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T cell circuits that sense antigen density with an ultrasensitive threshold bioRxiv. Synth. Biol. Pub Date : 2021-01-21 Rogelio A Hernandez-Lopez; Wei Yu; Katelyn Ashley Cabral; Olivia A Creasey; Maria del Pilar Lopez Pamino; Yurie Tonai; Arsenia De Guzman; Anna Makela; Kalle Saksela; Zev Jordan Gartner; Wendell Lim
Overexpressed tumor associated antigens (e.g. HER2 and EGFR) are attractive targets for therapeutic T cells, but toxic cross-reaction with normal tissues expressing low antigen levels has been observed with Chimeric Antigen Receptor (CAR) T cells targeting such antigens. Inspired by natural ultrasensitive response circuits, we engineer a two-step positive feedback circuit that allows T cells to discriminate
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Spatiotemporal manipulation of the mismatch repair system of Pseudomonas putida accelerates phenotype emergence bioRxiv. Synth. Biol. Pub Date : 2021-01-21 Lorena Fernandez-Cabezon; Antonin Cros; Pablo Ivan Nikel
Developing complex phenotypes in industrially-relevant bacteria is a major goal of metabolic engineering, which encompasses the implementation of both rational and random approaches. In the latter case, several tools have been developed towards increasing mutation frequencies - yet the precise spatiotemporal control of mutagenesis processes continues to represent a significant technical challenge.
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Harnessing the central dogma for stringent multi-level control of gene expression bioRxiv. Synth. Biol. Pub Date : 2021-01-19 F. Veronica Greco; Amir Pandi; Tobias J Erb; 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, but ideally without affecting the achievable range of expression. Here, we demonstrate how the central dogma offers a simple way to effectively
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Crosstalk between H2A variant-specific modifications impacts vital cell functions bioRxiv. Synth. Biol. Pub Date : 2021-01-18 Anna Schmuecker; Bingkun Lei; Zdravko Lorkovic; Matias Capella; Sigurd J Braun; Pierre Bourguet; Olivier Mathieu; Karl Mechtler; Frederic Berger
Histone variants are distinguished by specific substitutions and motifs that might be subject to post-translational modifications (PTMs). Compared with the high conservation of H3 variants, the N- and C-terminal tails of H2A variants are more divergent and are potential substrates for a more complex array of PTMs, which have remained largely unexplored. We used mass spectrometry to inventory the PTMs
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A fragment-based protein interface design algorithm for symmetric assemblies bioRxiv. Synth. Biol. Pub Date : 2021-01-14 Joshua Laniado; Kyle Meador; Todd O Yeates
Theoretical and experimental advances in protein engineering have led to the creation of precisely defined, novel protein assemblies of great size and complexity, with diverse applications. One powerful approach involves designing a new attachment or binding interface between two simpler symmetric oligomeric protein components. The required methods of design, which present both similarities and key
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A novel and effective Cre/lox-based genetic tool for repeated, targeted and markerless gene integration bioRxiv. Synth. Biol. Pub Date : 2021-01-14 Qinghua Zhou; Liangcheng Jiao; Wenjuan Li; Zhiming Hu; Yunchong Li; Houjin Zhang; Li Xu; Yunjun Yan
The unconventional yeast Yarrowia lipolytica is extensively applied in bioproduction fields owing to its excellent metabolite and protein production ability. Nonetheless, utilization of this promising host is still restricted by limited availability of precise and effective gene integration tools. In this study, a novel and efficient genetic tool was developed for targeted, repeated, and markerless
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Chemical probe-based Nanopore Sequencing to Selectively Assess the RNA modifications bioRxiv. Synth. Biol. Pub Date : 2021-01-12 Soundhar Ramasamy; Vinodh J Sahayasheela; Zutao Yu; Takuya Hidaka; Li Cai; Hiroshi Sugiyama; Ganesh N Pandian
Current methods to identify RNA modifications with short-read sequencing are laborious and direct RNA sequencing gets proclaimed as the viable alternative. Herein, we harness the selective reactivity of the acrylonitrile towards the Inosine (I) and pseudouridine (Ψ) modifications and developed a chemical probe-based direct RNA sequencing method. We first demonstrated that the chemical probe-induced
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Cell-Free Characterization of Coherent Feed-Forward Loop-Based Synthetic Genetic Circuit bioRxiv. Synth. Biol. Pub Date : 2021-01-11 Pascal A. Pieters; Bryan L. Nathalia; Ardjan J. van der Linden; Peng Yin; Jongmin Kim; Wilhelm T.S. Huck; Tom F.A. de Greef
