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Impact of Chemical Dynamics of Commercial PURE Systems on Malachite Green Aptamer Fluorescence bioRxiv. Synth. Biol. Pub Date : 2024-03-16 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 varies in commercial PURE systems, leading to inaccurate RNA calculations. MGapt fluorescence is not solely linked to waste products or buffering
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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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Pangenomic landscapes shape the genetic circuit performance in a Stutzerimonas biodesign toolkit bioRxiv. Synth. Biol. Pub Date : 2024-02-15 Dennis Tin Chat Chan, Hans C. Bernstein
Implementation of identical biodesign strategies into different species often results in different performance, a process called the chassis effect. Our current understanding of how cellular host context underpins its ability to be engineered is lacking and closing this knowledge gap will greatly improve the rational design of microorganisms. Here, we combined global differential gene expression analysis
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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
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A spring-loaded and leakage-tolerant synthetic gene switch for in-vitro detection of DNA and RNA bioRxiv. Synth. Biol. Pub Date : 2024-02-14 Krishna Gupta, Elisha Krieg
Nucleic acid tests (NATs) are essential for biomedical diagnostics. Traditional NATs, often complex and expensive, have prompted the exploration of Toehold-Mediated Strand Displacement (TMSD) circuits as an economical alternative. However, the wide application of TMSD-based reactions is limited by leakage-the spurious activation of the reaction leading to high background signals and false positives
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Mechanism Study on the Effect of Kidney Tonifying and Liver Soothing Methods on Mouse Oocyte Quality by Regulating OSFs and Smads Pathways bioRxiv. Synth. Biol. Pub Date : 2024-02-14 Ruijuan Zhang, Rui Jia, Jing Bai, Huilan Du, Liyun Yang
Objective: To observe the effects of kidney tonifying and liver soothing methods on the secretion of factor BMP-6, related receptors ALK-2/6, and downstream Smads pathway Smad1/5/844 in oocytes, and to explore the targeted mechanisms of their effects on mouse oocyte quality; Method: Healthy female mice aged 6-7 weeks were randomly divided into 6 groups, namely high and low dose kidney tonifying groups;
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Breakthrough in Dicot Prime Editing: Enabling Heritable Desired Edits in Tomato and Arabidopsis bioRxiv. Synth. Biol. Pub Date : 2024-02-11 Tien Van Vu, Ngan Thi Nguyen, Jihae Kim, Young Jong Song, Thu Hoai Nguyen, Jae-Yean Kim
Prime editing (PE) enables almost all types of precise genome editing in animals and plants. It has been successfully adapted to edit several plants at variable efficiency and versatility. However, this technique is inefficient for dicots for unknown reasons. Here, by employing novel combinations of PE components, including an RNA chaperone and modified epegRNAs driven by a PolII-PolIII composite promoter
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Infection-on-Chip: an in vitro human vessel to study Neisseria meningitidis colonization and vascular damages bioRxiv. Synth. Biol. Pub Date : 2024-02-10 Lea Pinon, Melanie Chabaud, Pierre Nivoit, Jerome Wong-Ng, Tri-Tho Nguyen, Vanessa Paul, Sylvie Goussard, Emmanuel Frachon, Dorian Obino, Samy Gobaa, Guillaume Dumenil
Bloodstream infections leading to sepsis, are a life-threatening condition and remain difficult to treat. Although necessary for the better understanding of the pathogenesis of this type of infection, in vitro experimental models that reflect their key features are still lacking. We here developed a photoablation-based 3-dimensional, microfluidic model of meningococcal vascular colonization, which
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Self-assembled cell-scale containers made from DNA origami membranes bioRxiv. Synth. Biol. Pub Date : 2024-02-09 Christoph Karfusehr, Markus Eder, Friedrich C. Simmel
DNA origami provides a methodology for the sequence-programmable generation of precisely defined molecular nanostructures with sizes of order 100 nm. A new frontier for the field is the generation of superstructures made from DNA origami subunits, which requires other self-assembly strategies than those used for DNA origami itself. Challenges faced by current approaches include the increasing complexity
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Model-guided metabolic engineering of curcuminoid Production in Pseudomonas putida bioRxiv. Synth. Biol. Pub Date : 2024-02-08 Maria Martin-Pascual, Sara Moreno-Paz, Rik P Van Rosmalen, Julia Dorigo, Francesca Demaria, Richard Van Kranenburg, Vitor Martins dos Santos, Maria S Suarez-Diez
Production of value-added, plant-derived compounds in microbes increasingly attracts commercially interest in food and pharmaceutical industries. However, plant metabolic pathways are complex, require a robust balance of enzymes, cofactors, ATP and other metabolites, and often result in low production when transplanted to bacteria. This is exemplified by the biosynthesis of curcuminoids from the Curcuma
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Thermogenetics for cardiac pacing bioRxiv. Synth. Biol. Pub Date : 2024-02-08 Alexander V. Balatskiy, Alexey M. Nesterenko, Aleksandr A. Lanin, Vera S. Ovechkina, Semyon S. Sabinin, Elena S. Fetisova, Alexander A. Moshchenko, David Jappy, Rostislav A. Sokolov, Diana Z. Biglova, Georgy M. Solius, Ekaterina M. Solius, Sergei V. Korolev, Oleg V. Podgorny, Ilya V Kelmanson, Andrei V. Rozov, Andrei B. Fedotov, Tobias Bruegmann, Alexei M. Zheltikov, Andrey A. Mozhaev, Vsevolod V.
