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| Open AccessEnhancing bioreactor arrays for automated measurements and reactive control with ReacSight
Small-scale bioreactors are increasingly used in quantitative biology. Here, the authors report ReacSight, a software solution to connect reactor arrays with sensitive measurement devices using low-cost pipetting robots and provide applications leveraging optogenetic control in yeast.
- François Bertaux
- , Sebastián Sosa-Carrillo
- & Gregory Batt
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Article
| Open AccessControlling synthetic membraneless organelles by a red-light-dependent singlet oxygen-generating protein
Membraneless organelles play vital cellular roles, and control over their formation and state could have varied applications. Here, the authors develop photoresponsive synthetic condensates whose activity can be controlled through the use of light to trigger liquid-to-solid phase transition.
- Manjia Li
- , Byung Min Park
- & Fei Sun
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Article
| Open AccessOrder-of-magnitude enhancement in photocurrent generation of Synechocystis sp. PCC 6803 by outer membrane deprivation
The low extracellular electron transfer activity hampers the application of cyanobacteria in biophotovoltaics. Here, the authors report an order-of-magnitude enhancement in photocurrent generation of the cyanobacterium by deprivation of the outer cell membrane.
- Shoko Kusama
- , Seiji Kojima
- & Shuji Nakanishi
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Article
| Open AccessDe novo biosynthesis of rubusoside and rebaudiosides in engineered yeasts
Rubusoside and rebaudiosides are considered the next generation of sugar substitutes. In this article, the authors report the engineering of Saccharomyces cerevisiae, remodelling the complex metabolic networks by a modular engineering approach, obtaining rubusoside and rebaudiosides at titers of around 1.4 g/L and 100 mg/L, respectively.
- Yameng Xu
- , Xinglong Wang
- & Long Liu
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| Open AccessCell-free prototyping enables implementation of optimized reverse β-oxidation pathways in heterotrophic and autotrophic bacteria
An attractive route for carbon-negative synthesis of biochemical products is the reverse β-oxidation pathway coupled to the Wood-Ljungdahl pathway. Here the authors use a high-throughput in vitro prototyping workflow to screen 762 unique pathway combinations using cell-free extracts tailored for r-BOX to identify enzyme sets for enhanced product selectivity.
- Bastian Vögeli
- , Luca Schulz
- & Michael C. Jewett
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Article
| Open AccessComputationally designed hyperactive Cas9 enzymes
The ability to alter the genomes of living cells is key to understanding how genes influence the functions of organisms and will be critical to modify living systems for useful purposes. Here, the authors use computational design to discover Cas9 enzymes with increased activity.
- Pascal D. Vos
- , Giulia Rossetti
- & Oliver Rackham
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Article
| Open AccessRewritable two-dimensional DNA-based data storage with machine learning reconstruction
Current DNA-based data storage platforms encode information only in the nucleotide sequence. Here, the authors report a 2DDNA platform that can store data in both sequence context and backbone structure, and has improved image inpainting and enhancement via automatic discoloration detection and deep learning.
- Chao Pan
- , S. Kasra Tabatabaei
- & Olgica Milenkovic
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| Open AccessA high-throughput multiparameter screen for accelerated development and optimization of soluble genetically encoded fluorescent biosensors
Fluorescent biosensors are important tools for studying cellular metabolism, but development and optimization are challenging. Koveal et al. present a high-throughput multiparameter screen for sensor performance, and used it to generate LiLac, a high-performance, quantitative lactate sensor.
- Dorothy Koveal
- , Paul C. Rosen
- & Gary Yellen
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Article
| Open AccessA framework to efficiently describe and share reproducible DNA materials and construction protocols
DNA constructs and their annotated sequence maps have been rapidly accumulating with the advancement of DNA cloning, synthesis, and assembly methods. Here the authors introduce QUEEN, a framework to describe and share DNA materials and construction protocols.
- Hideto Mori
- & Nozomu Yachie
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| Open AccessBroad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro
A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here the authors show that CRISPR-Cas13d can be used as a broad-spectrum antiviral to inhibit human coronaviruses, including new SARS-CoV-2 variants, combined with small molecule drugs for an enhanced antiviral effect in human primary cells.
