan Dasgupta

    In this paper, we report for the first time, the synthesis of a semiconducting biofilm. Photosynthetic bacterial biofilm has been used to weave together MoS2 nanosheets into an adherent film grown on interdigitated electrodes. Liquid-phase exfoliation of bulk MoS2 powder was used to obtain MoS2 nanosheets. A synchronous-fluorescence scan revealed the presence of two emission maxima at 682nm and 715nm

  • Some theoretical aspects of reprogramming the standard genetic code
    bioRxiv. Synth. Biol. Pub Date : 2020-09-13
    Kuba Nowak; Pawel Blazej; Malgorzata Wnetrzak; Dorota Mackiewicz; Pawel Mackiewicz

    Reprogramming of the standard genetic code in order to include non-canonical amino acids (ncAAs) opens a new perspective in medicine, industry and biotechnology. There are several methods of engineering the code, which allow us for storing new genetic information in DNA sequences and transmitting it into the protein world. Here, we investigate the problem of optimal genetic code extension from theoretical

  • Nanobody-mediated control of gene expression and epigenetic memory
    bioRxiv. Synth. Biol. Pub Date : 2020-09-10
    Mike V Van; Taihei Fujimori; Lacramioara Bintu

    Targeting chromatin regulators to specific genomic locations for gene control is emerging as a powerful method in basic research and synthetic biology. However, many chromatin regulators are large, making them difficult to deliver and combine in mammalian cells. Here, we developed a new strategy for gene control using small nanobodies that bind and recruit endogenous chromatin regulators to a gene

  • Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA base-editing activity, functionality and specificity
    bioRxiv. Synth. Biol. Pub Date : 2020-09-09
    Dhruva Katrekar; Nathan Palmer; Yichen Xiang; Anushka Saha; Dario Meluzzi; Prashant Mali

    Adenosine deaminases acting on RNA (ADARs) can be repurposed to enable programmable RNA editing, however their exogenous delivery leads to transcriptome-wide off-targeting, and additionally, enzymatic activity on certain RNA motifs, especially those flanked by a 5′ guanosine is very low thus limiting their utility as a transcriptome engineering toolset. To address this, we explored comprehensive ADAR2

  • Antisense inhibition of accA suppressed luxS expression that is essential for quorum sense signaling, biofilm formation, and virulence
    bioRxiv. Synth. Biol. Pub Date : 2020-09-08
    Tatiana Hillman

    Bacterial multiple drug resistance (MDR) is a major issue for the medical community. Gram-negative bacteria (GNB) exhibit higher rates of multidrug resistance. Gram-negative bacteria are more resistant to multiple antibiotics. The double membrane contains an outer membrane with a lipid bilayer of lipopolysaccharides (LPS) and an inner cytoplasmic membrane (IM). Gram-negative bacteria limit hydrophobic

  • Synthetic mammalian signaling circuits for robust cell population control
    bioRxiv. Synth. Biol. Pub Date : 2020-09-03
    Yitong Ma; Mark W Budde; Michaelle N Mayalu; Junqin Zhu; Richard M Murray; Michael B Elowitz

    In multicellular organisms, cells actively sense, respond to, and control their own population density. Synthetic mammalian quorum sensing circuits could provide insight into principles of population control and improve cell therapies. However, a key challenge is avoiding their inherent sensitivity to "cheater" mutations that evade control. Here, we repurposed the plant hormone auxin to enable orthogonal

  • Engineering high-yield biopolymer secretion creates an extracellular protein matrix for living materials
    bioRxiv. Synth. Biol. Pub Date : 2020-09-01
    Marimikel Charrier; Maria Teresa Orozco-Hidalgo; Nicholas Tjahjono; Dong Li; Sara Molinari; Kathleen R. Ryan; Paul D. Ashby; Behzad Rad; Caroline Ajo-Franklin

