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| Open AccessEngineering a scalable and orthogonal platform for synthetic communication in mammalian cells
The rational design and implementation of synthetic mammalian communication systems can unravel fundamental design principles of cell communication circuits and offer a framework for engineering of designer cell consortia with potential applications in cell therapeutics. Here the authors present a synthetic communication platform in mammalian cells based on diffusible dipeptide ligands and synthetic receptors, that is by design highly orthogonal, scalable, and programmable.
- Anna-Maria Makri Pistikou
- , Glenn A. O. Cremers
- & Tom F. A. de Greef
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Article
| Open AccessEngineering microbial division of labor for plastic upcycling
Plastic pollution is rapidly increasing worldwide, causing adverse impacts on the environment, wildlife and human health. Here the authors present a synthetic microbial consortium that efficiently degrades polyethylene terephthalate hydrolysate and upcycles it to desired chemicals through cellular division of labor.
- Teng Bao
- , Yuanchao Qian
- & Ting Lu
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Article
| Open AccessNext generation synthetic memory via intercepting recombinase function
Memory is a basic tenet of intelligent biological systems. Here the authors engineered a programmable and expandable iteration of recombinase-based synthetic memory (interception) that functions post-translation, resulting in faster recombination.
- Andrew E. Short
- , Dowan Kim
- & Corey J. Wilson
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Article
| Open AccessMultidimensional characterization of inducible promoters and a highly light-sensitive LOV-transcription factor
The ability to independently control the expression of different genes is important for quantitative biology. Here, the authors report kinetic parameters, noise scaling, impact on growth, and the fundamental leakiness of a wide range of inducible transcriptional systems, including a new, highly light sensitive LOV-transcription factor.
- Vojislav Gligorovski
- , Ahmad Sadeghi
- & Sahand Jamal Rahi
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Article
| Open AccessA cybergenetic framework for engineering intein-mediated integral feedback control systems
Homeostasis and robust perfect adaptation are remarkable features of living cells. Here, to synthetically achieve this, the authors present a theoretical and experimental framework using inteins to implement compact biomolecular integral feedback controllers.
- Stanislav Anastassov
- , Maurice Filo
- & Mustafa Khammash
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Article
| Open AccessIntegrase-mediated differentiation circuits improve evolutionary stability of burdensome and toxic functions in E. coli
Improving evolution stability of engineered functions is important for bioproduction and synthetic biology. Here, the authors developed an integrase-recombination-based terminal differentiation gene circuit in E. coli to improve the evolutionary stability of engineered function in a general manner.
- Rory L. Williams
- & Richard M. Murray
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Article
| Open AccessInducible plasmid copy number control for synthetic biology in commonly used E. coli strains
The ability to externally control gene expression has been important for all areas of biological research, especially for synthetic biology. Here the authors present plasmid TULIP which offers DNA copy number control via chemical induction to accelerate the design, prototyping, and reuse of gene circuits in diverse contexts.
- Shivang Hina-Nilesh Joshi
- , Chentao Yong
- & Andras Gyorgy
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Article
| Open AccessModel-guided engineering of DNA sequences with predictable site-specific recombination rates
Site-specific recombination (SSR) is an important tool in synthetic biology, but its applications are limited by the inability to predictably tune SSR reaction rates. Here, using quantitative high-throughput experiments and machine learning, the authors achieve rational control of a DNA attachment site sequence to predictably modulate site-specific recombination rates both in vitro and in cells.
- Qiuge Zhang
- , Samira M. Azarin
- & Casim A. Sarkar
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Article
| Open AccessA versatile active learning workflow for optimization of genetic and metabolic networks
Optimization of biological networks is often limited by wet lab labor and cost, and the lack of convenient computational tools. Here, aimed at democratization and standardization, the authors describe METIS, a modular and versatile active machine learning workflow with a simple online interface for the optimization of biological target functions with minimal experimental datasets.
