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Unlocking Precision Gene Therapy: Harnessing AAV Tropism with Nanobody Swapping at Capsid Hotspots bioRxiv. Synth. Biol. Pub Date : 2024-03-27 Mareike Daniela Hoffmann, Joseph P Gallant, Aaron M LeBeau, Daniel Schmidt
Adeno-associated virus has been remarkably successful in the clinic, but its broad tropism is a practical limitation of precision gene therapy. A promising path to engineer AAV tropism is the addition of binding domains to the AAV capsid that recognize cell surface markers present on a targeted cell type. We have recently identified two previously unexplored capsid regions near the 2-fold valley and
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Cyanamide-inducible expression of homing nuclease I-SceI for iterative genome engineering and parallel promoter characterisation in Saccharomyces cerevisiae bioRxiv. Synth. Biol. Pub Date : 2024-03-27 Liam McDonnell, Samuel Evans, Zeyu Lu, Mitch Suchoronczak, Jonah Leighton, Eugene Ordeniza, Blake Ritchie, Nik Valado, Niamh Walsh, James Peter Antoney, Chengqiang Wang, Colin Scott, Robert Speight, Claudia Vickers, Bingyin Peng
In synthetic biology, microbial chasses including yeast Saccharomyces cerevisiae are iteratively engineered with increasing complexity and scale. Wet-lab genetic engineering tools are developed and optimised to facilitate strain construction but are often incompatible with each other due to shared regulatory elements, such as the galactose-inducible (GAL) promoter in S. cerevisiae. Here, we prototyped
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Hybrid Sequencing Facilitates Robust De Novo Plasmid Assembly bioRxiv. Synth. Biol. Pub Date : 2024-03-26 Sarah I Hernandez, Casey-Tyler Berezin, Katie M Miller, Samuel J Peccoud, Jean Peccoud
Despite the wide use of plasmids in research and clinical production, the verification of plasmid sequences is a bottleneck that is too often overlooked in the manufacturing process. Although sequencing platforms continue to improve, the method and assembly pipeline chosen still influence the final plasmid assembly sequence. Furthermore, few dedicated tools exist for plasmid assembly, especially for
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A cell-free workflow for detecting and characterizing RiPP recognition element-precursor peptide interactions bioRxiv. Synth. Biol. Pub Date : 2024-03-26 Derek A. Wong, Maria D. Cabezas, Martin Daniel-Ivad, Deepali V. Prasanna, Regina Fernandez, Robert Nicol, Emily P. Balskus, Ashty S. Karim, Michael C. Jewett
Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent a promising class of new therapeutics and antimicrobials. Unfortunately, RiPP discovery efforts are hampered by low-throughput methods for characterizing RiPP recognition elements (RRE), which direct tailoring enzymes to their peptide substrates for RiPP maturation. To address this bottleneck, we report a high-throughput
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Pangenomic Landscapes Shape Performances of a Synthetic Genetic Circuit Across Stutzerimonas Species bioRxiv. Synth. Biol. Pub Date : 2024-03-26 Dennis Tin Chat Chan, Hans C. Bernstein
Engineering identical genetic circuits into different species typically results in large differences in performance due to the unique cellular environmental context of each host, a phenomenon known as the "chassis-effect". A better understanding of how genomic and physiological contexts underpin the chassis-effect will greatly improve biodesign strategies across diverse microorganisms. Here, we combined
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Impact of Chemical Dynamics of Commercial PURE Systems on Malachite Green Aptamer Fluorescence bioRxiv. Synth. Biol. Pub Date : 2024-03-26 Zoila Jurado, Richard M Murray
The malachite green aptamer (MGapt) is known for its utility in RNA measurement in vivo and lysate-based cell-free protein systems. However, MGapt fluorescence dynamics do not accurately reflect mRNA concentration. Our study finds that MGapt fluorescence is unstable in commercial PURE systems. We discovered that the chemical composition of the cell-free reaction strongly influences MGapt fluorescence
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An ultra-high-throughput method for measuring biomolecular activities bioRxiv. Synth. Biol. Pub Date : 2024-03-25 Boqiang Tu, Vikram Sundar, Kevin Esvelt
Large datasets of biomolecular activities are crucial for protein engineering, yet their scarcity due to limited experimental throughput hampers progress. We introduce Direct High-throughput Activity Recording and Measurement Assay (DHARMA), an innovative method enabling ultra-high-throughput measurement of biomolecular activities. DHARMA employs molecular recording techniques to link activity directly
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Oligonucleotide Library Assisted Sequence Mining Reveals Promoter Sequences With Distinct Temporal Expression Dynamics For Applications In Curvibacter SP. AEP1-3 bioRxiv. Synth. Biol. Pub Date : 2024-03-25 Maurice Mager, Lukas Becker, Nina Schulten, Sebastian Fraune, Ilka Maria Axmann