- Leiping Zeng
- , Yanxia Liu
- & Lei S. Qi
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Article
| Open AccessPERSIST platform provides programmable RNA regulation using CRISPR endoRNases
Gene circuits must resist epigenetic silencing for reliable therapeutic applications. Here the authors develop an RNA-level regulation platform using CRISPR endoRNases that is modular, scalable, and more stable than traditional transcriptional versions.
- Breanna DiAndreth
- , Noreen Wauford
- & Ron Weiss
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Article
| Open AccessMetabolite trafficking enables membrane-impermeable-terpene secretion by yeast
The engineering of metabolite secretion from microorganisms can lead to many applications in synthetic biology. In this article, the authors engineer a metabolite trafficking system for the secretion of medicinal terpenes.
- So-Hee Son
- , Jae-Eung Kim
- & Ju Young Lee
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Article
| Open AccessSynthetic cells with self-activating optogenetic proteins communicate with natural cells
Synthetic biology and engineering approaches are harnessed to incorporate new capabilities in synthetic cells. Here, the authors designed bioluminescent signaling mechanisms for intracellular and intercellular synthetic-to-natural cell communication.
- Omer Adir
- , Mia R. Albalak
- & Avi Schroeder
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Article
| Open AccessA reversibly gated protein-transporting membrane channel made of DNA
Artificial systems to control the transport of molecules across biomembranes can be useful for biosensing or drug delivery. Here, the authors assemble a DNA channel enabling the precisely timed, stimulus-controlled transport of functional proteins across bilayer membranes.
- Swarup Dey
- , Adam Dorey
- & Hao Yan
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Article
| Open AccessMachine learning-coupled combinatorial mutagenesis enables resource-efficient engineering of CRISPR-Cas9 genome editor activities
Screening combinatorial mutants is too massive for wet-lab experiment alone. Here the authors present a machine learning-coupled combinatorial mutagenesis approach to vastly reduce experimental burden for engineering Cas9 genome editing enzymes.
- Dawn G. L. Thean
- , Hoi Yee Chu
- & Alan S. L. Wong
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Article
| Open AccessDevelopment of NanoLuc-targeting protein degraders and a universal reporter system to benchmark tag-targeted degradation platforms
t ag-T argetedP roteinD egrader (tTPD) systems are powerful tools for preclinical target validation. Here the authors extend the tTPD platform by developing NanoTACs that degrade NanoLuc tagged substrates and benchmark each tTPD system using an interchangeable tag reporter system.- Christoph Grohmann
- , Charlene M. Magtoto
- & Rebecca Feltham
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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 AccessMetal-responsive regulation of enzyme catalysis using genetically encoded chemical switches
Dynamic control over protein function is a central challenge in synthetic biology. Here the authors present an integrated computational and experimental workflow for engineering reversible protein switches; metal-chelating unnatural amino acids genetically encoded into two conformationally dynamic enzymes to yield robust switches.
- Yasmine S. Zubi
- , Kosuke Seki
- & Jared C. Lewis
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Article
| Open AccessCryo-EM structures of a LptDE transporter in complex with Pro-macrobodies offer insight into lipopolysaccharide translocation
Lateral opening of the LptDE transporter in the outer membrane of Gram-negative bacteria is necessary for insertion of lipopolysaccharides. Here, Botte et al. report a cryo-EM structure of a partially opened LptDE transporter, in complex with rigid chaperones derived from nanobodies.
- Mathieu Botte
- , Dongchun Ni
- & Michael Hennig
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Article
| Open AccessStructure-based design of stabilized recombinant influenza neuraminidase tetramers
Influenza virus neuraminidase (NA) is a drug target and a potential vaccine antigen. Here, the authors provide a detailed analysis of the conformational stability of NA, and show how expression and stability of recombinant NA antigens can be strengthened through structure-based design.
