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Open Access
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Article
| Open AccessEngineering a synthetic gene circuit for high-performance inducible expression in mammalian systems
Inducible gene expression systems can be used to control the expression of a gene of interest by means of small molecules. Here the authors present CASwitch, a synthetic gene circuit platform enhancing inducible gene expression systems by reducing leakiness and boosting fold induction, for real world applications like gene therapy vector production and biosensors.
- Giuliano De Carluccio
- , Virginia Fusco
- & Diego di Bernardo
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Article
| Open AccessATP-free in vitro biotransformation of starch-derived maltodextrin into poly-3-hydroxybutyrate via acetyl-CoA
Several in vitro synthetic enzymatic biosystems (ivSEBs) to produce poly-3-hydroxybutyrate (PHB) via acetyl-coenzyme A (acetyl-CoA) have been reported, but suffer from complicated operation procedures, low yields, and/or dependence on costly ATP. Here, the authors report the design of an ATP-free ivSEB for one-pot, high-yield PHB biosynthesis via acetyl-CoA utilizing starch-derived maltodextrin as the sole substrate.
- Xinlei Wei
- , Xue Yang
- & Chun You
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Perspective
| Open AccessEngineering biology and climate change mitigation: Policy considerations
Engineering biology is a dynamic field that uses gene editing, synthesis, assembly, and engineering to design new or modified biological systems. Here the authors discuss the policy considerations and interventions needed to support a role for engineering biology in climate change mitigation.
- Jonathan Symons
- , Thomas A. Dixon
- & Isak S. Pretorius
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Article
| Open AccessEngineering intelligent chassis cells via recombinase-based MEMORY circuits
The unification of decision-making, communication, and memory would enable the programming of intelligent biotic systems. Here, the authors achieve this goal by engineering E. coli chassis cells with an array of inducible recombinases that mediate diverse genetic programs.
- Brian D. Huang
- , Dowan Kim
- & Corey J. Wilson
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Article
| Open AccessA universal system for boosting gene expression in eukaryotic cell-lines
Production of proteins at scale and affordable cost has been a major need of the biotech sector for the last several decades. Here the authors present a design algorithm called UNILIB for boosting gene expression in eukaryotic cells developed using an oligo-library and machine learning approach, validated in both yeast and mammalian cells using unseen sequences.
- Inbal Vaknin
- , Or Willinger
- & Roee Amit
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Article
| Open AccessSpyMask enables combinatorial assembly of bispecific binders
Bispecific antibody architecture is often important for function but rarely optimized. Here, authors present a modular approach to assemble bispecifics in varied formats using a SpyTag/SpyCatcher approach called SpyMask, and build anti-HER2 bispecifics whose activities depend on binder orientation and bispecific geometry.
- Claudia L. Driscoll
- , Anthony H. Keeble
- & Mark R. Howarth
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Article
| Open AccessAdvancing the scale of synthetic biology via cross-species transfer of cellular functions enabled by iModulon engraftment
Machine learning applied to large compendia of transcriptomic data has enabled the decomposition of bacterial transcriptomes to identify independently modulated sets of genes. Here the authors present iModulon-based engineering for precise identification of genes for cross-species function transfer to streamline synthetic biology for strain development and biomanufacturing.
- Donghui Choe
- , Connor A. Olson
- & Bernhard O. Palsson
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Article
| Open AccessCRISPR-powered quantitative keyword search engine in DNA data storage
Targeting the files containing content-of-interest is a challenge in DNA data storage. Here, the authors develop a CRISPR-powered search engine to quantitatively identify the keyword in files stored in DNA.
- Jiongyu Zhang
- , Chengyu Hou
- & Changchun Liu
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Article
| Open AccessA citric acid cycle-deficient Escherichia coli as an efficient chassis for aerobic fermentations
While tricarboxylic acid cycle (TCA cycle) is required for heterotrophic microbes, it reduces carbon yield of industrial products due to the release of excess CO2. Here, the authors construct an E. coli strain without a functional TCA cycle and demonstrate its feasibility as a chassis strain for production of four separate compounds.
- Hang Zhou
- , Yiwen Zhang
- & Baixue Lin
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Article
| Open AccessEdible mycelium bioengineered for enhanced nutritional value and sensory appeal using a modular synthetic biology toolkit
Fungi have the potential to produce sustainable foods for a growing population, but current products are based on a small number of strains with inherent limitations. Here, the authors develop genetic tools for an edible fungus and engineer its nutritional value and sensory appeal for alternative meat applications.