    The bacterial extracellular matrix forms autonomously, giving rise to complex material properties and multicellular behaviors. Synthetic matrix analogues can replicate these functions, but require exogenously added material or have limited programmability. Here we design a two-strain bacterial system that self-synthesizes and structures a synthetic extracellular matrix of proteins. We engineered Caulobacter

  • Low-N protein engineering with data-efficient deep learning
    bioRxiv. Synth. Biol. Pub Date : 2020-08-31
    Surojit Biswas; Grigory Khimulya; Ethan C Alley; Kevin M Esvelt; George M Church

    Protein engineering has enormous academic and industrial potential. However, it is limited by the lack of experimental assays that are consistent with the design goal and sufficiently high-throughput to find rare, enhanced variants. Here we introduce a machine learning-guided paradigm that can use as few as 24 functionally assayed mutant sequences to build an accurate virtual fitness landscape and

  • Biological Structure and Function Emerge from Scaling Unsupervised Learning to 250 Million Protein Sequences
    bioRxiv. Synth. Biol. Pub Date : 2020-08-31
    Alexander Rives; Joshua Meier; Tom Sercu; Siddharth Goyal; Zeming Lin; Demi Guo; Myle Ott; C. Lawrence Zitnick; Jerry Ma; Rob Fergus

    In the field of artificial intelligence, a combination of scale in data and model capacity enabled by unsupervised learning has led to major advances in representation learning and statistical generation. In the life sciences, the anticipated growth of sequencing promises unprecedented data on natural sequence diversity. Evolutionary-scale language modeling is a logical step toward predictive and generative

  • Advanced eMAGE for highly efficient combinatorial editing of a stable genome
    bioRxiv. Synth. Biol. Pub Date : 2020-08-31
    Zhuobin Liang; Eli Metzner; Farren J Isaacs

    Eukaryotic multiplex genome engineering (eMAGE) offers a powerful tool to generate precise combinatorial genome modifications in Saccharomyces cerevisiae. We optimize the design of synthetic oligonucleotides and enrichment of edited populations to increase editing frequencies up to 90%, reduce workflow time by 40%, and engineer a tunable mismatch repair system to lower the rate of spontaneous mutations

  • Two-stage Dynamic Deregulation of Metabolism Improves Process Robustness & Scalability in Engineered E. coli.
    bioRxiv. Synth. Biol. Pub Date : 2020-08-30
    Zhixia Ye; Shuai Li; Jennifer N Hennigan; Juliana Lebeau; Eirik A Moreb; Jacob Wolf; Michael D Lynch

    We report improved strain and bioprocess robustness as a result of the dynamic deregulation of central metabolism using two-stage dynamic control. Dynamic control is implemented using combinations of CRISPR interference and controlled proteolysis to reduce levels of central metabolic enzymes in the context of a standardized two-stage bioprocesses. Reducing the levels of key enzymes alters metabolite

  • Potential For Applying Continuous Directed Evolution To Plant Enzymes
    bioRxiv. Synth. Biol. Pub Date : 2020-08-27
    Jorge D Garcia-Garcia; Jaya Joshi; Jenelle A Patterson; Lidimarie Trujillo-Rodriguez; Christopher R Reisch; Alex A Javanpour; Chang C Liu; Andrew D Hanson

    Plant evolution has produced enzymes that may not be optimal for maximizing yield and quality in today's agricultural environments and plant biotechnology applications. By improving enzyme performance, it should be possible to alleviate constraints on yield and quality currently imposed by kinetic properties or enzyme instability. Enzymes can be optimized faster than naturally possible by applying

  • Heterologous expression of cryptomaldamide in a cyanobacterial host
    bioRxiv. Synth. Biol. Pub Date : 2020-08-26
    Arnaud Taton; Andrew Ecker; Brienna Diaz; Nathan A Moss; Brooke Anderson; Raphael Reher; Tiago F Leão; Ryan Simkovsky; Pieter C Dorrestein; Lena Gerwick; William H Gerwick; James W Golden