Cardiac arrhythmias are common disorders that can be fatal. Modern methods of treating bradyarrhythmias include the implantation of pacemakers and cardioverters - defibrillators. However, the implantable devices can cause various complications including infectious ones, related to the electrodes installed inside the heart. Less invasive heart rhythm modulation could be beneficial for some cohorts of
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Engineering an anti-CD206-synNotch receptor: insights into the development of novel synthetic receptors bioRxiv. Synth. Biol. Pub Date : 2024-02-07 Sofija Semeniuk, Binzhi Qian, Elise Cachat
Immune cells play a pivotal role in the establishment, growth and progression of tumors at primary and metastatic sites. Macrophages, in particular, play a critical role in suppressing immune responses and promoting an anti-inflammatory environment through both direct and indirect cell-cell interactions. However, our understanding of the mechanisms underlying such interactions is limited due to a lack
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DNA representations and generalization performance of sequence-to-expression models bioRxiv. Synth. Biol. Pub Date : 2024-02-07 Yuxin Shen, Grzegorz Kudla, Diego A Oyarzun
The increasing demand for biological products drives many efforts to engineer cells that produce heterologous proteins at maximal yield. Recent advances in massively parallel reporter assays can deliver data suitable for training machine learning models and support the design of microbial strains with optimized protein expression phenotypes. The best performing sequence-to-expression models have been
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Ultra-high efficiency T cell reprogramming at multiple loci with SEED-Selection bioRxiv. Synth. Biol. Pub Date : 2024-02-07 Christopher R Chang, Vivasvan S Vykunta, Daniel B Goodman, Joseph J Muldoon, William A Nyberg, Chang Liu, Vincent Allain, Allison Rothrock, Charlotte H Wang, Alexander Marson, Brian R Shy, Justin Eyquem
Multiplexed reprogramming of T cell specificity and function can generate powerful next-generation cellular therapies. However, current manufacturing methods produce heterogenous mixtures of partially engineered cells. Here, we develop a one-step process to enrich for unlabeled cells with knock-ins at multiple target loci using a family of repair templates named Synthetic Exon/Expression Disruptors
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Enhancing Plant Photosynthesis using Carbon Dots as Light Converter and Photosensitizer bioRxiv. Synth. Biol. Pub Date : 2024-02-07 Haitao Hu, Wenbo Cheng, Xueyun Wang, Yu Yang, Xuemeng Yu, Jianwei Ding, Yiliang Lin, Wei Zhao, Qiao Zhao, Rodrigo Ledesma-Amaro, Xihan Chen, Junzhong Liu, Chen Yang, Xiang Gao
Improving photosynthetic efficiency is pivotal for CO2-based biomanufacturing and agriculture purposes. Despite the progress on photosynthetic biohybrids integrating biocatalysts with synthetic materials, nanomaterials with improved optical and photoelectrochemical properties are still needed to increase the energy-conversion efficiency. Here, we present a novel approach using carbon dots (CDs) as
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Cellular distribution and motion of essential magnetosome proteins expressed in mammalian cells bioRxiv. Synth. Biol. Pub Date : 2024-02-07 Qin Sun, Cecile Fradin, R Terry Thompson, Frank S Prato, Donna E Goldhawk
Magnetosomes are organelle-like structures within magnetotactic bacteria that store iron biominerals in membrane-bound vesicles. In the bacteria, formation of these structures is highly regulated by approximately 30 genes which are conserved throughout different species. To compartmentalize iron in mammalian cells for magnetic resonance imaging using gene-based contrast, we are introducing key magnetosome
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Enhanced Sequence-Activity Mapping and Evolution of Artificial Metalloenzymes by Active Learning bioRxiv. Synth. Biol. Pub Date : 2024-02-06 Tobias Vornholt, Mojmír Mutný, Gregor W. Schmidt, Christian Schellhaas, Ryo Tachibana, Sven Panke, Thomas R. Ward, Andreas Krause, Markus Jeschek
Tailored enzymes hold great potential to accelerate the transition to a sustainable bioeconomy. Yet, enzyme engineering remains challenging as it relies largely on serendipity and is, therefore, highly laborious and prone to failure. The efficiency and success rates of engineering campaigns may be improved substantially by applying machine learning to construct a comprehensive representation of the