- Amir Pandi
- , Christoph Diehl
- & Tobias J. Erb
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Article
| Open AccessA genetic toolkit and gene switches to limit Mycoplasma growth for biosafety applications
Mycoplasmas are minimal cell model organisms but lack genetic tools. Here the authors provide a robust genetic toolkit for Mycoplasma demonstrating gene circuit engineering applications.
- Alicia Broto
- , Erika Gaspari
- & Mark Isalan
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Article
| Open AccessRobust and tunable signal processing in mammalian cells via engineered covalent modification cycles
Phosphorylation networks are frequently at the heart of complex cellular decision making. Here the authors engineer synthetic phosphorylation devices with feedback regulation in mammalian cells and demonstrate how to use these to achieve tunable and robust control of cell behaviours.
- Ross D. Jones
- , Yili Qian
- & Ron Weiss
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Article
| Open AccessIndependent control of mean and noise by convolution of gene expression distributions
Gene expression noise can reduce fitness but analysis is hampered by a scaling relationship between noise and expression level. Here the authors show that gene expression mean and noise can be independently controlled by expressing two copies of a gene from separate inducible promoters in the same cell.
- Karl P. Gerhardt
- , Satyajit D. Rao
- & Jeffrey J. Tabor
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Article
| Open AccessEngineering self-organized criticality in living cells
Biological systems are known to behave in optimal ways when poised close to critical points, right on the edge between order and disorder. Here the authors show how this state can be engineered in living cells.
- Blai Vidiella
- , Antoni Guillamon
- & Ricard Solé
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Article
| Open AccessDesigning an irreversible metabolic switch for scalable induction of microbial chemical production
A promising strategy to increase product synthesis from bacteria uses inducible systems to switch metabolism to production. Here, the authors use models to show how engineering positive feedback loops into the genetic circuitry creates a switch that requires only temporary induction with a cheap nutrient to switch metabolism irreversibly, and so drastically reduce inducer use and cost.
- Ahmad A. Mannan
- & Declan G. Bates
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Article
| Open AccessSynthetic neural-like computing in microbial consortia for pattern recognition
Complex biological systems have individual cells acting collectively to solve complex tasks. Here the authors implement neural network-like computing in a bacterial consortia to recognise patterns.
- Ximing Li
- , Luna Rizik
- & Ramez Daniel
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Article
| Open AccessHarnessing the central dogma for stringent multi-level control of gene expression
Inducible gene expression systems should minimise leaky output and offer a large achievable range of expression. Here, the authors regulate transcription and translation together to suppress noise and create digital-like responses, while maintaining a large expression range in vivo and in vitro.
- F. Veronica Greco
- , Amir Pandi
- & Thomas E. Gorochowski
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Article
| Open AccessEngineering advanced logic and distributed computing in human CAR immune cells
Most CAR designs lack control and computation features, limiting the sophistication of the engineered immune response. Here the authors leverage a split CAR design for engineering coordinated immune responses.
- Jang Hwan Cho
- , Atsushi Okuma
- & Wilson W. Wong
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Article
| Open AccessContextual dependencies expand the re-usability of genetic inverters
Genetic circuits can be engineered to generate predefined outcomes, however host context is a crucial factor in performance. Here the authors characterise twenty NOT gates in seven different bacteria to understand and predict interoperability and portability across hosts.
- Huseyin Tas
- , Lewis Grozinger
- & Ángel Goñi-Moreno
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Article
| Open AccessSynthetic protein-binding DNA sponge as a tool to tune gene expression and mitigate protein toxicity
Decoy binding sites are natural regulators of gene expression. Here the authors design synthetic DNA sponges that fine tune the performance of synthetic gene circuits in a simple yet systematic manner, expanding the synthetic biology toolkit for gene regulation.