    Filamentous marine cyanobacteria make a variety of bioactive molecules that are produced by polyketide synthases, non-ribosomal peptide synthetases, and hybrid pathways that are encoded by large biosynthetic gene clusters. These cyanobacterial natural products represent potential drugs leads; however, thorough pharmacological investigations have been impeded by the limited quantity of compound that

  • Attribution of genetic engineering: a practical and accurate machine learning toolkit for biosecurity
    bioRxiv. Synth. Biol. Pub Date : 2020-08-22
    Ethan C. Alley; Miles Turpin; Andrew Bo Liu; Taylor Kulp-McDowall; Jacob Swett; Rey Edison; Stephen E. Von Stetina; George M. Church; Kevin M. Esvelt

    The promise of biotechnology is tempered by its potential for accidental or deliberate misuse. Reliably identifying telltale signatures characteristic to different genetic designers, termed genetic engineering attribution, would deter misuse, yet is still considered unsolved. Here, we show that recurrent neural networks trained on DNA motifs and basic phenotype can reach 70% attribution accuracy distinguishing

  • De Novo Designed Peptide and Protein Hairpins Self-assemble into Sheets and Nanoparticles.
    bioRxiv. Synth. Biol. Pub Date : 2020-08-18
    Johanna M Galloway; Harriet E. V. Bray; Deborah K Shoemark; Lorna R Hodgson; Jennifer Coombs; Judith M Mantell; Ruth Sarah Rose; James F Ross; Caroline Morris; Robert L Harniman; Christopher Wells Wood; Christopher Arthur; Paul Verkade; Derek N Woolfson

    The design and assembly of peptide based materials has advanced considerably, leading to a variety of fibrous, sheet and nanoparticle structures. A remaining challenge is to account for and control different possible supramolecular outcomes accessible to the same or similar peptide building blocks. Here we present a de novo peptide system that forms nanoparticles or sheets depending on the strategic

  • Baculovirus-vectored precision delivery of large DNA cargoes in human genomes
    bioRxiv. Synth. Biol. Pub Date : 2020-08-17
    Francesco Aulicino; Martin Pelosse; Christine Toelzer; Julien Capin; Parisa Meysami; Mark S Dillingham; Christiane Schaffitzel; Imre Berger

    Precise gene editing and genome engineering by CRISPR technology requires simultaneous delivery of multiple DNA-encoded components into living cells rapidly exceeding the cargo capacity of currently utilized viral vector systems. Here we exploit the unmatched heterologous DNA cargo capacity of baculovirus to resolve this bottleneck. We implement hybrid DNA techniques (MultiMate) for rapid and error-free

  • A modular RNA interference system for multiplexed gene regulation
    bioRxiv. Synth. Biol. Pub Date : 2020-08-17
    Ari Dwijayanti; Marko Storch; Guy-Bart Stan; Geoff S Baldwin

    The rational design and realization of simple-to-use genetic control elements that are modular, orthogonal and robust is essential to the construction of predictable and reliable biological systems of increasing complexity. To this effect, we introduce modular Artificial RNA interference (mARi), a rational, modular and extensible design framework that enables robust, portable and multiplexed post-transcriptional

  • Programming animal physiology and behaviors through engineered bacteria
    bioRxiv. Synth. Biol. Pub Date : 2020-08-16
    Qing Sun; Baizhen Gao

    A central goal of synthetic biology is to predictably and efficiently reprogram living systems to perform computations and carry out specific biological tasks. Although there have been many advances in the bio-computational design of living systems, these advances have mainly been applied to microorganisms or cell lines; programming animal behavior and altering animal physiology remain challenges for

  • Multidimensional single-cell benchmarking of inducible promoters for precise dynamic control in budding yeast
    bioRxiv. Synth. Biol. Pub Date : 2020-08-16
    Vojislav Gligorovski; Ahmad Sadeghi; Sahand Jamal Rahi