The β-proteobacterial species Curvibacter sp. AEP1-3 is a model organism for the study of symbiotic interactions as it is the most abundant bacterial colonizer of the basal metazoan Hydra vulgaris. Yet, genetic tools for Curvibacter are still in an infancy: few promoters have been characterized for Curvibacter. Here we employ an oligonucleotide based strategy to find potential expression systems derived
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Conversion of natural cytokine receptors into orthogonal synthetic biosensors bioRxiv. Synth. Biol. Pub Date : 2024-03-24 Hailey I Edelstein, Amparo Cosio, Max L Ezekiel, William K Corcoran, Aaron H Morris, Joshua Nathaniel Leonard
Synthetic receptors enable bioengineers to build cell-based therapies that perform therapeutic functions in a targeted or conditional fashion to enhance specificity and efficacy. Although many synthetic receptors exist, it remains challenging to generate new receptors that sense soluble cues and relay that detection through orthogonal mechanisms independent of native pathways. Towards this goal, we
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Design of a sequestration-based network with tunable pulsing dynamics bioRxiv. Synth. Biol. Pub Date : 2024-03-24 Eiji Nakamura, Christian Cuba Samaniego, Franco Blanchini, Giulia Giordano, Elisa Franco
Incoherent feedforward networks exhibit the ability to generate temporal pulse behavior. However, exerting control over specific dynamic properties, such as amplitude and rise time, poses a challenge and is intricately tied to the network's implementation. In this study, we focus on analyzing sequestration-based networks capable of exhibiting pulse behavior. By employing time-scale separation in the
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Combining positive and negative regulation for modular and robust biomolecular control architectures bioRxiv. Synth. Biol. Pub Date : 2024-03-24 Kirill Sechkar, Harrison Steel
Engineered biotechnologies are powered by synthetic gene regulation and control systems, known as genetic circuits, which must be modular and robust to disturbances if they are to perform reliably. An emerging family of regulatory mechanisms is mediated by clustered interspaced palindromic repeats (CRISPR) that can both interfere with (downregulate) or activate (upregulate) a given gene's expression
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Neural networks built from enzymatic reactions can operate as linear and nonlinear classifiers bioRxiv. Synth. Biol. Pub Date : 2024-03-23 Christian Cuba Samaniego, Emily Wallace, Franco Blanchini, Elisa Franco, Giulia Giordano
The engineering of molecular programs capable of processing patterns of multi-input biomarkers holds great potential in applications ranging from in vitro diagnostics (e.g., viral detection, including COVID-19) to therapeutic interventions (e.g., discriminating cancer cells from normal cells). For this reason, mechanisms to design molecular networks for pattern recognition are highly sought after.
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Synthetic genome modules designed for programmable silencing of functions and chromosomes bioRxiv. Synth. Biol. Pub Date : 2024-03-22 Xinyu Lu, William M Shaw, Anima Sutradhar, Giovanni Stracquadanio, Tom Ellis
Unlike in bacteria, eukaryotes rarely cluster sets of genes in their genomes according to function, instead having most genes spread randomly across different chromosomes and loci. However, with the advent of genome engineering, synthetic co-location of genes that together encode a cell function has now become possible. Here, using Saccharomyces cerevisiae we demonstrate the feasibility of reorganising
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MOSAIC: a highly efficient, one-step recombineering approach to plasmid editing and diversification bioRxiv. Synth. Biol. Pub Date : 2024-03-22 Marijn van den Brink, Timotheus Y. Althuis, Christophe Danelon, Nico J. Claassens
The editing of plasmids and construction of plasmid libraries is paramount to the engineering of desired functionalities in synthetic biology. Typically, plasmids with targeted mutations are produced through time- and resource-consuming DNA amplification and/or cloning steps. In this study, we establish MOSAIC, a highly efficient protocol for the editing of plasmids and generation of combinatorial
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Genetic designs for stochastic and probabilistic biocomputing bioRxiv. Synth. Biol. Pub Date : 2024-03-22 Lewis Grozinger, Jesus Miro-Bueno, Angel Goni-Moreno
The programming of computations in living cells can be done by manipulating information flows within genetic networks. Typically, a single bit of information is encoded by a single gene's steady state expression. Expression is discretized into high and low levels that correspond to 0 and 1 logic values, analogous to the high and low voltages in electronic logic circuits. However, the processes of molecular
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Functional characterization of fatty acid synthase in Hermetia illucens bioRxiv. Synth. Biol. Pub Date : 2024-03-22 Yuguo Jiang, Wei Xue, Zhidan Peng, Peng Hou, Zongqing Kou, Bihui Chen, Huimeng Lu, Yongping Huang