Regulatory pathways inside living cells employ feed-forward architectures to fulfill essential signal processing functions that aide in the interpretation of various types of inputs through noise-filtering, fold-change detection and adaptation. Although it has been demonstrated computationally that a coherent feed-forward loop (CFFL) can function as noise filter, a property essential to decoding complex
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Double drives and private alleles for localised population genetic control bioRxiv. Synth. Biol. Pub Date : 2021-01-09 Katie Willis; Austin Burt
Synthetic gene drive constructs could, in principle, provide the basis for highly efficient interventions to control disease vectors and other pest species. This efficiency derives in part from leveraging natural processes of dispersal and gene flow to spread the construct and its impacts from one population to another. However, sometimes (for example, with invasive species) only specific populations
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Strong and tunable anti-CRISPR/Cas9 activity of AcrIIA4 in plants bioRxiv. Synth. Biol. Pub Date : 2021-01-08 Camilo Calvache; Marta Vazquez-Vilar; Sara Selma; Mireia Uranga; Jose Antonio Daros; Diego Orzaez
This study describes the strong anti-CRISPR activity of the bacterial AcrIIA4 protein in Nicotiana benthamiana, a model plant used as molecular farming platform. The results demonstrate that AcrIIA4 abolishes site-directed mutagenesis in leaves when transiently co-expressed with CRISPR/Cas9. We also show that AcrIIA4 represses CRISPR/dCas9-based transcriptional activation (CRISPRa) of both reporter
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Synthetic gene networks recapitulate dynamic signal decoding and differential gene expression bioRxiv. Synth. Biol. Pub Date : 2021-01-07 Dirk Benzinger; Serguei Ovinnikov; Mustafa Khammash
Cells live in constantly changing environments and employ dynamic signaling pathways to transduce information about the signals they encounter. However, the mechanisms by which dynamic signals are decoded into appropriate gene expression patterns remain poorly understood. Here, we devise networked optogenetic pathways that achieve novel dynamic signal processing functions that recapitulate cellular
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An engineered lactate responding promoter system operating in glucose-rich and anaerobic environments. bioRxiv. Synth. Biol. Pub Date : 2021-01-06 Ana Zuniga; Hung-Ju Chang; Elsa Fristot; Jerome Bonnet
Bacteria equipped with genetically-encoded lactate biosensors would support several applications in biopharmaceutical production, diagnosis, or therapeutics. However, many applications involve glucose-rich and anaerobic environments, in which current whole-cell lactate biosensors have low performance. Here we engineered a synthetic lactate biosensor system by repurposing the natural LldPRD promoter
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In-depth computational analysis of natural and artificial carbon fixation pathways bioRxiv. Synth. Biol. Pub Date : 2021-01-06 Hannes Löwe; Andreas Kremling
In the recent years, engineering new-to-nature CO2 and C1 fixing metabolic pathways made a leap forward. These new, artificial pathways promise higher yields and activity than natural ones like the Calvin-Benson-Bassham cycle. The question remains how to best predict their in vivo performance and what actually makes one pathway "better" than another. In this context, we explore aerobic carbon fixation
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Mapping the dynamic transfer functions of epigenome editing bioRxiv. Synth. Biol. Pub Date : 2021-01-05 Jessica B Lee; Leandra M Caywood; Jennifer Y Lo; Nicholas B Levering; Albert J Keung
Biological information can be encoded in the dynamics of signaling components which has been implicated in a broad range of physiological processes including stress response, oncogenesis, and stem cell differentiation. To study the complexity of information transfer across the eukaryotic promoter, we screened 119 dynamic conditions-modulating the frequency, intensity, and pulse width of light-regulating
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Massively parallel characterization of engineered transcript isoforms using direct RNA sequencing bioRxiv. Synth. Biol. Pub Date : 2021-01-03 Matthew J Tarnowski; Thomas E Gorochowski
Transcriptional terminators signal where transcribing RNA polymerases (RNAPs) should halt and disassociate from DNA. However, because termination is stochastic, two different forms of transcript could be produced: one ending at the terminator and the other reading through. An ability to control the abundance of these transcript isoforms would offer bioengineers a mechanism to regulate multi-gene constructs
