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Design of a self-regulating mRNA gene circuit bioRxiv. Synth. Biol. Pub Date : 2024-04-23 Eric C Dykeman
Protein expression from mRNA in vivo is predominately controlled via regulatory feedback mechanisms that adjust the level of mRNA transcription. However, for positive sense single-stranded RNA viruses, protein expression is often controlled via secondary structural elements, such as internal ribosomal entry sites, that are encoded within the mRNA. The self-regulation of mRNA translation observed in
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Towards AI-designed genomes using a variational autoencoder bioRxiv. Synth. Biol. Pub Date : 2024-04-23 Natasha K Dudek, Doina Precup
Genomes serve as the blueprints for life, encoding elaborate networks of genes whose products must seamlessly interact to support living organisms. Humans' capacity to understand biological systems from scratch is limited by their sheer size and complexity. In this work, we develop a framework for training a machine learning algorithm to learn the basic genetic principles that underlie genome composition
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Structure-Infused Protein Language Models bioRxiv. Synth. Biol. Pub Date : 2024-04-23 Daniel Penaherrera, David Ryan Koes
Embeddings from protein language models (PLMs) capture intricate patterns for protein sequences, enabling more accurate and efficient prediction of protein properties. Incorporating protein structure information as direct input into PLMs results in an improvement on the predictive ability of protein embeddings on downstream tasks. In this work we demonstrate that indirectly infusing structure information
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Synthetic bacteria with programmed cell targeting and protein injection suppress tumor growth in vivo bioRxiv. Synth. Biol. Pub Date : 2024-04-22 Alejandro Asensio-Calavia, Carmen Manas, Alba Cabrera-Fisac, Eva Pico-Sanchez, Elena M. Seco, Starsha Kolodziej, Daniel S. Leventhal, Jose Lora, Beatriz Alvarez, Luis Angel Fernandez
Bacterial living therapeutics (BLTs) hold promise for treating cancer and other human diseases because they can be engineered and transported into the microbiota (e.g., of tumors, gastrointestinal tract) to deliver therapeutic payloads. Current approaches rely on the natural tropism of the bacterial chassis used and trigger the local release of protein cargoes, typically through active extracellular
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Simulations Predict Stronger CRISPRi Transcriptional Repression in Plants for Identical than Heterogeneous gRNA Target Sites bioRxiv. Synth. Biol. Pub Date : 2024-04-22 Helen G Scott, Alessandro Occhialini, Scott C Lenaghan, Jacob Beal
Plant synthetic biologists have been working to adapt the CRISPRa and CRISPRi promoter regulation methods for applications such as improving crops or installing other valuable pathways. With other organisms, strong transcriptional control has typically required multiple gRNA target sites, which poses a critical engineering choice between heterogeneous sites, which allow each gRNA to target existing
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Design of highly functional genome editors by modeling the universe of CRISPR-Cas sequences bioRxiv. Synth. Biol. Pub Date : 2024-04-22 Jeffrey A. Ruffolo, Stephen Nayfach, Joseph Gallagher, Aadyot Bhatnagar, Joel Beazer, Riffat Hussain, Jordan Russ, Jennifer Yip, Emily Hill, Martin Pacesa, Alexander J. Meeske, Peter Cameron, Ali Madani
Gene editing has the potential to solve fundamental challenges in agriculture, biotechnology, and human health. CRISPR-based gene editors derived from microbes, while powerful, often show significant functional tradeoffs when ported into non-native environments, such as human cells. Artificial intelligence (AI) enabled design provides a powerful alternative with potential to bypass evolutionary constraints
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Systematic genome-wide discovery of host factors governing bacteriophage infectivity bioRxiv. Synth. Biol. Pub Date : 2024-04-21 Chutikarn Chitboonthavisuk, Cody Martin, Phil Huss, Jason M. Peters, Karthik Anantharaman, Srivatsan Raman