- Daniel Ellis
- , Julia Lederhofer
- & Masaru Kanekiyo
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Article
| Open AccessSelective optogenetic control of Gq signaling using human Neuropsin
Gq proteins are one of four major classes of G proteins; optogenetic receptors for selective and repetitive activation of Gq proteins with fast kinetics are lacking. Here the authors report UV light-dependent Gq signalling using human Neuropsin (hOPN5) and demonstrate its potential as an optogenetic tool.
- Ahmed Wagdi
- , Daniela Malan
- & Tobias Bruegmann
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Article
| Open AccessDe novo design and directed folding of disulfide-bridged peptide heterodimers
Peptide heterodimers are prevalent in nature, which are not only functional macromolecules but molecular tools for chemical and synthetic biology. Here the authors report de novo design and directed folding of peptide heterodimers crosslinked through multiple disulfide bonds, which can be explored as chemical tools for orthogonal labeling of proteins and preparing protein hybrids.
- Sicong Yao
- , Adam Moyer
- & Chuanliu Wu
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Article
| Open AccessPrecise tumor immune rewiring via synthetic CRISPRa circuits gated by concurrent gain/loss of transcription factors
“Reinvigoration of antitumor immunity has recently become the central theme for the development of cancer therapies. Here the authors present an adaptable gene circuit to harness the CRISPRa for tumorlocalized immune activation.”
- Yafeng Wang
- , Guiquan Zhang
- & Jianghuai Liu
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Article
| Open AccessGenetically stable CRISPR-based kill switches for engineered microbes
Biocontainment is a key to developing safe genetically-engineered microbes (GEMs). Here the authors demonstrate genetically stable CRISPR-based kill switches that control GEMs’ viability in animal hosts, enabling their safe biomedical applications.
- Austin G. Rottinghaus
- , Aura Ferreiro
- & Tae Seok Moon
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Article
| Open AccessImproved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage
Some DNA sequences are refractory to CRISPR-Cas9 cleavage, partially due to gRNA misfolding. Here the authors engineer gRNAs to prevent misfolding and further enhanced their stability by chemical modifications allowing robust genome editing regardless of target sequence.
- Stephan Riesenberg
- , Nelly Helmbrecht
- & Svante Pääbo
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| Open AccessPandemic preparedness: synthetic biology and publicly funded biofoundries can rapidly accelerate response time
Synthetic biology has played a key role in responding to the current pandemic. Biofoundries are critical synthetic biology infrastructure which should be available to all nations as a part of their independent bioengineering, biosecurity, and countermeasure response systems.
- Claudia E. Vickers
- & Paul S. Freemont
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Article
| Open AccessNuclear and mitochondrial DNA editing in human cells with zinc finger deaminases
Base editing in nuclear DNA and mitochondrial DNA (mtDNA) is broadly useful for biomedical research, medicine, and biotechnology. Here the authors present zinc finger deaminases which catalyze targeted C-to-T base conversions without inducing unwanted indels in human cells.
- Kayeong Lim
- , Sung-Ik Cho
- & Jin-Soo Kim
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Article
| Open AccessTowards a generic prototyping approach for therapeutically-relevant peptides and proteins in a cell-free translation system
Generic approach for rapid prototyping is essential for the progress of synthetic biology. Here the authors modify the cell-free translation system to control protein aggregation and folding and validate the approach by using single conditions for prototyping of various disulfide-constrained polypeptides.
- Yue Wu
- , Zhenling Cui
- & Sergey Mureev
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Article
| Open AccessMechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography
Shark antibodies (Variable New Antigen Receptors, VNARs) are the smallest naturally occurring antibody fragments. Here, the authors screen a VNAR phage display library against the SARS-CoV2 receptor binding domain (RBD) and identify VNARs that neutralize the SARSCoV-2 virus and discuss their mechanisms of viral neutralization.
- Obinna C. Ubah
- , Eric W. Lake
- & Caroline J. Barelle
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| Open AccessExploiting a Y chromosome-linked Cas9 for sex selection and gene drive
CRISPR-based engineering can be used to bias sex ratios. Here the authors develop a transgenic line of Drosophila melanogaster expressing Cas9 from the Y chromosome and functionally characterize the utility of this strain for both sex selection and gene drive.