- Vayu Maini Rekdal
- , Casper R. B. van der Luijt
- & Jay D. Keasling
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Article
| Open AccessPHEIGES: all-cell-free phage synthesis and selection from engineered genomes
Bacteriophages have great potential in both medicine and biotechnology. Here the authors present PHEIGES, a cell-free method for phage genome engineering, synthesis and selection based on T7, which allows direct selection of engineered and mutant phages without compartmentalization.
- Antoine Levrier
- , Ioannis Karpathakis
- & Vincent Noireaux
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Article
| Open AccessDeep model predictive control of gene expression in thousands of single cells
Gene expression is inherently dynamic, due to complex regulation and stochastic biochemical events. Here the authors train a deep neural network to predict and dynamically control gene expression in thousands of individual bacteria in real-time which they then apply to control antibiotic resistance and study single-cell survival dynamics.
- Jean-Baptiste Lugagne
- , Caroline M. Blassick
- & Mary J. Dunlop
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Article
| Open AccessCas9-assisted biological containment of a genetically engineered human commensal bacterium and genetic elements
Engineered biosensing bacteria can potentially probe the human gut microbiome to prevent, diagnose, or treat disease. Here the authors present a robust biocontainment assisted by Cas9 and an engineered gene expression control combined in a genetically engineered human commensal bacterium that successfully functioned in a mouse intestinal tract as well as cell culture condition.
- Naoki Hayashi
- , Yong Lai
- & Timothy K. Lu
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Article
| Open AccessBiosensor and machine learning-aided engineering of an amaryllidaceae enzyme
Amaryllidaceae alkaloids, such as the Alzheimer’s medication galantamine, are currently extracted from low-yielding daffodils. Here, authors pair biosensor-assisted screening with machine learning-guided protein design to rapidly engineer an improved Amaryllidaceae enzyme in a microbial host.
- Simon d’Oelsnitz
- , Daniel J. Diaz
- & Andrew D. Ellington
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Article
| Open AccessMachine learning-aided design and screening of an emergent protein function in synthetic cells
Here, the authors introduce a pipeline to screen machine learning generated variants of a protein that forms intracellular spatiotemporal patterns in E. coli, demonstrating the best variants can substitute the wildtype gene.
- Shunshi Kohyama
- , Béla P. Frohn
- & Petra Schwille
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Article
| Open AccessA coarse-grained bacterial cell model for resource-aware analysis and design of synthetic gene circuits
Competition for the host cell’s resources influences synthetic biology circuit behavior. Here the authors present an E. coli cell model that combines insights into bacterial resource allocation with a simplified model of competition, facilitating resource-aware circuit design.
- Kirill Sechkar
- , Harrison Steel
- & Guy-Bart Stan
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Article
| Open AccessSynthetic microbe-to-plant communication channels
The soil microbiome communicates with plant roots using a chemical language. Here, using p-coumaroyl-homoserine lactone as the synthetic communication signal, the authors demonstrate programmable microbe-to-plant communication from the sender in the soil bacteria to a receiver in the plant.
- Alice Boo
- , Tyler Toth
- & Christopher A. Voigt
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Article
| Open AccessMotility of an autonomous protein-based artificial motor that operates via a burnt-bridge principle
Inspired by biology, great progress has been made in creating artificial molecular motors. Here the authors report the synthesis and characterization of the Lawnmower, an autonomous, protein-based artificial molecular motor and show their design is capable of track-guided motion.
- Chapin S. Korosec
- , Ivan N. Unksov
- & Nancy R. Forde
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Article
| Open AccessProtein design using structure-based residue preferences
Recent protein design methods rely on large neural networks, yet it is unclear which dependencies are critical for determining function. Here, authors show that learning the per residue mutation preferences, without considering interactions, enables design of functional and diverse protein variants.
- David Ding
- , Ada Y. Shaw
- & Debora S. Marks
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Article
| Open AccessOrthogonal inducible control of Cas13 circuits enables programmable RNA regulation in mammalian cells
The lack of control over Cas13 activity has limited its utility. Here the authors report Control of RNA with Inducible SpliT CAs13 Orthologs and Exogenous Ligands (CRISTAL), controlled by orthogonal split inducible Cas13 effectors that can be turned ON or OFF, providing precise temporal control.