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A genome-scale yeast library with inducible expression of individual genes bioRxiv. Synth. Biol. Pub Date : 2021-01-01 Yuko Arita; Griffin Kim; Zhijian Li; Helena Friesen; Gina Turco; Rebecca Y. Wang; Dale Climie; Matej Usaj; Manuel Hotz; Emily Stoops; Anastasia Baryshnikova; Charles Boone; David Botstein; Brenda J. Andrews; R. Scott McIsaac
The ability to switch a gene from off to on and monitor dynamic changes provides a powerful approach for probing gene function and elucidating causal regulatory relationships, including instances of feedback control. Here, we developed and characterized YETI (Yeast Estradiol strains with Titratable Induction), a collection in which 5,687 yeast genes are engineered for transcriptional inducibility with
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Heterologous expression of methylxanthine synthesis enzymes in mammalian cells and their use as reporter proteins bioRxiv. Synth. Biol. Pub Date : 2020-12-29 Brandon Cisneros; Neal Devaraj
This work demonstrates the reconstitution of active methylxanthine synthesis enzymes in human cells and their potential use as inducible reporter enzymes. A variety of plant enzymes involved in caffeine synthesis have been characterized in vitro and several of these methylxanthine synthesis enzymes have been heterologously-expressed in yeast or bacteria. In this work, enzymes from Coffea arabica, Camellia
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An open platform for high-resolution light-based control of microscopic collectives bioRxiv. Synth. Biol. Pub Date : 2020-12-28 Ana Rubio Denniss; Thomas E Gorochowski; Sabine Hauert
Engineering microscopic collectives of cells or microrobots is challenging due to the often-limited capabilities of the individual agents, our inability to program their motion and local interactions, and difficulties visualising their behaviours. Here, we present a low-cost, modular and open-source Dynamic Optical MicroEnvironment (DOME) and demonstrate its ability to augment microagent capabilities
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Enhancing multi-bioreactor platforms for automated measurements and reactive experiment control bioRxiv. Synth. Biol. Pub Date : 2020-12-27 Francois Bertaux; Sebastian Sosa Carillo; Achille Fraisse; Chetan Aditya; Mariela Furstenheim; Gregory Batt
New small-scale, low-cost bioreactor designs provide researchers with exquisite control of environmental parameters of microbial cultures over long durations, allowing them to perform sophisticated, high-quality experiments that are particularly useful in systems biology, synthetic biology and bioengineering. However, existing setups are limited in their automated measurement capabilities, primarily
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A GenoChemetic strategy for derivatization of the violacein natural product scaffold bioRxiv. Synth. Biol. Pub Date : 2020-12-27 Hung-En Lai; Alan MC Obled; Soo Mei Chee; Rhodri M Morgan; Rosemary Lynch; Sunil V Sharma; Simon J Moore; Karen M Polizzi; Rebecca JM Goss; Paul S Freemont
Natural products and their analogues are often challenging to synthesise due to their complex scaffolds and embedded functional groups. Solely relying on engineering the biosynthesis of natural products may lead to limited compound diversity. Integrating synthetic biology with synthetic chemistry allows rapid access to much more diverse portfolios of xenobiotic compounds which may accelerate the discovery
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Standardization of inducer-activated broad host range expression modules: Debugging and refactoring an alkane-responsive AlkS/PalkB device bioRxiv. Synth. Biol. Pub Date : 2020-12-26 Alejandro Arce-Rodriguez; Ilaria Benedetti; Rafael Silva-Rocha; Victor de Lorenzo
Although inducible heterologous expression systems have been available since the birth of recombinant DNA technology, the diversity of devices and genetic architectures of the corresponding vectors have often resulted in a lack of reproducibility and interoperability. In an effort to increase predictability of expression of genes of interest in a variety of possible bacterial hosts we propose a composition
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Optogenetic Amplification Circuits for Light-Induced Metabolic Control bioRxiv. Synth. Biol. Pub Date : 2020-12-23 Evan M Zhao; Makoto A Lalwani; Jhong-Min Chen; Paulina Orillac; Jared E Toettcher; Jose L Avalos
Dynamic control of microbial metabolism is an effective strategy to improve chemical production in fermentations. While dynamic control is most often implemented using chemical inducers, optogenetics offers an attractive alternative due to the high tunability and reversibility afforded by light. However, a major concern of applying optogenetics in metabolic engineering is the risk of insufficient light