- Stephanie Gamez
- , Duverney Chaverra-Rodriguez
- & Omar S. Akbari
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Article
| Open AccessProbing ion channel functional architecture and domain recombination compatibility by massively parallel domain insertion profiling
Here, the authors perform a large-scale, high-throughput biochemical assay to determine the compatibility of over 300,000 domain recombination variants of the inward rectifier K+ channel Kir2.1. They derive rules for designing domain insertion variants that fold and traffic to the cell surface and conclude that the insertion of domains at protein termini is evolutionary favoured.
- Willow Coyote-Maestas
- , David Nedrud
- & Daniel Schmidt
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Article
| Open AccessA genetically encoded fluorescent biosensor for extracellular l-lactate
l-lactate is an important intercellular energy currency. Here the authors report a genetically encoded biosensor eLACCO1.1 to monitor extracellular l-lactate; they use eLACCO1.1 to image extracellular l-lactate in cultured mammalian cells and brain tissue.
- Yusuke Nasu
- , Ciaran Murphy-Royal
- & Robert E. Campbell
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Article
| Open AccessDirected evolution of and structural insights into antibody-mediated disruption of a stable receptor-ligand complex
Facilitated dissociation is a mechanism where antibody-mediated disruption of high-affinity complexes can enhance the therapeutic effects of a drug. Here the authors present a yeast display approach to select and engineer omalizumab variants that dissociate receptor-bound IgE to accelerate its inhibition of the allergic response.
- Luke F. Pennington
- , Pascal Gasser
- & Theodore S. Jardetzky
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Article
| Open AccessVersatile selective evolutionary pressure using synthetic defect in universal metabolism
Rational design of enzymes with new or improved properties is rarely straightforward, and artificial selection pressure approaches that link an improvement in the target to cell growth are an alternative. Here, the authors show that diverse enzymes sharing the ubiquitous cofactor NAD(P)+ can substitute for defective NAD+ regeneration, representing a very broadly-applicable artificial selection.
- Lara Sellés Vidal
- , James W. Murray
- & John T. Heap
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| Open AccessGene editing enables rapid engineering of complex antibiotic assembly lines
Engineering biosynthetic assembly lines is a powerful path to new natural products but is challenging with current methods. Here the authors use CRISPR-Cas9 to exchange subdomains within NRPS to alter substrate selectivity.
- Wei Li Thong
- , Yingxin Zhang
- & Jason Micklefield
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| Open AccessHomecoming: rewinding the reductive evolution of the chloroplast genome for increasing crop yields
Developing more productive and sustainable crops will be essential to achieving food security in coming decades. A core process in plant evolution has been the transfer of chloroplast-encoded genes to the nuclear genome. We propose reverting this process as a new approach to improve plant disease resistance and photosynthesis in future crops.
- Briardo Llorente
- , María Eugenia Segretin
- & Nicolás E. Blanco
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| Open AccessMechanistic principles of an ultra-long bovine CDR reveal strategies for antibody design
Certain bovine antibodies have ultra-long long complementarity-determining regions (CDRs) that contain a knob for antigen interaction, which is connected to the antibody through a stalk. Here, the authors combine biophysical experiments and MD simulations and show that the stalk length is critical for the folding and stability of these antibodies. The authors also demonstrate that ultra-long bovine CDRs can be grafted into human antibodies, and furthermore show that de novo designed mini-domains that bind to the SARS-CoV-2 spike protein with high affinity can be integrated as a knob in ultra-long CDRs in bovine and human antibodies, which neutralize SARS-CoV-2.
- Hristo L. Svilenov
- , Julia Sacherl
- & Johannes Buchner
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Article
| Open AccessTwo-input protein logic gate for computation in living cells
Traditional synthetic biology tools operate by complex re-programming of DNA, requiring significant amount of ‘nucleotide-based code’ to implement instructions that are transcribed at the protein level. Here the authors demonstrate the direct regulation of cellular phenotype at the single-protein level by creating a two-input logic gate for biological computation using ‘allosteric wiring’.