- Xinyi Wan
- , Filipe Pinto
- & Baojun Wang
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Article
| Open AccessAn endoribonuclease-based feedforward controller for decoupling resource-limited genetic modules in mammalian cells
Accurately predicting the behaviour of a genetic circuit remains difficult due to the lack of modularity. Here the authors quantify the effects of resource loading in mammalian systems and develop an endoribonuclease-based feedfoward controller to adapt gene expression to the effects of resource loading.
- Ross D. Jones
- , Yili Qian
- & Domitilla Del Vecchio
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Perspective
| Open AccessExploring the synthetic biology potential of bacteriophages for engineering non-model bacteria
Non-model bacteria offer unique and versatile metabolisms for synthetic biology. In this Perspective, the authors explore the limited availability of well-characterised biological parts in these species and argue that bacteriophages represent a diverse trove of orthogonal parts.
- Eveline-Marie Lammens
- , Pablo Ivan Nikel
- & Rob Lavigne
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Article
| Open AccessCharacterization and mitigation of gene expression burden in mammalian cells
The design of genetic networks in mammalian cells is still slow and often fails. Here the authors show that miRNA-based incoherent feedforward loop circuits can be used to alleviate cellular burden.
- Timothy Frei
- , Federica Cella
- & Velia Siciliano
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Article
| Open AccessEngineered systems of inducible anti-repressors for the next generation of biological programming
Transcriptional anti-repressors have been largely absent in the design of regulated genetic circuits. Here, the authors present a workflow of the engineering of non-natural anti-reperssors that can be built into NOT oriented logic gates.
- Thomas M. Groseclose
- , Ronald E. Rondon
- & Corey J. Wilson
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Article
| Open AccessMultistable and dynamic CRISPRi-based synthetic circuits
Synthetic circuits based on CRISPRi have not achieved multistable and dynamic behaviors. Here the authors build an oscillator, a toggle switch and an incoherent feed-forward loop using CRISPRi, and provide a mathematical model suggesting that unspecific binding in CRISPRi enables multistability.
- Javier Santos-Moreno
- , Eve Tasiudi
- & Yolanda Schaerli
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Article
| Open AccessTunable genetic devices through simultaneous control of transcription and translation
Synthetic genetic circuits are sensitive to their environment and host cell, requiring many rounds of physical reassembly to achieve a desired function. Here the authors use a multi-level regulatory motif to dynamically tune the function of genetic parts as a step towards robust adaptive circuits.
- Vittorio Bartoli
- , Grace A. Meaker
- & Thomas E. Gorochowski
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Article
| Open AccessEffective CRISPRa-mediated control of gene expression in bacteria must overcome strict target site requirements
The use of CRISPRa in bacteria lacks predictive rules for identifying effective gRNA target sites. Here the authors define features of bacterial promoters that impose stringent requirements on effective sites.
- Jason Fontana
- , Chen Dong
- & Jesse G. Zalatan
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Article
| Open AccessCaliciviral protein-based artificial translational activator for mammalian gene circuits with RNA-only delivery
Synthetic RNA-based circuits allow for gene regulation without the risk of mutagenesis. Here the authors demonstrate a Caliciviral VPg-based Translational activator which allows translational activation of synthetic mRNAs.
- Hideyuki Nakanishi
- & Hirohide Saito
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Article
| Open AccessInducible cell-to-cell signaling for tunable dynamics in microbial communities
Biotechnology innovations require the precise control over microbial dynamics. Here the authors engineer an inducible quorum sensing system to fine tune population and community level behaviour.
- Arianna Miano
- , Michael J. Liao
- & Jeff Hasty
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Article
| Open AccessHigh-performance chemical- and light-inducible recombinases in mammalian cells and mice
The availability of high performance recombinases with low basal activity and high dynamic range is limited. Here the authors present a library of over 20 orthogonal split recombinases that can be induced by small molecules, light and temperature in vivo.