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In Silico Design and Analysis of Genetic Circuit-Based Whole-Cell Biosensors for Detecting Groundwater Contaminants bioRxiv. Synth. Biol. Pub Date : 2020-12-22 Samuel Fajemilua; Solomon Bada; M. Ahsanul Islam
Contaminants of emerging concern (CEC) such as tetracycline, erythromycin, and salicylic acid in groundwater can seriously endanger the environment and human health due to their widespread and everlasting harmful effects. Thus, continuous monitoring of various CEC concentrations in groundwater is essential to ensure the safety, security, and biodiversity of natural habitats. CECs can be detected using
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Rapid 40 kb genome construction from 52 parts bioRxiv. Synth. Biol. Pub Date : 2020-12-22 John M Pryor; Vladimir Potapov; Nilisha Pokhrel; Gregory J S Lohman
Large DNA constructs (>10 kb), including small genomes and artificial chromosomes, are invaluable tools for genetic engineering and vaccine development. However, the manufacture of these constructs is laborious. To address this problem, we applied new design insights and modified protocols to Golden Gate assembly. While this methodology is routinely used to assemble 5-10 DNA parts in one-step, we found
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Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations bioRxiv. Synth. Biol. Pub Date : 2020-12-21 Shane Miersch; Zhijie Li; Reza Saberianfar; Mart Ustav; James Brett Case; Levi Blazer; Chao Chen; Wei Ye; Alevtina Pavlenco; Maryna Gorelik; Julia Garcia Perez; Suryasree Subramania; Serena Singh; Lynda Ploder; Safder Ganaie; Rita Chen; Daisy Leung; Pier Paolo Pandolfi; Giuseppe Novelli; Giulia Matusali; Francesca Colavita; Maria R. Copabianchi; Suresh Jain; J.B. Gupta; Gaya K. Amarasinghe; Michael
Neutralizing antibodies (nAbs) hold promise as effective therapeutics against COVID-19. Here, we describe protein engineering and modular design principles that have led to the development of synthetic bivalent and tetravalent nAbs against SARS-CoV-2. The best nAb targets the host receptor binding site of the viral S-protein and its tetravalent versions can block entry with a potency that exceeds the
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Analysis of Circuits for Dosage Control in Microbial Populations bioRxiv. Synth. Biol. Pub Date : 2020-12-20 Sophie J. Walton; Samuel E. Clamons; Richard M Murray
Designing genetic circuits to control the behaviors of microbial populations is an ongoing challenge in synthetic biology. Here we analyze circuits which implement dosage control by controlling levels of a global signal in a microbial population in face of varying cell density, growth rate, and environmental dilution. We utilize the Lux quorum sensing system to implement dosage control circuits, and
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Designing an irreversible metabolic switch for scalable induction of microbial chemical production bioRxiv. Synth. Biol. Pub Date : 2020-12-18 Ahmad A Mannan; Declan G Bates
Bacteria can be harnessed for the synthesis of high-value chemicals. A promising strategy for increasing productivity uses inducible control systems to switch metabolism from growth to chemical synthesis once a large population of cell factories are generated. However, chemical induction via IPTG and other gratuitous inducers is extremely expensive, limiting the scaleability of this approach for biotechnological
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Genotype-phenotype map of an RNA-ligand complex bioRxiv. Synth. Biol. Pub Date : 2020-12-17 Olga Puchta; Grzegorz Sobczyk; Vanessa Smer-Barreto; Hollie Ireland; Marc Vendrell; Diego A Oyarzun; Janusz M A Bujnicki; Graeme Whyte; Grzegorz Kudla
RNA-ligand interactions play important roles in biology and biotechnology, but they often involve complex three-dimensional folding of RNA and are difficult to predict. To systematically explore the phenotypic landscape of an RNA-ligand complex, we used microarrays to investigate all possible single and double mutants of the 49-nt RNA aptamer Broccoli bound to the fluorophore DFHBI-1T. We collected
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Optimized gene expression from bacterial chromosome by high-throughput integration and screening bioRxiv. Synth. Biol. Pub Date : 2020-12-15 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 and high-throughput screening. We demonstrate multiplexed gene integration and expression-level
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Biological structure and function emerge from scaling unsupervised learning to 250 million protein sequences bioRxiv. Synth. Biol. Pub Date : 2020-12-15 Alexander Rives; Joshua Meier; Tom Sercu; Siddharth Goyal; Zeming Lin; Jason Liu; 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. Protein language modeling at the scale of evolution is a logical step toward predictive