Fatty acid synthase (FAS) is a key role in de novo lipogenesis (DNL) in most lives. In insects, type I FAS functions in homodimer, activated by a phosphopantetheine transferase (PPT), to de novo produce essential saturated fatty acid for cellular processes. In a nova insect species Hermetia illucens (Diptera: Stratiomyidae), different from most animals and insects, lauric acid (LA) accumulates to a
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Bioinformatic prediction and high throughput in vivo screening to identify cis-regulatory elements for the development of algal synthetic promoters bioRxiv. Synth. Biol. Pub Date : 2024-03-21 Yasin Torres-Tiji, Harish Sethuram, Abhishek Gupta, Joshua McCauley, Joao-Vitor Dutra-Molino, Ruchi Pathania, Lisa Saxton, Kalisa Kang, Nathan J Hillson, Stephen P Mayfield
Algae biotechnology holds immense promise for revolutionizing the bioeconomy through the sustainable and scalable production of various bioproducts. However, its development has been hindered by the lack of advanced genetic tools. This study introduces a synthetic biology approach to develop such tools, focusing on the construction and testing of synthetic promoters. By analyzing conserved DNA motifs
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Orthogonality of shell proteins across BMC subclasses in cyanobacteria bioRxiv. Synth. Biol. Pub Date : 2024-03-20 Joshua S MacCready, Matthew E Dwyer, Cheryl A. Kerfeld, Daniel C. Ducat
Bacterial microcompartments (BMC) are protein-based organelles broadly distributed across all bacterial phyla and subclassified into ≥60 functional variants. Despite their evolutionary and metabolic diversity, shell proteins that structurally compose the BMC surface are closely related across BMC classes. Herein, we sought to identify molecular and physiological features that could promote independent
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Reprogramming genetic circuits using space bioRxiv. Synth. Biol. Pub Date : 2024-03-20 Lorea Alejaldre, Jesus Miro-Bueno, Angeles Hueso-Gil, Lewis Grozinger, Huseyin Tas, Sina Geibler, Angel Goni-Moreno
Genetic circuits confer computing abilities to living cells, performing novel transformations of input stimuli into output responses. These genetic circuits are routinely engineered for insertion into bacterial plasmids and chromosomes, using a design paradigm whose only spatial consideration is a linear ordering of the individual components. However, chromosomal DNA has a complex three dimensional
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Allele Sails: launching traits and fates into wild populations with DNA sequence modifiers bioRxiv. Synth. Biol. Pub Date : 2024-03-19 Michelle L Johnson, Bruce Hay, Maciej Maselko
Population-scale genome editing can be used to alter the composition or fate of wild populations. One approach to achieving these aims utilizes a synthetic gene drive element a multi-gene cassette-to bring about an increase in the frequency of an existing allele. However, the use of gene drives is complicated by the multiple scientific, regulatory, and social issues associated with transgene persistence
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CRISPR-Cas9-assisted genome editing in E. coli elevates the frequency of unintended mutations bioRxiv. Synth. Biol. Pub Date : 2024-03-19 Karl A Widney, Dong-dong Yang, Leo M Rusch, Shelley D Copley
Cas-assisted lambda Red recombineering techniques have rapidly become a mainstay of bacterial genome editing. Such techniques have been used to construct both individual mutants and massive libraries to assess the effects of genomic changes. We have found that a commonly used Cas9-assisted editing method results in unintended mutations elsewhere in the genome in 26% of edited clones. The unintended
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Developmental assembly of multi-component polymer systems through interconnected gene networks in vitro bioRxiv. Synth. Biol. Pub Date : 2024-03-14 Daniela Sorrentino, Simona Ranallo, Francesco Ricci, Elisa Franco
Living cells regulate the dynamics of developmental events through interconnected signaling systems that activate and deactivate inert precursors. This suggests that similarly, synthetic biomaterials could be designed to develop over time by using chemical reaction networks to regulate the availability of assembling components. Here we demonstrate how the sequential activation or deactivation of distinct
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Synthetic dosage-compensating miRNA circuits for quantitative gene therapy bioRxiv. Synth. Biol. Pub Date : 2024-03-14 Michael James Flynn, Acacia M. Mayfield, Rongrong Du, Viviana Gradinaru, Michael B Elowitz
A longstanding challenge in gene therapy is expressing a dosage-sensitive gene within a tight therapeutic window. For example, loss of MECP2 function causes Rett syndrome, while its duplication causes MECP2 duplication syndrome. Viral gene delivery methods generate variable numbers of gene copies in individual cells, creating a need for gene dosage-invariant expression systems. Here, we introduce a