    For quantitative systems biology, simultaneous readout of multiple cellular processes as well as precise, independent control over different genes' activities are needed. In contrast to readout systems such as fluorescent proteins, control systems such as inducible transcription-factor-promoter systems have not been characterized systematically, impeding reliable modeling and precise system-level probing

  • Elucidation and refinement of synthetic receptor mechanisms
    bioRxiv. Synth. Biol. Pub Date : 2020-08-14
    Hailey I Edelstein; Patrick S Donahue; Joseph J Muldoon; Anthony K Kang; Taylor B Dolberg; Lauren M Battaglia; Everett R Allchin; Mihe Hong; Joshua Nathaniel Leonard

    Synthetic receptors are powerful tools for engineering mammalian cell-based devices. These biosensors enable cell-based therapies to perform complex tasks such as regulating therapeutic gene expression in response to sensing physiological cues. Although multiple synthetic receptor systems now exist, many aspects of receptor performance are poorly understood. In general, it would be useful to understand

  • An antiviral self-replicating molecular heterotroph
    bioRxiv. Synth. Biol. Pub Date : 2020-08-14
    Anastasia Shapiro; Alexander Rosenberg; Adva Levy-Zamir; Liron Bassali; Shmulik Ittah; Almogit Abu-Horowitz; Ido Bachelet

    We report the synthesis of a molecular machine, fabricated from nucleic acids, which is capable of digesting viral RNA and utilizing it to assemble additional copies of itself inside living cells. The machine's body plan combines several parts that build upon the target RNA, assembling an immobile, DNA:RNA 4-way junction, which contains a single gene encoding a hammerhead ribozyme (HHR). Full assembly

  • Rational engineering of Kluyveromyces marxianus to create a chassis for the production of aromatic products
    bioRxiv. Synth. Biol. Pub Date : 2020-08-12
    Arun S. Rajkumar; John P. Morrissey

    The yeast Kluyveromyces marxianus offers unique potential for industrial biotechnology because of useful features like rapid growth, thermotolerance and a wide substrate range. As an emerging alternative platform, K. marxianus requires the development and validation of metabolic engineering strategies to best utilize its metabolism as a basis for bio-based production. To illustrate the synthetic biology

  • Systematic Engineering of Artificial Metalloenzymes for New-to-Nature Reactions
    bioRxiv. Synth. Biol. Pub Date : 2020-08-11
    Tobias Vornholt; Fadri Christoffel; Michela Pellizzoni; Sven Panke; Thomas R. Ward; Markus Jeschek

    Artificial metalloenzymes (ArMs) catalyzing new-to-nature reactions under mild conditions could play an important role in the transition to a sustainable, circular economy. While ArMs have been created for a variety of bioorthogonal transformations, attempts at optimizing their performance by enzyme engineering have been case-specific and resulted only in modest improvements. To realize the full potential

  • dCas9 regulator to neutralize competition in CRISPRi circuits
    bioRxiv. Synth. Biol. Pub Date : 2020-08-11
    Hsin-Ho Huang; Massimo Bellato; Yili Qian; Pablo Cárdenas; Lorenzo Pasotti; Paolo Magni; Domitilla Del Vecchio

    CRISPRi-mediated gene repression allows simultaneous control of many genes. However, despite highly specific sgRNA-promoter binding, multiple sgRNAs still interfere with one another by competing for dCas9. We created a dCas9 regulator that adjusts dCas9 concentration based on sgRNAs' demand, mitigating competition in CRISPRi-based logic gates. The regulator's performance is demonstrated on both single-stage

  • Engineered sex distortion in the global agricultural pest Ceratitis capitata
    bioRxiv. Synth. Biol. Pub Date : 2020-08-07
    Angela Meccariello; Flavia Krsticevic; Rita Colonna; Giuseppe Del Corsano; Barbara Fasulo; Philippos Aris Papathanos; Nikolai Windbichler