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Transformer protein language models are unsupervised structure learners bioRxiv. Synth. Biol. Pub Date : 2020-12-15 Roshan M Rao; Joshua Meier; Tom Sercu; Sergey Ovchinnikov; Alexander Rives
Unsupervised contact prediction is central to uncovering physical, structural, and functional constraints for protein structure determination and design. For decades, the predominant approach has been to infer evolutionary constraints from a set of related sequences. In the past year, protein language models have emerged as a potential alternative, but performance has fallen short of state-of-the-art
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Engineering clinically-approved drug gated CAR circuits bioRxiv. Synth. Biol. Pub Date : 2020-12-14 Hui-Shan Li; Nicole M. Wong; Elliot Tague; John T. Ngo; Ahmad S. Khalil; Wilson W. Wong
Chimeric antigen receptor (CAR) T cell immunotherapy has the potential to revolutionize cancer medicine. However, excessive CAR activation, lack of tumor-specific surface markers, and anti-gen escape have limited the safety and efficacy of CAR T cell therapy. A multi-antigen targeting CAR system that is regulated by safe, clinically-approved pharmaceutical agents is urgently needed, yet only a few
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Optogenetic inhibition and activation of Rac and Rap1 using a modified iLID system bioRxiv. Synth. Biol. Pub Date : 2020-12-11 Nick R. Elston; Michael Pablo; Fred Pimenta; Klaus M. Hahn; Takashi Watanabe
The small GTPases Rac1 and Rap1 can fulfill multiple cellular functions because their activation kinetics and localization are precisely controlled. To probe the role of their spatiotemporal dynamics, we generated optogenetic tools that activate or inhibit endogenous Rac and Rap1 in living cells. An improved version of the light induced dimerization (iLID) system [1] was used to control plasma membrane
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A Synthetic Transcription Platform for Programmable Gene Expression in Mammalian Cells bioRxiv. Synth. Biol. Pub Date : 2020-12-11 William C.W. Chen; Leonid Gaidukov; Ming-Ru Wu; Jicong Cao; Gigi C.G. Choi; Rachel P. Utomo; Ying-Chou Chen; Liliana Wroblewska; Lin Zhang; Ron Weiss; Timothy K. Lu
Precise, scalable, and sustainable control of genetic and cellular activities in mammalian cells is key to developing precision therapeutics and smart biomanufacturing. We created a highly tunable, modular, versatile CRISPR-based synthetic transcription system for the programmable control of gene expression and cellular phenotypes in mammalian cells. Genetic circuits consisting of well-characterized
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Improved Photocleavable Proteins with Faster and More Efficient Dissociation bioRxiv. Synth. Biol. Pub Date : 2020-12-10 Xiaocen Lu; Yurong Wen; Shuce Zhang; Wei Zhang; Yilun Chen; Yi Shen; Joanne Lemieux; Robert E. Campbell
The photocleavable protein (PhoCl) is a green-to-red photoconvertible fluorescent protein that, when illuminated with violet light, undergoes main chain cleavage followed by spontaneous dissociation of the resulting fragments. The first generation PhoCl (PhoCl1) exhibited a relative slow rate of dissociation, potentially limiting its utilities for optogenetic control of cell physiology. In this work
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Coiled-coil heterodimers with increased stability for cellular regulation and sensing SARS-CoV-2 spike protein-mediated cell fusion bioRxiv. Synth. Biol. Pub Date : 2020-12-10 Tjasa Plaper; Jana Aupic; Petra Dekleva; Fabio Lapenta; Mateja Mancek; Roman Jerala; Mojca Bencina
Coiled-coil (CC) dimer-forming peptides are attractive designable modules for mediating protein association. Highly stable CCs are desired for biological activity regulation and assay. Here, we report the design and versatile applications of orthogonal CC dimer-forming peptides with a dissociation constant in the low nanomolar range. In vitro stability and specificity was confirmed in mammalian cells
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Integrating continuous hypermutation with high-throughput screening for optimization of cis,cis-muconic acid production in yeast bioRxiv. Synth. Biol. Pub Date : 2020-12-09 Emil Damgaard Jensen; Francesca Ambri; Marie Blatt Bendtsen; Alex Javanpour; Chang Liu; Michael Krogh Jensen; Jay D. Keasling
Directed evolution is a powerful method to optimize proteins and metabolic reactions towards user-defined goals. It usually involves subjecting genes or pathways to iterative rounds of mutagenesis, selection, and amplification. While powerful, systematic searches through large sequence-spaces is a labor-intensive task, and can be further limited by a priori knowledge about the optimal initial search
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Genetic Encoding of Three Distinct Noncanonical Amino Acids Using Reprogrammed Initiator and Nonsense Codons bioRxiv. Synth. Biol. Pub Date : 2020-12-08 Jeffery M Tharp; Oscar Vargas-Rodriguez; Alanna Schepartz; Dieter Söll