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Host evolution improves genetic circuit function in complex growth environments bioRxiv. Synth. Biol. Pub Date : 2024-03-13 Joanna T Zhang, Andrew Lezia, Philip Emmanuele, Muyao Wu, Connor A Olson, Adam T Feist, Jeff Hasty
Genetically engineered bacteria have become an attractive platform for numerous biomedical and industrial applications. Despite genetic circuitry functioning predictably under favorable growth conditions in the lab, the same cannot be said when placed in more complex environments for eventual deployment. Here, we used a combination of evolutionary and rational engineering approaches to enhance E. coli
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Scalable design of repeat protein structural dynamics via probabilistic coarse-grained models bioRxiv. Synth. Biol. Pub Date : 2024-03-13 Seeralan Sarvaharman, Timon E. Neary, Thomas E Gorochowski, Fabio Parmeggiani
Computational protein design has emerged as a powerful tool for creating proteins with novel functionalities. However, most existing methods ignore structural dynamics even though they are known to play a central role in many protein functions. Furthermore, methods like molecular dynamics that are able to simulate protein movements are computationally demanding and do not scale for the design of even
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Small molecule and cell contact-inducible systems for controlling expression and differentiation in stem cells bioRxiv. Synth. Biol. Pub Date : 2024-03-12 Sarah S. Soliman, Devan H. Shah, Hana El-Samad, Zara Y. Weinberg
Synthetic developmental biology uses engineering approaches to understand multicellularity with goals ranging from recapitulating development to building synthetic organisms. Current approaches include engineering multicellular patterning, controlling differentiation, and implementing cooperative cellular behaviors in model systems. Synthetic biology tools enable these pursuits with genetic circuits
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miRNA circuit modules for precise, tunable control of gene expression bioRxiv. Synth. Biol. Pub Date : 2024-03-12 Rongrong Du, Michael J. Flynn, Monique Honsa, Ralf Jungmann, Michael B Elowitz
The ability to express transgenes at specified levels is critical for understanding cellular behaviors, and for applications in gene and cell therapy. Transfection, viral vectors, and other gene delivery methods produce varying protein expression levels, with limited quantitative control, while targeted knock-in and stable selection are inefficient and slow. Active compensation mechanisms can improve
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Light-driven synchronization of optogenetic clocks bioRxiv. Synth. Biol. Pub Date : 2024-03-11 Maria Cristina Cannarsa, Filippo Liguori, Nicola Pellicciotta, Giacomo Frangipane, Roberto Di Leonardo
Synthetic genetic oscillators can serve as internal clocks within engineered cells to program periodic expression. However, cell-to-cell variability introduces a dispersion in the characteristics of these clocks that drives the population to complete desynchronization. Here we introduce the optorepressilator, an optically controllable genetic clock that combines the repressilator, a three-node synthetic
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Reconfigurable multi-component nanostructures built from DNA origami voxels bioRxiv. Synth. Biol. Pub Date : 2024-03-11 Minh Tri Luu, Jonathan Berengut, Jasleen Kaur Daljit Singh, Kanako Coffi Dit Glieze, Matthew Turner, Karuna Skipper, Sreelakshmi Meppat, Ali Abbas, Hannah Fowler, William Close, Jonathan P K Doye, Shelley F.J. Wickham
In cells, proteins rapidly self-assemble into sophisticated nanomachines. Bio-inspired self-assembly approaches, such as DNA origami, have achieved complex 3D nanostructures and devices. However, current synthetic systems are limited by lack of structural diversity, low yields in hierarchical assembly, and challenges in reconfiguration. Here, we develop a modular system of DNA origami voxels with programmable
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Enhancing Gastrodin Production in Yarrowia lipolytica by Metabolic Engineering bioRxiv. Synth. Biol. Pub Date : 2024-03-11 Yuanqing Wu, Shuocheng Li, Baijian Sun, Jingyi Guo, Meiyi Zheng, Aitao Li
Gastrodin, 4-hydroxybenzyl alcohol-4-O-β-D-glucopyranoside, has been widely used in the treatment of neurogenic and cardiovascular diseases. Currently, gastrodin biosynthesis has been achieved in model microorganisms. However, the production levels are insufficient for industrial applications. In this study, we successfully engineered a Yarrowia lipolytica strain to overproduce gastrodin through metabolic
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Highly Dynamic and Sensitive NEMOer Calcium Indicators for Imaging ER Calcium Signals in Excitable Cells bioRxiv. Synth. Biol. Pub Date : 2024-03-09 Wenjia Gu, Jia-Hui Chen, Yiyin Zhang, Zhirong Wang, Jia Li, Sijia Wang, Hanhan Zhang, Amin Jiang, Ziyi Zhong, Jiaxuan Zhang, Chao Xi, Tingting Hou, Donald L. Gill, Dong Li, Yu Mu, Shi-Qiang Wang, Ai-Hui Tang, Youjun Wang