Bacterial host factors regulate the infection cycle of bacteriophages. Except for some well-studied host factors (e.g., receptors or restriction-modification systems), the contribution of the rest of the host genome on phage infection remains poorly understood. We developed PHAGEPACK, a pooled assay that systematically and comprehensively measures each host-gene impact on phage fitness. PHAGEPACK combines
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Highly multiplexed design of an allosteric transcription factor to sense novel ligands bioRxiv. Synth. Biol. Pub Date : 2024-04-21 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
Allosteric transcription factors (aTF), widely used as biosensors, have proven challenging to design for detecting novel molecules because mutation of ligand-binding residues often disrupts allostery. We developed Sensor-seq, a high-throughput platform to design and identify aTF biosensors that bind to non-native ligands. We screened a library of 17,737 variants of the aTF TtgR, a regulator of a multidrug
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Evolution engineering of methylotrophic E. coli enables faster growth than native methylotrophs bioRxiv. Synth. Biol. Pub Date : 2024-04-18 Liang-Yu Nieh, Frederic Y.-H. Chen, Hsin-Wei Jung, Kuan-Yu Su, Chao-Yin Tsuei, Chun Ting Lin, Yue-Qi Lee, James C. Liao
As methanol can be derived from either CO2 or methane, methanol economy may play a role in combating climate change. In this scenario, rapid utilization of methanol by an industrial microorganism is the first and crucial step for efficient utilization of the C1 feedstock chemical. Here, we report the development of a methylotrophic E. coli strain (SM6) with a doubling time of 3.5 hours, outpacing that
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A cell-free system for functional studies of small membrane proteins bioRxiv. Synth. Biol. Pub Date : 2024-04-18 Shan Jiang, Gulce Celen, Timo Glatter, Henrike Niederholtmeyer, Jing Yuan
Numerous small proteins have been discovered across all domains of life, among which many are hydrophobic and predicted to localize to the cell membrane. Based on a few that are well-studied, small membrane proteins are regulators involved in various biological processes, such as cell signaling, nutrient transport, drug resistance, and stress response. However, the function of most identified small
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Targeted protein degradation systems to enhance Wnt signaling bioRxiv. Synth. Biol. Pub Date : 2024-04-17 Parthasarathy Sampathkumar, Heekyung Jung, Hui Chen, Zhengjian Zhang, Nicholas Suen, Yiran Yang, Zhong Huang, Tom Lopez, Robert Benisch, Sung-Jin Lee, Jay Ye, Wen-Chen Yeh, Yang Li
Molecules that facilitate targeted protein degradation (TPD) offer great promise as novel therapeutics. The human hepatic lectin asialoglycoprotein receptor (ASGR) is selectively expressed on hepatocytes. We have previously engineered an anti-ASGR1 antibody-mutant RSPO2 (RSPO2RA) fusion protein (called SWEETSTM) to drive tissue-specific degradation of ZNRF3/RNF43 E3-ubiquitin ligases, which achieved
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Synthetic G protein-coupled receptors for programmable sensing and control of cell behavior bioRxiv. Synth. Biol. Pub Date : 2024-04-15 Nicholas A Kalogriopoulos, Reika Tei, Yuqi Yan, Matthew Ravalin, Yulong Li, Alice Y Ting
Synthetic receptors that mediate antigen-dependent cell responses are transforming therapeutics, drug discovery, and basic research. However, established technologies such as chimeric antigen receptors (CARs) can only detect immobilized antigens, have limited output scope, and lack built-in drug control. Here, we engineer synthetic G protein-coupled receptors (GPCRs) capable of driving a wide range
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Engineering sequestration-based biomolecular classifiers with shared resources bioRxiv. Synth. Biol. Pub Date : 2024-04-15 Hossein Moghimianavval, Ignacio Gispert, Santiago R Castillo, Olaf B. W. H. Corning, Allen P Liu, Christian Cuba Samaniego