We recently described an orthogonal initiator tRNA (itRNATy2) that can initiate protein synthesis with noncanonical amino acids (ncAAs) in response to the UAG nonsense codon. Here we report that a mutant of itRNATy2 (itRNATy2AUA) can efficiently initiate translation in response to the UAU tyrosine codon, giving rise to proteins with an ncAA at their N-terminus. We show that, in cells expressing itRNATy2AUA
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Genetically Engineered Integral Feedback Controllers for Robust Perfect Adaptation in Mammalian Cells bioRxiv. Synth. Biol. Pub Date : 2020-12-06 Timothy Frei; Ching-Hsiang Chang; Maurice Filo; Mustafa Khammash
Mammalian cells collectively maintain a consistent internal milieu that supports their host's survival in varying and uncertain environments. This homeostasis is often achieved through negative feedback loops that act at various levels of biological organization, from the system and organ levels down to gene expression at the molecular scale. Recently, a molecular regulatory motif has been discovered
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CFPU: a cell-free processing unit for high-throughput, automated in vitro circuit characterization bioRxiv. Synth. Biol. Pub Date : 2020-12-04 Zoe Swank; Sebastian Josef Maerkl
Forward engineering synthetic circuits is at the core of synthetic biology. Automated solutions will be required to facilitate circuit design and implementation. Circuit design is increasingly being automated with design software, but innovations in experimental automation are lagging behind. Microfluidic technologies made it possible to perform in vitro transcription-translation (tx-tl) reactions
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Design of smart antibody mimetics with photosensitive switches bioRxiv. Synth. Biol. Pub Date : 2020-12-04 Lian He; Peng Tan; Yun Huang; Yubin Zhou
As two prominent examples of intracellular single-domain antibodies or antibody mimetics derived from synthetic protein scaffolds, monobodies and nanobodies are gaining wide applications in cell biology, structural biology, synthetic immunology, and theranostics. We introduce herein a generally-applicable method to engineer light-controllable monobodies and nanobodies, designated as moonbody and sunbody
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Lyophilized yeast powder for adjuvant free thermostable vaccine delivery bioRxiv. Synth. Biol. Pub Date : 2020-12-03 Ravinder Kumar; Bhushan N Kharbikar
Thermolabile nature of commercially available vaccines necessitates their storage, transportation and dissemination under refrigerated condition. Maintenance of continuous cold chain at every step increases the final cost of vaccines. Any breach in the cold chain, even for a short duration results in the need to discard the vaccine. As a result, there is a pressing need for the development of thermostable
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Anti-CRISPR RNAs: designing universal riboregulators with deep learning of Csy4-mediated RNA processing bioRxiv. Synth. Biol. Pub Date : 2020-12-01 Haotian Guo; Xiaohu Song; Ariel B Lindner
RNA-based regulation offers a promising alternative of protein-based transcriptional networks. However, designing synthetic riboregulators with desirable functionalities using arbitrary sequences remains challenging, due in part to insufficient exploration of RNA sequence-to-function landscapes. Here we report that CRISPR-Csy4 mediates a nearly all-or-none processing of precursor CRISPR RNAs (pre-crRNAs)
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Intein-assisted bisection mapping systematically splits proteins for Boolean logic and inducibility engineering bioRxiv. Synth. Biol. Pub Date : 2020-12-01 Trevor Y. H. Ho; Alexander Shao; Zeyu Lu; Harri Savilahti; Filippo Menolascina; Lei Wang; Neil Dalchau; Baojun Wang
Split inteins are powerful tools for seamless ligation of synthetic split proteins. Yet, their use remains limited because the already intricate split site identification problem is often complicated by the requirement of extein junction sequences. To address this, we augmented a mini-Mu transposon-based screening approach and devised the intein-assisted bisection mapping (IBM) method. IBM robustly
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Regulating enzymatic reactions in Escherichia coli utilizing light-responsive cellular compartments based on liquid-liquid phase separation bioRxiv. Synth. Biol. Pub Date : 2020-11-29 Zikang Huang; Lize Sun; Genzhe Lu; Hongrui Liu; Zihan Zhai; Site Feng; Ji Gao; Chunyu Chen; Chuheng Qing; Meng Fang; Bowen Chen; Jiale Fu; Xuan Wang; Guo-Qiang Chen