Endoplasmic/sarcoplasmic reticulum (ER/SR) sits at the heart of the calcium (Ca2+) signaling machinery, yet current genetically encoded Ca2+ indicators (GECIs) lack the ability to detect elementary Ca2+ release events from ER/SR, particularly in muscle cells. Here we report a set of organellar GECIs, termed NEMOer, to efficiently capture ER Ca2+ dynamics with increased sensitivity and responsiveness
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Specific Modulation of CRISPR Transcriptional Activators through RNA-Sensing Guide RNAs in Mammalian Cells and Zebrafish Embryos bioRxiv. Synth. Biol. Pub Date : 2024-03-09 Oana Pelea, Sarah Mayes, Quentin RV. Ferry, Tudor A. Fulga, Tatjana Sauka-Spengler
Cellular transcripts encode important information regarding cell identity and disease status. The activation of CRISPR in response to RNA biomarkers holds the potential for controlling CRISPR activity with spatiotemporal precision. This would enable the restriction of CRISPR activity to specific cell types expressing RNA biomarkers of interest while preventing unwanted activity in other cells. Here
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Ratiometric control of two microbial populations in a dual chamber bioreactor bioRxiv. Synth. Biol. Pub Date : 2024-03-08 Sara Maria Brancato, Davide Salzano, Davide Fiore, Giovanni Russo, Mario di Bernardo
Ensuring stable coexistence between different species in microbial consortia is a challenging problem, especially when the species are not complementary, i.e. one species always outgrowing the other when left uncontrolled. Here, we present a novel control architecture based on the use of two bioreactors in which the slower species is separately grown and can be added to the main mixing chamber. After
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Highly multiplexed design of an allosteric transcription factor to sense novel ligands bioRxiv. Synth. Biol. Pub Date : 2024-03-08 Kyle K Nishikawa, Jackie Chen, Justin F Acheson, Svetlana V Harbaugh, Phil Huss, Max Frenkel, Nathan Novy, Hailey R Sieren, Ella C Lodewyk, Daniel H Lee, Jorge L Chavez, Brian G Fox, Srivatsan Raman
Designing biosensors to detect novel molecules is important in biotechnology. Allosteric transcription factors (aTF), which are widely used as biosensors, have proven challenging to design because mutating ligand-binding residues often disrupt allostery. Moreover, existing screening methods lack the sensitivity to identify rare, functional variants amidst a large pool of non-functional designs. We
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Thermodynamically-consistent, reduced models of gene regulatory networks bioRxiv. Synth. Biol. Pub Date : 2024-03-07 Michael Pan, Peter J. Gawthrop, Matthew Faria, Stuart T. Johnston
Synthetic biology aims to engineer novel functionalities into biological systems. While the approach has to date been predominantly applied to single cells, a richer set of biological phenomena can be engineered by applying synthetic biology to cell populations. To rationally design cell populations, we require mathematical models that link between intracellular biochemistry and intercellular interactions
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Sequence modeling and design from molecular to genome scale with Evo bioRxiv. Synth. Biol. Pub Date : 2024-03-06 Eric Nguyen, Michael Poli, Matthew G Durrant, Armin W Thomas, Brian Kang, Jeremy Sullivan, Madelena Y Ng, Ashley Lewis, Aman Patel, Aaron Lou, Stefano Ermon, Stephen A Baccus, Tina Hernandez-Boussard, Christopher Re, Patrick D Hsu, Brian L Hie
The genome is a sequence that completely encodes the DNA, RNA, and proteins that orchestrate the function of a whole organism. Advances in machine learning combined with massive datasets of whole genomes could enable a biological foundation model that accelerates the mechanistic understanding and generative design of complex molecular interactions. We report Evo, a genomic foundation model that enables
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Expanding the genetic toolbox for the obligate human pathogen Streptococcus pyogenes bioRxiv. Synth. Biol. Pub Date : 2024-03-04 Nina Lautenschlaeger, Katja Schmidt, Carolin Schiffer, Thomas F. Wulff, Karin Hahnke, Knut Finstermeier, Moise Mansour, Alexander K. W. Elsholz, Emmanuelle Charpentier
Genetic tools form the basis for the study of molecular mechanisms. Despite many recent advances in the field of genetic engineering in bacteria, genetic toolsets remain scarce for non-model organisms, such as the obligatory human pathogen Streptococcus pyogenes. In this study, we set out to develop a comprehensive set of plasmids, promoters and reporters for S. pyogenes. We present an expansion to
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Light-directed evolution of dynamic, multi-state, and computational protein functionalities bioRxiv. Synth. Biol. Pub Date : 2024-03-02 Sahand Jamal Rahi, Vojislav Gligorovski, Marco Labagnara
Directed evolution is a powerful method in biological engineering. Current approaches were devised for evolving steady-state properties such as enzymatic activity or fluorescence intensity. A fundamental problem remains how to evolve dynamic, multi-state, or computational functionalities, e.g., folding times, on-off kinetics, state-specific activity, stimulus-responsiveness, or switching and logic