- Yage Ding
- , Cristina Tous
- & Wilson W. Wong
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Article
| Open AccessA hybrid transistor with transcriptionally controlled computation and plasticity
Interfacing living systems with electronics for biosensing and biocomputing applications is challenging. Here, Gao et al. present hybrid transistors with electroactive bacteria capable of extracellular electron transfer, enabling transduction of biological computations to electrical readouts.
- Yang Gao
- , Yuchen Zhou
- & Benjamin K. Keitz
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Review Article
| Open AccessChoreographing root architecture and rhizosphere interactions through synthetic biology
Engineering the form and function of root systems and their associated microbiota could provide a means to mitigate adverse climate-driven effects. Here, the authors review the recent developments in plant and rhizobacterial synthetic biology and highlight engineering targets for applications in root systems and rhizosphere.
- Carin J. Ragland
- , Kevin Y. Shih
- & José R. Dinneny
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Article
| Open AccessOrthogonal LoxPsym sites allow multiplexed site-specific recombination in prokaryotic and eukaryotic hosts
Site-specific recombinases such as the Cre-LoxP system are routinely used for genome engineering in both prokaryotes and eukaryotes. Here the authors develop 63 symmetrical LoxP variants and test 1192 pairwise combinations to determine their cross-reactivity and specificity upon Cre activation.
- Charlotte Cautereels
- , Jolien Smets
- & Kevin J. Verstrepen
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Article
| Open AccessCombinatorial optimization of gene expression through recombinase-mediated promoter and terminator shuffling in yeast
Fine-tuning the expression of biosynthetic pathway genes is crucial to improve microbial production titres. Here, the authors present GEMbLeR, an optimization strategy to balance the expression of multiple genes simultaneously over a wide range.
- Charlotte Cautereels
- , Jolien Smets
- & Kevin J. Verstrepen
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Article
| Open AccessAAV-delivered muscone-induced transgene system for treating chronic diseases in mice via inhalation
Long-term control of therapeutic transgene expression is needed. Here the authors report a muscone-induced transgene system packaged into AAVs based on a G protein-coupled murine olfactory receptor and a synthetic cAMP-responsive promoter: they show dose- and exposure-time-dependent gene expression control in mice.
- Xin Wu
- , Yuanhuan Yu
- & Haifeng Ye
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Article
| Open AccessEngineering stringent genetic biocontainment of yeast with a protein stability switch
Comprehensive safety measures are lacking to employ engineered microorganisms in open-environment applications. Here the authors introduce a genetically encoded biocontainment system for engineered microorganisms based on conditional protein stability.
- Stefan A. Hoffmann
- & Yizhi Cai
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Article
| Open AccessLogical design of synthetic cis-regulatory DNA for genetic tracing of cell identities and state changes
Descriptive data in biomedical research are expanding rapidly, but functional validation methods lag behind. Here, authors present Logical Synthetic cis-regulatory DNA, a framework to design reporters that mark cellular states and pathways, showcasing its applicability to complex phenotypic states.
- Carlos Company
- , Matthias Jürgen Schmitt
- & Gaetano Gargiulo
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Article
| Open AccessAnti-CRISPR Anopheles mosquitoes inhibit gene drive spread under challenging behavioural conditions in large cages
CRISPR-based gene drives have the potential to spread within populations and are considered as promising vector control tools. Here the authors show an anti-drive mosquito strain that prevents the spread and collapse of a population suppression gene drive in laboratory Anopheles mosquito large cage trials in complex ecological and behavioral conditions.
- Rocco D’Amato
- , Chrysanthi Taxiarchi
- & Ruth Müller
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Article
| Open AccessA genetic circuit on a single DNA molecule as an autonomous dissipative nanodevice
Achieving genetic circuits on single DNA molecules could have varied applications. Here, authors observed proteins emerging from single DNA molecules through coupled transcription-translation complexes, and show that nascent proteins lingered on DNA, regulating cascaded reactions on the same DNA and allowing the design of a pulsatile genetic circuit.