Constructing molecular classifiers that enable cells to recognize linear and non-linear input patterns would expand the biocomputational capabilities of engineered cells, thereby unlocking their potential in diagnostics and therapeutic applications. While several biomolecular classifier schemes have been designed, the effect of biological constraints such as resource limitation and competitive binding
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Tunable Universal OR-gated CAR T cells for AML bioRxiv. Synth. Biol. Pub Date : 2024-04-14 Menna Y Siddiqui, Jingyao Chen, Madeline Loffredo, Seunghee Lee, Han Deng, Yongshuai Li, Nelia Leemans, Tim Lu, Brian S Garrison, Marcela Guzmán Ayala, Nicholas W Frankel, Wilson W Wong
Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by antigen heterogeneity and poor prognosis. A potential therapeutic approach to address this heterogeneity is targeting multiple surface antigens to prevent antigen escape and relapse. Chimeric antigen receptor (CAR) T cells are an adoptive cell therapy that have demonstrated remarkable clinical success in the treatment of B
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Towards a Dataset for State of the Art Protein Toxin Classification bioRxiv. Synth. Biol. Pub Date : 2024-04-14 Chance A Challacombe, Nikhil S Haas
In-silico toxin classification assists in industry and academic endeavors and is critical for biosecurity. For instance, proteins and peptides hold promise as therapeutics for a myriad of conditions, and screening these biomolecules for toxicity is a necessary component of synthesis. Additionally, with the expanding scope of biological design tools, improved toxin classification is essential for mitigating
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Engineering inducible signaling receptors to enable erythropoietin-free erythropoiesis bioRxiv. Synth. Biol. Pub Date : 2024-04-12 Aadit P. Shah, Kiran R. Majeti, Freja K. Ekman, Sridhar Selvaraj, Eric Soupene, Prathamesh Chati, Roshani Sinha, Sofia E. Luna, Carsten T. Charlesworth, Travis McCreary, Benjamin J. Lesch, Devesh Sharma, Simon N. Chu, Matthew H. Porteus, M. Kyle Cromer
Blood transfusion plays a vital role in modern medicine. However, availability is contingent on donated blood, and frequent shortages pose a significant healthcare challenge. Ex vivo manufacturing of red blood cells (RBCs) derived from universal donor O-negative pluripotent stem cells emerges as a solution, yet the high cost of recombinant cytokines required for ex vivo erythroid differentiation remains
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A molecular proximity sensor based on an engineered, dual-component guide RNA bioRxiv. Synth. Biol. Pub Date : 2024-04-10 Junhong Choi, Wei Chen, Hanna Liao, Xiaoyi Li, Jay Shendure
One of the goals of synthetic biology is to enable the design of arbitrary molecular circuits with programmable inputs and outputs. Such circuits bridge the properties of electronic and natural circuits, processing information in a predictable manner within living cells. Genome editing is a potentially powerful component of synthetic molecular circuits, whether for modulating the expression of a target
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Allele Sails: launching traits and fates into wild populations with Mendelian DNA sequence modifiers bioRxiv. Synth. Biol. Pub Date : 2024-04-10 Michelle L. Johnson, Bruce A. 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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Modelling dynamic host-pathway interactions at the genome scale bioRxiv. Synth. Biol. Pub Date : 2024-04-09 Charlotte Merzbacher, Oisin Mac Aodha, Diego A. Oyarzún
Pathway engineering offers a promising avenue for sustainable chemical production. The design of efficient production systems requires understanding complex host-pathway interactions that shape the metabolic phenotype. While genome-scale metabolic models are widespread tools for studying static host-pathway interactions, it remains a challenge to predict dynamic effects such as metabolite accumulation