Enzymatic reactions in cells are well organized into different compartments, among which protein-based membraneless compartments formed through liquid-liquid phase separation (LLPS) are believed to play important roles1,2. Hijacking them for our own purpose has promising applications in metabolic engineering3. Yet, it is still hard to precisely and dynamically control target enzymatic reactions in
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Cell-Free Biosynthesis to Evaluate Lasso Peptide Formation and Enzyme-Substrate Tolerance bioRxiv. Synth. Biol. Pub Date : 2020-11-27 Yuanyuan Si; Ashley M Kretsch; Laura M Daign; Mark J Burk; Douglas A Mitchell
Lasso peptides are ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that display a unique lariat-like structure. Owing to a rigid topology, lasso peptides are unusually stable towards heat and proteolytic degradation. Some lasso peptides have been shown to bind human cell-surface receptors and exhibit anticancer properties, while others display antibacterial
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Cyber-yeast: Automatic synchronisation of the cell cycle in budding yeast through closed-loop feedback control bioRxiv. Synth. Biol. Pub Date : 2020-11-27 Giansimone Perrino; Sara Napolitano; Francesca Galdi; Antonella La Regina; Davide Fiore; Teresa Giuliano; Mario di Bernardo; Diego di Bernardo
The cell cycle is the process by which eukaryotic cells replicate. Yeast cells cycle asynchronously with each cell in the population budding at a different time. Although there are several experimental approaches to "synchronise" cells, these work only in the short-term. Here, we built a cyber-genetic system to achieve long-term synchronisation of the cell population, by interfacing genetically modified
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A two-state ribosome and protein model can robustly capture the chemical reaction dynamics of gene expression bioRxiv. Synth. Biol. Pub Date : 2020-11-26 Ayush Pandey; Richard M Murray
We derive phenomenological models of gene expression from a mechanistic description of chemical reactions using an automated model reduction method. Using this method, we get analytical descriptions and computational performance guarantees to compare the reduced dynamics with the full models. We develop a new two-state model with the dynamics of the available free ribosomes in the system and the protein
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Generating complex patterns of gene expression without regulatory circuits bioRxiv. Synth. Biol. Pub Date : 2020-11-25 Sahil B Shah; Alexis M Hill; Claus O Wilke; Adam J Hockenberry
Synthetic biology has successfully advanced our ability to design complex, time-varying genetic circuits executing precisely specified gene expression patterns. However, such circuits usually require regulatory genes whose only purpose is to regulate the expression of other genes. When designing very small genetic constructs, such as viral genomes, we may want to avoid introducing such auxiliary gene
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Automated Rational Strain Construction Based on High-Throughput Conjugation bioRxiv. Synth. Biol. Pub Date : 2020-11-24 Niklas Tenhaef; Robert Stella; Julia Frunzke; Stephan Noack
Molecular cloning is the core of Synthetic Biology, as it comprises the assembly of DNA and its expression in target hosts. At present, however, cloning is most often a manual, time-consuming and repetitive process that highly benefits from automation. The automation of a complete rational cloning procedure, i.e., from DNA part creation to expression in the target host, involves the integration of
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Hybrid Living Capsules Autonomously Produced by Engineered Bacteria bioRxiv. Synth. Biol. Pub Date : 2020-11-24 Daniel P Birnbaum; Avinash Manjula-Basavanna; Anton Kan; Neel S Joshi
Bacterial cellulose (BC) has excellent material properties and can be produced cheaply and sustainably through simple bacterial culture, but BC-producing bacteria lack the extensive genetic toolkits of model organisms such as Escherichia coli. Here, we describe a simple approach for producing highly programmable BC materials through incorporation of engineered E. coli. The acetic acid bacterium Gluconacetobacter
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A Synthetic Defective Interfering SARS-CoV-2 bioRxiv. Synth. Biol. Pub Date : 2020-11-23 Shun Yao; Anoop Narayanan; Sydney Majowicz; Joyce Jose; Marco Archetti
Viruses thrive by exploiting the cells they infect but must also produce viral proteins to replicate and infect other cells. As a consequence, they are also susceptible to exploitation by defective versions of themselves that do not produce such proteins. A defective viral genome with deletions in protein-coding genes could still replicate in cells coinfected with full-length viruses, and even replicate