    Genetic sex ratio distorters have potential for the area-wide control of harmful insect populations. Endonucleases targeting the X-chromosome and whose activity is restricted to male gametogenesis have recently been pioneered as a means to engineer such traits. Here we enabled endogenous CRISPR/Cas9 and CRISPR/Cas12a activity during spermatogenesis of the Mediterranean fruit fly Ceratitis capitata

  • An extension of Shannon's entropy to explain taxa diversity and human diseases
    bioRxiv. Synth. Biol. Pub Date : 2020-08-07
    Farzin Kamari; Sina Dadmand

    In this study, with the use of the information theory, we have proposed and proved a mathematical theorem by which we argue the reason for the existence of human diseases. To introduce our theoretical frame of reference, first, we put forward a modification of Shannon's entropy, computed for all available proteomes, as a tool to compare systems complexity and distinguish between the several levels

  • Engineering Yarrowia lipolytica as a chassis for de novo synthesis of five aromatic-derived natural products and chemicals
    bioRxiv. Synth. Biol. Pub Date : 2020-08-06
    Yang Gu; Jingbo Ma; Yonglian Zhu; Xinyu Ding; Peng Xu

    Yarrowia lipolytica is a novel microbial chassis to upgrade renewable low-cost carbon feedstocks to high-value commodity chemicals and natural products. In this work, we systematically characterized and removed the rate-limiting steps of the shikimate pathway and achieved de novo synthesis of five aromatic chemicals in Y. lipolytica. We determined that eliminating amino acids formation and engineering

  • Cheetah: a computational toolkit for cybergenetic control
    bioRxiv. Synth. Biol. Pub Date : 2020-08-05
    Elisa Pedone; Irene de Cesare; Criseida Zamora; David Haener; Lorena Postiglione; Barbara Shannon; Nigel Savery; Claire S Grierson; Mario di Barnardo; Thomas E Gorochowski; Lucia Marucci

    Advances in microscopy, microfluidics and optogenetics enable single cell monitoring and environmental regulation and offer the means to control cellular phenotypes. The development of such systems is challenging and often results in bespoke setups that hinder reproducibility. To address this, we introduce Cheetah - a flexible computational toolkit that simplifies the integration of real-time microscopy

  • A MATLAB Toolbox for Modeling Genetic Circuits in Cell-Free Systems
    bioRxiv. Synth. Biol. Pub Date : 2020-08-05
    Vipul Singhal; Zoltan A. Tuza; Zachary Sun; Richard M Murray

    We introduce a MATLAB based simulation toolbox, called txtlsim, for an E. coli based Transcription-Translation (TX- TL) system. This toolbox accounts for several cell-free related phenomena, such as resource loading, consumption, and degradation, and in doing so, models the dynamics of TX-TL reactions for the entire duration of batch-mode experiments. We use a Bayesian parameter inference approach

  • A novel prokaryotic CRISPR-Cas12a based tool for programmable transcriptional activation and repression
    bioRxiv. Synth. Biol. Pub Date : 2020-08-05
    Christoph Schilling; Mattheos A Koffas; Volker Sieber; Jochen Schmid

    Transcriptional perturbation using inactivated CRISPR-nucleases (dCas) is a common method in eukaryotic organisms. While rare examples of dCas9 based tools for prokaryotes have been described, multiplexing approaches are limited due to the used effector nuclease. For the first time, a dCas12a derived broad host range tool for the targeted activation and repression of genes was developed. Therefore

  • Distinguishing Critical, Beneficial, Neutral and Harmful Mutations Uncovered in the Directed Evolution of a Yeast Membrane Receptor
    bioRxiv. Synth. Biol. Pub Date : 2020-08-05
    Adebola Adeniran; Keith Edward Jaggard Tyo

    We present a reversion analysis of mutations introduced during the directed evolution of the yeast G-protein coupled receptor (GPCR) Ste2p to detect a peptide biomarker of chronic kidney disease. Two mutated receptors are analyzed in this study. Mutations acquired during directed evolution were reverted one at a time to the wild-type residue to assess the mutation's contribution to receptor function

  • 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

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