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A general strategy to engineer high-performance mammalian Whole-Cell Biosensors bioRxiv. Synth. Biol. Pub Date : 2024-03-01 Alessio Mallozzi, Virginia Fusco, Francesco Ragazzini, Arne Praznik, Roman Jerala, Diego di Bernardo
Transcription-based whole-cell biosensors (WCBs) are cells engineered with an analyte-responsive promoter driving the transcription of a reporter gene. WCBs can sense and report on bioactive molecules (analytes) relevant to human health. Designing an analyte-sensitive promoter requires a cumbersome trial-and-error approach and usually results in biosensors with poor performance. Here, we integrated
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Engineering Material Properties of Transcription Factor Condensates to Control Gene Expression in Mammalian Cells and Mice bioRxiv. Synth. Biol. Pub Date : 2024-03-01 Alexandra A.M. Fischer, Hanah B. Robertson, Deqiang Kong, Merlin M. Grimm, Jakob Grether, Johanna Groth, Carsten Baltes, Manfred Fliegauf, Franziska Lautenschlaeger, Bodo Grimbacher, Haifeng Ye, Volkhard Helms, Wilfried Weber
Phase separation of biomolecules into condensates is a key mechanism in the spatiotemporal organization of biochemical processes in cells. However, the impact of the material properties of biomolecular condensates on important processes, such as the control of gene expression, remains largely elusive. Here, we systematically tune the material properties of optogenetically induced transcription factor
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Statistical design of a synthetic microbiome that clears a multi-drug resistant gut pathogen bioRxiv. Synth. Biol. Pub Date : 2024-02-29 Rita Oliveira, Bipul Pandey, Kiseok Lee, Mahmoud Yousef, Robert Y. Chen, Conrad Triebold, Emma McSpadden, Fidel Haro, Valeryia Aksianiuk, Ramaswamy Ramanujam, Seppe Kuehn, Arjun Raman
Microbiomes perform critical functions across many environments on Earth1,2,3. However, elucidating principles of their design is immensely challenging4,5,6,7. Using a diverse bank of human gut commensal strains and clearance of multi-drug resistant Klebsiella pneumoniae as a target, we engineered a functional synthetic microbiome using a process that was agnostic to mechanism of action, bacterial
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Designing a hybrid in silico/in-cell controller robust to process-model mismatch associated with dynamically regulated enzyme overexpression bioRxiv. Synth. Biol. Pub Date : 2024-02-29 Tomoki Ohkubo, Yuichi Sakumura, Fuzhong Zhang, Katsuyuki Kunida
Discrepancy between model predictions and actual processes, known as process-model mismatch (PMM), remains a serious challenge in bioprocess optimization. Previously, we proposed the hybrid in silico/in-cell controller (HISICC) concept combining model-based optimization with cell-based feedback to address the PMM problem. Herein, this approach was advanced to regulate intracellular concentrations of
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Effect of translation enhancing nascent SKIK peptide on the arrest peptides containing consecutive Proline bioRxiv. Synth. Biol. Pub Date : 2024-02-29 Yuma Nishikawa, Riko Fujikawa, Hideo Nakano, Teruyo Ojima-Kato
Ribosome arrest peptides (RAPs) consisting of specific amino acid sequences such as SecM AP and WPPP containing consecutive proline, are known to cause translational stalling in Escherichia coli. This work shows that the translation enhancing SKIK peptide tag cancels the translational arrest by WPPP, and the closer the physical distance between the SKIK and WPPP, the greater the canceling effect in
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High-accuracy crRNA array assembly strategy for multiplex CRISPR bioRxiv. Synth. Biol. Pub Date : 2024-02-29 Xiangtong Zhao, Lixian Yang, Peng Li, Zijing Cheng, Yongshi Jia, Limin Luo, Aihong Bi, Hanchu Xiong, Haibo Zhang, Jinrui Zhang, Yaodong Zhang
Simultaneous targeting of multiple loci with CRISPR system, a tool known as multiplex CRISPR, offers us more feasibility to manipulate and elucidate the intricate and redundant endogenous networks underlying complex cellular functions. Owing to the versatility of the continuously emerging Cas nucleases and the utilization of CRISPR arrays, multiplex CRISPR has been implemented by numerous in vitro
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Dissecting the role of CAR signaling architectures on T cell activation and persistence using pooled screening and single-cell sequencing bioRxiv. Synth. Biol. Pub Date : 2024-02-26 Rocio Castellanos-Rueda, Kai-Ling K. Wang, Juliette L. Forster, Alice Driessen, Jessica A. Frank, Maria Rodriguez Martinez, Sai T. Reddy
Chimeric antigen receptor (CAR) T cells represent a promising approach for cancer treatment, yet challenges remain such as limited efficacy due to a lack of T cell persistence. Given its critical role in promoting and modulating T cell responses, it is crucial to understand how alterations in the CAR signaling architecture influence T cell function. Here, we designed a combinatorial CAR signaling domain