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Measuring the burden of hundreds of BioBricks defines an evolutionary limit on constructability in synthetic biology bioRxiv. Synth. Biol. Pub Date : 2024-04-08 Noor Radde, Genevieve A. Mortensen, Diya Bhat, Shireen Shah, Joseph J. Clements, Sean P. Leonard, Matthew J. McGuffie, Dennis M. Mishler, Jeffrey E. Barrick
Engineered DNA will slow the growth of a host cell if it redirects limiting resources or otherwise interferes with homeostasis. Populations of engineered cells can rapidly become dominated by “escape mutants” that evolve to alleviate this burden by inactivating the intended function. Synthetic biologists working with bacteria rely on genetic parts and devices encoded on plasmids, but the burden of
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Microdroplet screening rapidly profiles a biocatalyst to enable its AI-assisted engineering bioRxiv. Synth. Biol. Pub Date : 2024-04-08 Maximilian Gantz, Simon V. Mathis, Friederike E. H. Nintzel, Paul J. Zurek, Tanja Knaus, Elie Patel, Daniel Boros, Friedrich-Maximilian Weberling, Matthew R. A. Kenneth, Oskar J. Klein, Elliot J. Medcalf, Jacob Moss, Michael Herger, Tomasz S. Kaminski, Francesco G. Mutti, Pietro Lio, Florian Hollfelder
Engineering enzyme biocatalysts for higher efficiency is key to enabling sustainable, ‘green’ production processes for the chemical and pharmaceutical industry. This challenge can be tackled from two angles: by directed evolution, based on labor-intensive experimental testing of enzyme variant libraries, or by computational methods, where sequence-function data are used to predict biocatalyst improvements
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RNA language models predict mutations that improve RNA function bioRxiv. Synth. Biol. Pub Date : 2024-04-06 Yekaterina Shulgina, Marena I. Trinidad, Conner J. Langeberg, Hunter Nisonoff, Seyone Chithrananda, Petr Skopintsev, Amos J. Nissley, Jaymin Patel, Ron S. Boger, Honglue Shi, Peter H. Yoon, Erin E Doherty, Tara Pande, Aditya M. Iyer, Jennifer A. Doudna, Jamie H. D. Cate
Structured RNA lies at the heart of many central biological processes, from gene expression to catalysis. While advances in deep learning enable the prediction of accurate protein structural models, RNA structure prediction is not possible at present due to a lack of abundant high-quality reference data. Furthermore, available sequence data are generally not associated with organismal phenotypes that
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Ribozyme-mediated gene-fragment complementation for non-destructive reporting of DNA transfer within soil bioRxiv. Synth. Biol. Pub Date : 2024-04-06 Malyn A. Selinidis, Andrew C Corliss, James Chappell, Jonathan J Silberg
Enzymes that produce volatile metabolites can be coded into genetic circuits to report non-disruptively on microbial behaviors in hard-to-image soils. However, these enzyme reporters remain challenging to apply in gene transfer studies due to leaky off states that can lead to false positives. To overcome this problem, we designed a reporter that uses ribozyme-mediated gene-fragment complementation
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A conserved phenylalanine motif among Teleost fish provides insight for improving electromagnetic perception. bioRxiv. Synth. Biol. Pub Date : 2024-04-05 Brianna Ricker, E. Alejandro Castellanos Franco, Gustavo de los Campos, Galit Pelled, Assaf A. Gilad
Magnetoreceptive biology as a field remains relatively obscure; compared to the breadth of species believed to sense magnetic fields, it remains under-studied. Here, we present grounds for the expansion of magnetoreception studies among Teleosts. We begin with the electromagnetic perceptive gene (EPG) from Kryptopterus vitreolus and expand to identify 72 Teleosts with homologous proteins containing
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Biology System Description Language (BiSDL): a modeling language for the design of multicellular synthetic biological systems bioRxiv. Synth. Biol. Pub Date : 2024-04-05 Leonardo Giannantoni, Roberta Bardini, Alessandro Savino, Stefano Di Carlo