- Benjamin H. Weinberg
- , Jang Hwan Cho
- & Wilson W. Wong
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Article
| Open AccessOncolytic adenovirus programmed by synthetic gene circuit for cancer immunotherapy
It is difficult to improve the efficacy of oncolytic virotherapy due to immune system responses and limited understanding of population dynamics. Here the authors use synthetic biology gene circuits to control adenoviral replication and release of immunomodulators in hepatocellular carcinoma cells.
- Huiya Huang
- , Yiqi Liu
- & Zhen Xie
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Article
| Open AccessGene networks that compensate for crosstalk with crosstalk
Crosstalk between genetic circuits is a major challenge for engineering sophisticated networks. Here the authors design networks that compensate for crosstalk by integrating, not insulating, pathways.
- Isaak E. Müller
- , Jacob R. Rubens
- & Timothy K. Lu
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Article
| Open AccessProgrammable biomolecular switches for rewiring flux in Escherichia coli
Current flux rewiring technologies in metabolic engineering are mainly transcriptional regulation. Here, the authors build two sets of controllable protein units using engineered viral proteases and proteolytic signals, and utilize for increasing titers of shikimate and D-xylonate in E. coli.
- Cong Gao
- , Jianshen Hou
- & Liming Liu
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Article
| Open AccessA modular degron library for synthetic circuits in mammalian cells
One method of controlling protein degradation is through the use of degrons. Here the authors present a toolbox of characterised degrons as a library to fine-tune biological gene-expression systems. Its application is demonstrated by a set of tunable synthetic pulse generators in mammalian cells.
- Hélène Chassin
- , Marius Müller
- & Martin Fussenegger
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Article
| Open AccessA quasi-integral controller for adaptation of genetic modules to variable ribosome demand
Competition for shared cellular resources often renders genetic circuits poorly predictable. Here the authors design a biomolecular quasi-integral controller that allows gene expression to adapt to variable demand in translation resources.
- Hsin-Ho Huang
- , Yili Qian
- & Domitilla Del Vecchio
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Article
| Open AccessCell fate potentials and switching kinetics uncovered in a classic bistable genetic switch
Bistable switches are a common regulatory motif in cell fate decision-making circuits with two mutually exclusive expression states. Here the authors develop a bistable reporter system and report two additional expression states.
- Xiaona Fang
- , Qiong Liu
- & Jie Xiao
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Article
| Open AccessCell-free prediction of protein expression costs for growing cells
The translation of heterologous proteins places a burden on host cell resources, affecting growth and productivity. Here the authors develop a cell-free assay to measure resource consumption and predict in vivo burden.
- Olivier Borkowski
- , Carlos Bricio
- & Tom Ellis
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Article
| Open AccessDrug-tunable multidimensional synthetic gene control using inducible degron-tagged dCas9 effectors
Deactivated Cas9 fused to transactivation domains can be used to control gene expression, however its presence can prevent rapid switching between different regulatory states. Here the authors generate conditionally degradable dCas9 and Cpf1 proteins for multidimensional control of functional activity.
- Dirk A. Kleinjan
- , Caroline Wardrope
- & Susan J. Rosser
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Article
| Open AccessHierarchical control of enzymatic actuators using DNA-based switchable memories
Naturally evolved regulatory circuits have hierarchical layers of signal generation and processing. Here, the authors emulate these networks using feedback-controlled DNA circuits that convert upstream signaling to downstream enzyme activity in a switchable memories circuit.
- Lenny H. H. Meijer
- , Alex Joesaar
- & Tom F. A. de Greef
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Article
| Open AccessEngineering species-like barriers to sexual reproduction
Genetic isolation of a genetically modified organism represents a useful strategy for biocontainment. Here the authors use dCas9-VP64-driven gene expression to construct a ‘species-like’ barrier to reproduction between two otherwise compatible populations.