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Design and construction towards a pan-microbial toolkit bioRxiv. Synth. Biol. Pub Date : 2024-02-24 Charlie Gilbert, Alexander Crits-Christoph, Elise Ledieu-Dherbécourt, Shinyoung Clair Kang, Stephanie L Brumwell, Henry H Lee, Nili Ostrov
Establishing genetic tractability in non-model microbes requires identifying genetic parts that function in a target host. However, the paucity and purported narrow host range of available parts means that successful identification is governed by serendipity. Instead, a more comprehensive and scalable process would be desirable. Here, we describe the design principles for a pan-microbial genetic toolkit
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ProT-Diff: A Modularized and Efficient Approach to De Novo Generation of Antimicrobial Peptide Sequences through Integration of Protein Language Model and Diffusion Model bioRxiv. Synth. Biol. Pub Date : 2024-02-23 Xue-Fei Wang, Jing-Ya Tang, Han Liang, Jing Sun, Sonam Dorje, Bo Peng, Xu-Wo Ji, Zhe Li, Xian-En Zhang, Dian-Bing Wang
Antimicrobial Peptides (AMPs) represent a promising class of antimicrobial agents crucial for combating antibiotic-resistant pathogens. Despite the emergence of deep learning approaches for AMP discovery, there remains a gap in efficiently generating novel AMPs across various amino acid lengths without prior knowledge of peptide structures or sequence alignments. Here we introduce ProT-Diff, a modularized
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Optimising the production of dsRNA biocontrols in microbial systems using multiple transcriptional terminators. bioRxiv. Synth. Biol. Pub Date : 2024-02-23 Sebastian James Ross, Gareth Owen, James Hough, Annelies Philips, Wendy Maddelein, John Ray, Peter Kilby, Mark J. Dickman
Crop pests and pathogens annually cause over $100 billion in global crop damage, with insects consuming 5-20% of major grain crops. Current crop pest and disease control strategies rely on insecticidal and fungicidal sprays, plant genetic resistance, transgenes and agricultural practices. dsRNA is emerging as a novel sustainable method of plant protection as an alternative to traditional chemical pesticides
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Rapid generation and screening of transgenic black soldier fly (Hermetia illucens) bioRxiv. Synth. Biol. Pub Date : 2024-02-22 Chandran Pfitzner, Kate Tepper, Sheemal Kumar, Carly Retief, Justin McNab, Robert A Harrell, Maciej Maselko
Background: The black soldier fly (BSF), Hermetia illucens is a widely used, and mass-produced insect that fulfils an important role in both the management of organic waste and as a component of animal feed formulations. They also have significant potential as a platform for converting organic waste into high-value proteins, and lipids for the production of biofuels. Applying synthetic biology to BSF
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Ligify: Automated genome mining for ligand-inducible transcription factors bioRxiv. Synth. Biol. Pub Date : 2024-02-21 Simon d'Oelsnitz, Andrew D Ellington, David Ross
Prokaryotic transcription factors can be repurposed into biosensors for the ligand-inducible control of gene expression, but the landscape of chemical ligands for which biosensors exist is extremely limited. To expand this landscape, we developed Ligify, a web application that leverages information in enzyme reaction databases to predict transcription factors that may be responsive to user-defined
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Development of a whole-cell biosensor for ethylene oxide and ethylene bioRxiv. Synth. Biol. Pub Date : 2024-02-21 Claudia F Moratti, Sui Nin Nicholas Yang, Colin Scott, Nicholas V Coleman
Ethylene and ethylene oxide are widely used in the chemical industry, and ethylene is also important for its role in fruit ripening. Better sensing systems would assist risk management of these chemicals. Here, we characterise the ethylene regulatory system in Mycobacterium strain NBB4 and use these genetic parts to create a biosensor. The regulatory genes etnR1 and etnR2 and cognate promoter Petn
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Essential magnetosome proteins MamI and MamL from magnetotactic bacteria interact in mammalian cells bioRxiv. Synth. Biol. Pub Date : 2024-02-20 Qin Sun, Liu Yu, Sarah C Donnelly, Cecile Fradin, R Terry Thompson, Frank S Prato, Donna E Goldhawk
To detect cellular activities deep within the body using magnetic resonance platforms, magnetosomes are the ideal model of genetically-encoded nanoparticles. These membrane-bound iron biominerals produced by magnetotactic bacteria are highly regulated by approximately 30 genes; however, only a few magnetosome genes are essential and may constitute the root structure upon which biominerals form. To
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Optogenetic Regulation of EphA1 RTK Activation and Signaling bioRxiv. Synth. Biol. Pub Date : 2024-02-20 Anna I Wurz, Kevin S Zheng, Robert M Hughes