- Ferdinand Greiss
- , Nicolas Lardon
- & Roy Bar-Ziv
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Article
| Open AccessLarge-scale genomic rearrangements boost SCRaMbLE in Saccharomyces cerevisiae
Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) is a promising tool to study genomic rearrangements. Here the authors present an engineered yeast strain with 83 sparsely distributed loxPsym sites across the genome can genrerate large-scale genomic rearrangements, which benefits cell fitness under stress and boosts the SCRaMbLE system when combined with synthetic chromosomes.
- Li Cheng
- , Shijun Zhao
- & Junbiao Dai
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Article
| Open AccessA multiplexed, confinable CRISPR/Cas9 gene drive can propagate in caged Aedes aegypti populations
Aedes aegypti is the main vector of several major pathogens including dengue, Zika and chikungunya viruses. Here the authors find that a CRISPR/Cas9 based split gene drive in Aedes aegypti could successfully bias inheritance up to 89% over successive generations in a multi-cage trial with further deep sequencing suggesting that the multiplexing design could mitigate resistance allele formation.
- Michelle A. E. Anderson
- , Estela Gonzalez
- & Luke Alphey
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Article
| Open AccessRational strain design with minimal phenotype perturbation
No consensus exists on the computationally tractable use of dynamic models for strain design. To tackle this, the authors report a framework, nonlinear-dynamic-model-assisted rational metabolic engineering design, for efficiently designing robust, artificially engineered cellular organisms.
- Bharath Narayanan
- , Daniel Weilandt
- & Vassily Hatzimanikatis
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Comment
| Open AccessA bumpy road ahead for genetic biocontainment
While the research community continues to develop novel proposals for intrinsic biocontainment of genetically engineered organisms, translation to real-world deployment faces several challenges.
- Dalton R. George
- , Mark Danciu
- & Emma K. Frow
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Article
| Open AccessGene drive and genetic sex conversion in the global agricultural pest Ceratitis capitata
Homing-based gene drives are novel interventions promising the area-wide, species-specific genetic control of harmful insect populations. Here the authors demonstrate the feasibility of a gene drive approach for the genetic control of the agricultural pest, the medfly, based on complete female-to-male sex conversion.
- Angela Meccariello
- , Shibo Hou
- & Nikolai Windbichler
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Article
| Open AccessDe novo biosynthesis of the hops bioactive flavonoid xanthohumol in yeast
Xanthohumol is a prenylated flavonoid produced by hops and is an important flavor substance in beer. Here, the authors engineer brewing yeast for the de novo biosynthesis of xanthohumol from glucose by balancing the three parallel biosynthetic pathways.
- Shan Yang
- , Ruibing Chen
- & Yongjin J. Zhou
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Article
| Open AccessModular assembly of an artificially concise biocatalytic cascade for the manufacture of phenethylisoquinoline alkaloids
Plant alkaloids – an important class of pharmaceuticals - are still largely acquired through phytoextraction. Here, the authors develop an artificial and concise four-enzyme biocatalytic cascade for synthesizing various phenethylisoquinoline alkaloids from readily available starting materials.
- Yue Gao
- , Fei Li
- & Yijian Rao
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Article
| Open AccessDipeptide coacervates as artificial membraneless organelles for bioorthogonal catalysis
Coacervate droplets have potential as components for cell-like materials, but are limited by complex molecular structure and control of the internal microenvironment. Here, the authors report stable dipeptide-based coacervates with a microenvironment for the encapsulation of hydrophobic species.
- Shoupeng Cao
- , Tsvetomir Ivanov
- & Lucas Caire da Silva
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Article
| Open AccessMammalian cell growth characterisation by a non-invasive plate reader assay
Automated and non-invasive mammalian cell analysis is currently lagging behind due to a lack of methods suitable for a variety of cell lines and applications. Here the authors develop a high throughput non-invasive method for tracking suspension and adhesion mammalian cell growth based on plate reader measures to characterize engineered cell lines.
- Alice Grob
- , Chiara Enrico Bena
- & Francesca Ceroni
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Article
| Open AccessBuilding synthetic chromosomes from natural DNA
Building synthetic chromosomes from natural components is an unexplored alternative to de novo chromosome synthesis that may have many potential applications. In this paper, the authors report CReATiNG, a method for constructing synthetic chromosomes from natural components in yeast.