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Resources allocation explains the differential roles of RBS and promoter strengths in cell mass distribution and optimal protein expression productivity bioRxiv. Synth. Biol. Pub Date : 2020-11-22 Jesus Pico; Fernando N. Santos-Navarro
Design of synthetic genetic circuits without considering the impact of host-circuit interactions results in an inefficient design process and lengthy trial-and-error iterations to appropriately tune the expression levels. Microorganisms have evolved to reach an optimal use of cellular resources. This balance is perturbed by circuit-host interactions resulting from the interaction among the cell environment
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Optogenetic control of infection signaling cascade of bacteria by an engineered light-responsive protein bioRxiv. Synth. Biol. Pub Date : 2020-11-20 Xinyi Cheng; Lu Pu; Shengwei Fu; Aiguo Xia; Shuqiang Huang; Lei Ni; Xiaochen Xing; Shuai Yang; Fan JIN
Bacterial pathogens operate by tightly controlling the virulence to facilitate invasion and survival in host. Although pathways regulating virulence have been defined in detail and signals modulating these processes are gradually understood, a lack of controlling infection signaling cascades of pathogens when and whereabouts specificity limits deeper investigating of host-pathogen interactions. Here
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A Hybrid Cellular and Heterogeneous Catalyst Strategy for the Production of Olefins from Glucose bioRxiv. Synth. Biol. Pub Date : 2020-11-18 Zhen Q. Wang; Heng Song; Noritaka Hara; Dae Sung Park; Gaurav Kumar; Yejin Min; Paul J. Dauenhauer; Michelle C. Y. Chang
Living systems provide a promising approach to chemical synthesis, having been optimized by evolution to convert renewable carbon sources such as glucose to an enormous range of small molecules. However, a large number of synthetic structures can still be difficult to obtain solely from cells, such as unsubstituted hydrocarbons. In this work, we demonstrate the use of a hybrid cellular-heterogeneous
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A Streptomyces venezuelae Cell-Free Toolkit for Synthetic Biology bioRxiv. Synth. Biol. Pub Date : 2020-11-17 Simon Moore; Hung-En Lai; Soo-Mei Chee; Ming Toh; Seth Coode; Patrick Capel; Christophe Corre; Emmanuel de los Santos; Paul Freemont
Prokaryotic cell-free coupled transcription-translation (TX-TL) systems are emerging as a powerful tool to examine natural product biosynthetic pathways in a test-tube. The key advantages of this approach are the reduced experimental timescales and controlled reaction conditions. In order to realise this potential, specialised cell-free systems in organisms enriched for biosynthetic gene clusters,
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Multiomics data collection, visualization, and utilization for guiding metabolic engineering bioRxiv. Synth. Biol. Pub Date : 2020-11-16 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
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Light-inducible Deformation of Mitochondria in Live Cells bioRxiv. Synth. Biol. Pub Date : 2020-11-15 Yutong Song; Peiyuan Huang; Xiaoying Liu; Bianxiao Cui; Liting Duan
Mitochondria, the powerhouse of the cell, are dynamic organelles that undergo constant morphological changes. Increasing evidence indicates that mitochondria morphologies and functions can be modulated by mechanical cues. However, the mechano-sensing and -responding properties of mitochondria and the correlation between mitochondrial morphologies and functions are unclear due to the lack of methods
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A novel design of transcriptional factor-mediated dynamic control of DNA recombination. bioRxiv. Synth. Biol. Pub Date : 2020-11-13 Jiayang Li; Yihao Zhang; Yeqing Zong
Genetic regulation is achieved by monitoring multiple levels of gene expression, from transcription to protein interaction. Unlike common temporary transcription regulation methods such as the use of inducible promoters, integrases permanently edit DNA sequences. Integrases, however, require especially strict regulation when implemented in synthetic genetic systems because of the irreversible result
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Genetic and process engineering strategies for enhanced recombinant N-glycoprotein production in bacteria bioRxiv. Synth. Biol. Pub Date : 2020-11-12 Fenryco Pratama; Dennis Linton; Neil Dixon
Background: The production of N-linked glycoproteins in genetically amenable bacterial hosts offers great potential for reduced cost, faster/simpler bioprocesses, greater customisation and utility for distributed manufacturing of glycoconjugate vaccines and glycoprotein therapeutics. Efforts to optimize production hosts have included heterologous expression of glycosylation enzymes, metabolic engineering
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