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In-vivo distributed multicellular control of gene expression in microbial consortia bioRxiv. Synth. Biol. Pub Date : 2024-02-05 Davide Salzano, Barbara Shannon, Claire Grierson, Lucia Marucci, Nigel Savery, Mario di Bernardo
This paper presents an innovative approach in synthetic biology, focusing on the engineering of biomolecular feedback control systems within microbial consortia to achieve robust and precise regulation of desired phenotypes. Traditional biomolecular control strategies, while effective, are predominantly confined to single-cell applications, limiting their complexity and adaptability due to metabolic
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Functional Modification of Cyanobacterial Phycobiliprotein and Phycobilisomes through Bilin Metabolism Control bioRxiv. Synth. Biol. Pub Date : 2024-02-04 Mizuho Sato, Takeshi Kawaguchi, Kaisei Maeda, Mai Watanabe, Masahiko Ikeuchi, Rei Narikawa, Satoru Watanabe
The light-harvesting antenna complexes in cyanobacteria are known as phycobilisomes (PBSs), and they can adapt to a diverse range of light environments owing to the deployment of different phycobiliproteins within the PBS structures. Freshwater cyanobacteria such as Synechococcus elongatus PCC 7942 thrive under red light due to phycocyanin (PC), along with phycocyanobilin (PCB), in PBS. Conversely
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DNA-Diffusion: Leveraging Generative Models for Controlling Chromatin Accessibility and Gene Expression via Synthetic Regulatory Elements bioRxiv. Synth. Biol. Pub Date : 2024-02-01 Lucas Ferreira DaSilva, Simon Senan, Zain Munir Patel, Aniketh Janardhan Reddy, Sameer Gabbita, Zach Nussbaum, Cesar Miguel Valdez Cordova, Aaron Wenteler, Noah Weber, Tin M. Tunjic, Talha Ahmad Khan, Zelun Li, Cameron Ray Smith, Matei Bejan, Lithin Karmel Louis, Paola Cornejo, Will Connell, Emily S. Wong, Wouter Meuleman, Luca Pinello
The challenge of systematically modifying and optimizing regulatory elements for precise gene expression control is central to modern genomics and synthetic biology. Advancements in generative AI have paved the way for designing synthetic sequences with the aim of safely and accurately modulating gene expression. We leverage diffusion models to design context-specific DNA regulatory sequences, which
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AC-BioSD : A biomolecular signal differentiator module with enhanced performance bioRxiv. Synth. Biol. Pub Date : 2024-01-30 Emmanouil Alexis, Jose 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 signal differentiation by introducing a novel biomolecular topology, termed AC-BioSD. Its structure allows for insensitivity to input signal changes and high precision in terms of signal differentiation
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CRISPR-Enabled Autonomous Transposable Element (CREATE) for RNA-based gene editing and delivery bioRxiv. Synth. Biol. Pub Date : 2024-01-30 Yuxiao Wang, Ruei-Zeng Lin, Meghan Harris, Bianca Lavayen, Bruce Mccreedy, Daniel Getts
To realize the potential of genome editing for broad therapeutic applications, new tools are needed for delivery of multi-kilobase (kb) payloads at desired genomic sites with high precision. Here we introduce the CRISPR-Enabled Autonomous Transposable Element (CREATE), an RNA-based genome editing system that merges CRISPR/Cas9 with the human L1 retrotransposon to insert gene-sized payloads without
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The integration of tandem gene repeats via a bacterial type-II toxin-antitoxin-mediated gene amplification (ToxAmp) system and stability visualisation in Saccharomyces cerevisiae bioRxiv. Synth. Biol. Pub Date : 2024-01-30 Samuel Evans, Zeyu Lu, Liam McDonnell, Will Anderson, Francisco Peralta, Tyson Watkins, Hafna Ahmed, Carlos Horacio Luna-Flores, Thomas D Loan, Laura Navone, Matt Trau, Colin Scott, Robert Speight, Claudia Vickers, Bingyin Peng
Tandem gene repeats naturally occur as important genomic features and determine many traits in living organisms, like human diseases and microbial productivities of target bioproducts. Here, we develop a bacterial type-II toxin-antitoxin-mediated method to manipulate genomic integration of tandem gene repeats in Saccharomyces cerevisiae and further visualise the evolutionary trajectories of gene repeats