Background. The Biology System Description Language (BiSDL) is an accessible, easy-to-use computational language for multicellular synthetic biology. It allows synthetic biologists to represent spatiality and multi-level cellular dynamics inherent to multicellular designs, filling a gap in the state of the art. Developed for designing and simulating spatial, multicellular synthetic biological systems
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Massively parallel identification of single-cell immunophenotypes bioRxiv. Synth. Biol. Pub Date : 2024-04-04 Martin Cienciala, Laura Alvarez, Laura Berne, David Chena, Pavel Fikar, Monika Holubova, Hynek Kasl, Daniel Lysak, Mona Luo, Zuzana Novackova, Sheyla Ordonez, Zuzana Sramkova, Tomas Vlas, Daniel Georgiev
Translating insights from single-cell analysis into actionable indicators of health and disease requires large-scale confirmatory studies. We introduce biocytometry, a novel method utilizing engineered bioparticles for multiparametric immunophenotyping in suspension, enabling simultaneous measurement across thousands of assays with single-cell sensitivity and a wide dynamic range (1 to 1,000 target
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Alternate conformational trajectories in protein synthesis bioRxiv. Synth. Biol. Pub Date : 2024-04-04 Jose L Alejo, Dylan Girodat, Michael J Hammerling, Jessica A Willi, Michael C Jewett, Aaron E Engelhart, Katarzyna P Adamala
Translocation in protein synthesis entails the efficient and accurate movement of the mRNA-[tRNA]2 substrate through the ribosome after peptide bond formation. An essential conformational change during this process is the swiveling of the small subunit head domain about two rRNA hinge elements. Using directed evolution and molecular dynamics simulations, we derive alternate hinge elements capable of
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AC-BioSD : A biomolecular signal differentiator module with enhanced performance (extended version) bioRxiv. Synth. Biol. Pub Date : 2024-04-03 Emmanouil Alexis, José L. Avalos, Luca Cardelli, Antonis Papachristodoulou
Temporal gradient estimation is a pervasive phenomenon in natural biological systems and holds great promise for synthetic counterparts with broad-reaching applications. Here, we advance the concept of BioSD (Biomolecular Signal Differentiators) by introducing a novel biomolecular topology, termed Autocatalytic-BioSD or AC-BioSD. Its structure allows for insensitivity to input signal changes and high
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Towards self-regeneration: exploring the limits of protein synthesis in the PURE cell-free transcription-translation system bioRxiv. Synth. Biol. Pub Date : 2024-04-03 Ragunathan Bava Ganesh, Sebastian J. Maerkl
Self-regeneration is a key function of living systems that needs to be recapitulated in vitro to create a living synthetic cell. A major limiting factor for protein self-regeneration in the PURE cell-free transcription-translation system is its high protein concentration, which far exceed the system’s protein synthesis rate. Here we were able to drastically reduce the non-ribosomal PURE protein concentration
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Agrochemical control of gene expression using evolved split RNA polymerase. II bioRxiv. Synth. Biol. Pub Date : 2024-04-02 Yuan Yuan, Jin Miao
Agrochemical inducible gene expression system provides cost-effective and orthogonal control of energy and information flow in bacterial cells. However, the previous version of Mandipropamid inducible gene expression system (Mandi-T7) became constitutively active at room temperature. We moved the split site of the eRNAP from position LYS179 to position ILE109. This new eRNAP showed proximity dependence
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Random adversarial threshold search enables automated DNA screening bioRxiv. Synth. Biol. Pub Date : 2024-04-02 Dana Gretton, Brian Wang, Rey Edison, Leonard Foner, Jens Berlips, Theia Vogel, Martin Kysel, Walther Chen, Francesca Sage-Ling, Lynn Van Hauwe, Stephen Wooster, Benjamin Weinstein-Raun, Erika A. DeBenedictis, Andrew B. Liu, Emma Chory, Hongrui Cui, Xiang Li, Jiangbin Dong, Andres Fabrega, Christianne Dennison, Otilia Don, Cassandra Tong Ye, Kaveri Uberoy, Ronald L. Rivest, Mingyu Gao, Yu Yu, Carsten