- Alessandro L. V. Coradini
- , Christopher Ne Ville
- & Ian M. Ehrenreich
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Article
| Open AccessOptimized design and in vivo application of optogenetically functionalized Drosophila dopamine receptors
Designing optogenetically functionalized G protein-coupled receptors (optoXRs) to mimic endogenous signaling in vivo is challenging. Here, the authors optimize the design of optoXRs by considering evolutionary conserved protein interactions, and they employ this approach in fruit flies.
- Fangmin Zhou
- , Alexandra-Madelaine Tichy
- & Peter Soba
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Review Article
| Open AccessCustomizing cellular signal processing by synthetic multi-level regulatory circuits
As synthetic biology permeates society, the signal processing circuits in engineered living systems must be customized to meet practical demands. In this review, the authors outline design strategies for the DNA, RNA, and protein-level circuits and the hybrid “multi-level” circuits.
- Yuanli Gao
- , Lei Wang
- & Baojun Wang
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Article
| Open AccessCompetition and evolutionary selection among core regulatory motifs in gene expression control
Regulators represent a bioenergetic cost in gene expression control. Here, the author shows how functionally equivalent regulatory motifs have fundamentally different impacts on population structure, growth dynamics, and evolutionary outcomes.
- Andras Gyorgy
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Article
| Open AccessA unified Watson-Crick geometry drives transcription of six-letter expanded DNA alphabets by E. coli RNA polymerase
Here the authors present the structural mechanism of recognition of unnatural nucleobases in a six-letter expanded genetic system by E. coli RNA polymerase, and provide structural evidence for tautomerization during transcription.
- Juntaek Oh
- , Zelin Shan
- & Dong Wang
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Article
| Open AccessUniKP: a unified framework for the prediction of enzyme kinetic parameters
Prediction of enzyme kinetic parameters is essential for designing and optimising enzymes for various biotechnological and industrial applications. Here, authors presented a prediction framework (UniKP), which improves the accuracy of predictions for three enzyme kinetic parameters.
- Han Yu
- , Huaxiang Deng
- & Xiaozhou Luo
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Article
| Open AccessEngineering artificial photosynthesis based on rhodopsin for CO2 fixation
Microbial rhodopsins are major contributors to global light harvesting on Earth, but their role in carbon fixation is unclear. Here, the authors construct an artificial photosynthesis system by combining rhodopsin with an extracellular electron uptake mechanism for photoelectrosynthetic CO2 fixation in Ralstonia eutropha.
- Weiming Tu
- , Jiabao Xu
- & Wei E. Huang
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Article
| Open AccessProgrammable de novo designed coiled coil-mediated phase separation in mammalian cells
Membraneless liquid compartments based on phase-separating biopolymers have been observed in diverse cell types and attributed to weak multivalent interactions predominantly based on intrinsically disordered domains. Here the authors design protein liquid condensates from tunable concatenated coiled-coil dimer modules, unraveling the principles for coexisting condensates, chemical regulation, formation from either one or two polypeptide components in mammalian cells.
- Maruša Ramšak
- , Dominique A. Ramirez
- & Roman Jerala
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Article
| Open AccessBuilding a eukaryotic chromosome arm by de novo design and synthesis
In Saccharomyces cerevisiae, the left arm of chromosome XII only requires 12 genes to maintain cell viability, whereas 25 genes are needed for robust fitness. Here the authors demonstrate that the entire arm can be replaced by a neochromosome with completely artificial sequences.
- Shuangying Jiang
- , Zhouqing Luo
- & Junbiao Dai
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Article
| Open AccessA dynamic partitioning mechanism polarizes membrane protein distribution
Different membrane proteins dynamically polarize to organize signal transduction, but the underlying mechanism is unclear. Here, the authors show that a differential diffusion mediated partitioning process is sufficient to drive such spatiotemporal patterning of membrane-associated signaling proteins.
- Tatsat Banerjee
- , Satomi Matsuoka
- & Pablo A. Iglesias
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Article
| Open AccessInterrogating ligand-receptor interactions using highly sensitive cellular biosensors
The interaction of membrane-resident proteins plays an essential role in biological processes. Here the authors describe cellular biosensors based on chimeric receptors, as a tool to study the interaction of receptor-ligand pairs such as immune checkpoint molecules or virus attachment proteins and their receptors.
- Maximilian A. Funk
- , Judith Leitner
- & Peter Steinberger