Eph receptors are ubiquitous class of transmembrane receptors that mediate cell-cell communication, proliferation, differentiation, and migration. EphA1 receptors specifically play an important role in angiogenesis, fetal development, and cancer progression; however, studies of this receptor can be challenging as its ligand, ephrinA1, binds and activates several EphA receptors simultaneously. Optogenetic
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Directed evolution of a bacterial leucyl tRNA in mammalian cells for enhanced noncanonical amino acid mutagenesis bioRxiv. Synth. Biol. Pub Date : 2024-02-20 Rachel L Huang, Delilah Jewel, Rachel E Kelemen, Quan Pham, Shu Wang, Soumya Jyoti Singha Roy, Zeyi Huang, Samantha D Levinson, Bharathi Sundaresh, Suyen Espinoza Miranda, Tim van Opijnen, Abhishek Chatterjee
The E. coli leucyl-tRNA synthetase (EcLeuRS)/tRNAEcLeu pair has been engineered to genetically encode a structurally diverse group of enabling noncanonical amino acids (ncAAs) in eukaryotes, including those with bioconjugation handles, environment-sensitive fluorophores, photocaged amino acids, and native post-translational modifications. However, the scope of this toolbox in mammalian cells is limited
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Chloroplast Cell-Free Systems from Different Plant Species as a Rapid Prototyping Platform bioRxiv. Synth. Biol. Pub Date : 2024-02-19 Clemens V. Boehm, Rene Inckemann, Michael Burgis, Jessica Baumann, Cedric K. Brinkmann, Katarzyna E. Lipinska, Sara Gilles, Jonas Freudigmann, Vinca Seiler, Lauren G. Clark, Michael C. Jewett, Lars M. Voll, Henrike Niederholtmeyer
Climate change poses a significant threat to global agriculture, necessitating innovative solutions. Plant synthetic biology, particularly chloroplast engineering, holds promise as a viable approach to this challenge. Chloroplasts present a variety of advantageous traits for genetic engineering, but the development of genetic tools and genetic part characterization in these organelles is hindered by
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A temperature-inducible protein module for control of mammalian cell fate bioRxiv. Synth. Biol. Pub Date : 2024-02-19 William Benman, Zikang Dennis Huang, Pavan Iyengar, Delaney Wilde, Thomas Mumford, Lukasz Bugaj
Inducible protein switches are used throughout the biosciences to allow on-demand control of proteins in response to chemical or optical inputs. However, these inducers either cannot be controlled with precision in space and time or cannot be applied in optically dense settings, limiting their application in tissues and organisms. Here we introduce a protein module whose active state can be reversibly
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A chloroplast cell-free system for measuring ribosome binding site strengths bioRxiv. Synth. Biol. Pub Date : 2024-02-18 Lauren Clark, Christopher Voigt, Michael C Jewett
Plastid engineering offers the potential to carry multi-gene traits in plants, however, it requires reliable genetic parts to balance expression. The difficulty of chloroplast transformation and slow plant growth make it challenging to build plants just to characterize genetic parts. To address these limitations, we developed a cell-free system from Nicotiana tabacum chloroplast extracts for prototyping
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Degron-based bioPROTACs for controlling signaling in CAR T cells bioRxiv. Synth. Biol. Pub Date : 2024-02-17 Matthew S Kim, Hersh K Bhargava, Gavin E Shavey, Wendell A Lim, Hana El-Samad, Andrew H Ng
Chimeric antigen receptor (CAR) T cells have made a tremendous impact in the clinic, but potent signaling through the CAR can be detrimental to treatment safety and efficacy. The use of protein degradation to control CAR signaling can address these issues in pre-clinical models. Existing strategies for regulating CAR stability rely on small molecules to induce systemic degradation. In contrast to small
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Ultra-sensitive water contaminant detection with transcription factor interfaced microcantilevers bioRxiv. Synth. Biol. Pub Date : 2024-02-15 Dilip K Agarwal, Tyler J Lucci, Jaeyoung K Jung, Ghajendra S Shekhawat, Julius B Lucks, Vinayak P B Dravid
Water contamination is a growing global concern, creating a need to develop technologies that can detect a range of target compounds at the required thresholds. Here, we address this need by merging biological allosteric transcription factors with DNA coated nanomechanical microcantilevers to detect chemicals in water with digital readout. After proof-of-concept demonstration and optimization to detect
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Development of a signal-integrating reporter to monitor mitochondria-ER contacts bioRxiv. Synth. Biol. Pub Date : 2024-02-14 Zheng Yang, David Chan
Mitochondria-ER contact sites (MERCS) serve as hotspots for important cellular processes, including calcium homeostasis, phospholipid homeostasis, mitochondria dynamics, and mitochondrial quality control. MERCS reporters based on complementation of GFP fragments have been designed to visualize MERCS in real-time, but we find that they do not accurately respond to changes in MERCS content. Here, we