Custom DNA synthesis underpins modern biology, but hazardous genes in the wrong hands could threaten many lives and public trust in science. In 1992, a virology-trained mass murderer tried and failed to obtain physical samples of Ebola; today, viruses can be assembled from synthetic DNA fragments. Screening orders for hazards is unreliable and expensive because similarity search algorithms yield false
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Synthetic coevolution reveals adaptive mutational trajectories of neutralizing antibodies and SARS-CoV-2 bioRxiv. Synth. Biol. Pub Date : 2024-04-01 Roy A. Ehling, Mason Minot, Max D. Overath, Daniel J. Sheward, Jiami Han, Beichen Gao, Joseph M. Taft, Margarita Pertseva, Cédric R. Weber, Lester Frei, Thomas Bikias, Ben Murrell, Sai T. Reddy
The Covid-19 pandemic showcases a coevolutionary race between the human immune system and SARS-CoV-2, mirroring the Red Queen hypothesis of evolutionary biology. The immune system generates neutralizing antibodies targeting the SARS-CoV-2 spike protein’s receptor binding domain (RBD), crucial for host cell invasion, while the virus evolves to evade antibody recognition. Here, we establish a synthetic
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Efficient Simulation of Viral Transduction and Propagation for Biomanufacturing bioRxiv. Synth. Biol. Pub Date : 2024-03-31 Francesco Destro, Richard D. Braatz
Viral transduction is a main route for gene transfer to producer cells in biomanufacturing. Designing a transduction-based biomanufacturing process poses significant challenges, due to the complex dynamics of viral infection and virus-host interaction. This article introduces a software toolkit composed of a multiscale model and an efficient numeric technique that can be leveraged for determining genetic
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The CRISPRaTOR: a biomolecular circuit for Automatic Gene Regulation in Mammalian Cells with CRISPR technology bioRxiv. Synth. Biol. Pub Date : 2024-03-30 Alessio Mallozzi, Virginia Fusco, Francesco Ragazzini, Diego di Bernardo
We introduce the CRISPRaTOR, a biomolecular circuit for precise control of gene expression in mammalian cells. The CRISPRaTOR leverages the stochiometric interaction between the artificial transcription factor VPR-dCas9, and the anti-CRISPR protein AcrIIA4, enhanced with synthetic coiled-coil domains to boost their interaction, to maintain the expression of a reporter protein constant across diverse
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Effect of translation enhancing nascent SKIK peptide on the arrest peptides containing consecutive Proline bioRxiv. Synth. Biol. Pub Date : 2024-03-29 Yuma Nishikawa, Riko Fujikawa, Hideo Nakano, Takashi Kanamori, Teruyo Ojima-Kato
Ribosome arrest peptides (RAPs) such as SecM arrest peptide (SecM AP) and WPPP with consecutive Pro residues, are known to induce translational stalling in Escherichia coli. We demonstrate that the translation enhancing SKIK peptide tag, which consisted of four amino acid residues Ser-Lys-Ile-Lys, effectively alleviate translational arrest caused by WPPP. Moreover, the proximity between SKIK and WPPP
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From resonance to chaos: modulating spatiotemporal patterns through a synthetic optogenetic oscillator bioRxiv. Synth. Biol. Pub Date : 2024-03-29 Jung Hun Park, Gábor Holló, Yolanda Schaerli
Oscillations are a recurrent phenomenon in biological systems across scales, including circadian clocks, metabolic oscillations and embryonic genetic oscillators. Despite their fundamental significance in biology, deciphering core principles of biological oscillators is very challenging due to the multiscale complexity of genetic networks and the difficulty in perturbing organisms in vivo. In this
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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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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