- Maciej Maselko
- , Stephen C. Heinsch
- & Michael J. Smanski
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Article
| Open AccessInsulated transcriptional elements enable precise design of genetic circuits
Unwanted interactions between cellular components can complicate rational engineering of biological systems. Here the authors design insulated minimal promoters and operators that enable biophysical modeling of bacterial transcription without free parameters for precise circuit design.
- Yeqing Zong
- , Haoqian M. Zhang
- & Chunbo Lou
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Article
| Open AccessDigital logic circuits in yeast with CRISPR-dCas9 NOR gates
The leakiness of commonly used genetic components can make the construction of complex synthetic circuits difficult. Here the authors construct NOR gate architecture, using dCas9 fused to the chromatin remodeller Mxi1, that can be wired together into complex circuits.
- Miles W. Gander
- , Justin D. Vrana
- & Eric Klavins
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Article
| Open AccessDesign of synthetic epigenetic circuits featuring memory effects and reversible switching based on DNA methylation
Recording systems would allow synthetic organisms to store a ‘memory’ of a past event for future reference. Here the authors design an epigenetic memory system inE. colithat methylates DNA in response to exogenous and endogenous signals.
- Johannes A. H. Maier
- , Raphael Möhrle
- & Albert Jeltsch
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Article
| Open AccessProgramming mRNA decay to modulate synthetic circuit resource allocation
Synthetic circuits in host cells compete with endogenous processes for limited resources. Here the authors use MazF to funnel cellular resources to a synthetic circuit to increase product production and demonstrate how resource allocation can be manipulated.
- Ophelia S. Venturelli
- , Mika Tei
- & Adam P Arkin
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Article
| Open AccessEngineering orthogonal dual transcription factors for multi-input synthetic promoters
Genetic circuits usually employ the same set of transcription factors which can act via repression or activation of the target promoter. Here the authors present dual activator-repressor switches, designed via directed evolution, for orthogonal logic gates and multi-input circuit architectures.
- Andreas K. Brödel
- , Alfonso Jaramillo
- & Mark Isalan
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Article
| Open AccessTwister ribozymes as highly versatile expression platforms for artificial riboswitches
Twister ribozymes are small endonucleolytic RNA motifs. Here the authors develop twister ribozymes into RNA logic gates and cross-species synthetic genetic regulators.
- Michele Felletti
- , Julia Stifel
- & Jörg S. Hartig
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Article |
Highly modular bow-tie gene circuits with programmable dynamic behaviour
Engineering gene expression systems that can be programmed to respond to specific environmental conditions is challenging. Here, the authors develop a synthetic bow-tie circuit that is able to sense signals from microRNA molecules and affect a change in protein dynamics in mammalian cells.
- Laura Prochazka
- , Bartolomeo Angelici
- & Yaakov Benenson
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Article
| Open AccessA unified design space of synthetic stripe-forming networks
Constructing gene circuits with predefined behaviours is typically done on a case-by-case basis. Schaerli et al.instead computationally explore the design space for 3-node networks that generate a stripe in response to a morphogen gradient, and build networks based on their simplest possible forms.
- Yolanda Schaerli
- , Andreea Munteanu
- & Mark Isalan
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Article |
Sensitive detection of proteasomal activation using the Deg-On mammalian synthetic gene circuit
Proteasome activity can be monitored using a fluorescent substrate; however, screening for proteasome activators using this technique is challenging as signal loss is intrinsically more difficult to detect. Zhao et al.design a genetic inverter circuit that reports increased proteasome activity as signal gain.
- Wenting Zhao
- , Matthew Bonem
- & Laura Segatori
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Article |
Programming a Pavlovian-like conditioning circuit in Escherichia coli
Synthetic gene circuits can be programmed in living cells to perform diverse cellular functions. Here, the authors program a genetic circuit that performs a Pavlovian-like learning and recall function in E. coli, and demonstrate the dynamic nature of this conditioning process at a population level.
- Haoqian Zhang
- , Min Lin
- & Qi Ouyang