- Yashavantha L. Vishweshwaraiah
- , Jiaxing Chen
- & Nikolay V. Dokholyan
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Article
| Open AccessAsymmetric requirement of Dpp/BMP morphogen dispersal in the Drosophila wing disc
Morphogens disperse to pattern tissues and control their growth during development, allowing for the specification of multiple fates across space. Here the authors block dispersal of a morphogen Dpp (BMP2/4) and show that the requirement for Dpp dispersal is much lower than previously thought.
- Shinya Matsuda
- , Jonas V. Schaefer
- & Markus Affolter
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Article
| Open AccessSelection for constrained peptides that bind to a single target protein
Peptide secondary metabolites have a diverse range of functions. Here the authors present a method to design and screen a large library of modified peptides in E. coli against a target of interest.
- Andrew M. King
- , Daniel A. Anderson
- & Christopher A. Voigt
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Article
| Open AccessImprovement of a synthetic live bacterial therapeutic for phenylketonuria with biosensor-enabled enzyme engineering
PKU patients have elevated phenylalanine levels which can result in neurological impairment. Here the authors utilize biosensor-based ultra-high-throughput screening to optimize PAL activity in a synthetic biotic platform for improved in vivo performance.
- Kristin J. Adolfsen
- , Isolde Callihan
- & Vincent M. Isabella
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| Open AccessDe novo biosynthesis of bioactive isoflavonoids by engineered yeast cell factories
Isoflavonoids are a class of industrially important plant natural products, but their low abundance and structural complexity limits their availability. Here, the authors engineer Saccharomyces cerevisiae metabolism to become a platform for efficient production of daidzein which is core chemical scaffold for isoflavonoid biosynthesis, and show its application for production of bioactive glucosides from glucose.
- Quanli Liu
- , Yi Liu
- & Jens Nielsen
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Article
| Open AccessA dual-reporter system for investigating and optimizing protein translation and folding in E. coli
Heterologous expression of recombinant proteins often results in misfolding, aggregation and degradation. Here, we show an in vivo dual-biosensor system that simultaneously assesses protein translation and protein folding, thereby enabling rapid screening of expression strains as well as mutant libraries.
- Ariane Zutz
- , Louise Hamborg
- & Alex Toftgaard Nielsen
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Article
| Open AccessDisulfide-compatible phage-assisted continuous evolution in the periplasmic space
The directed evolution of antibodies yields important tools for research and therapy. Here the authors develop a periplasmic phage-assisted continuous evolution platform for improvement of protein-protein interactions in the disulfidecompatible E. coli periplasm.
- Mary S. Morrison
- , Tina Wang
- & David R. Liu
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Article
| Open AccessA standardized genome architecture for bacterial synthetic biology (SEGA)
Genome engineering is challenging compared to plasmid DNA manipulation. Here the authors create a simple methodology called SEGA that enables genome engineering by combining DNA and bacterial cells followed by identification of recombinant clones by a change in colour when grown on agar plates.
- Carolyn N. Bayer
- , Maja Rennig
- & Morten H. H. Nørholm
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Article
| Open AccessMultiplex suppression of four quadruplet codons via tRNA directed evolution
Genetic code expansion strategies are limited to specific codons that can be reassigned to new amino acids. Here the authors show that quadruplet-decoding tRNAs (qtRNAs) can be rapidly discovered and evolved to decode new quadruplet codons, enabling four independent decoding events in a single protein in living cells.
- Erika A. DeBenedictis
- , Gavriela D. Carver
- & Ahmed H. Badran
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Article
| Open AccessComprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules
Proteins evolve through the modular rearrangement of domains. Here the authors introduce MISER, a minimization by iterative size-exclusion and recombination method to make all possible deletions of a protein, uncovering functions for Cas9 domains involved in DNA binding.
- Arik Shams
- , Sean A. Higgins
- & David F. Savage
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| Open AccessDirected evolution of rRNA improves translation kinetics and recombinant protein yield
Ribosome kinetics are rate-limiting for protein synthesis. Here the authors evolve diverse 16S rRNAs for enhanced protein synthesis rates and genetic code expansion efficiencies in vivo.
- Fan Liu
- , Siniša Bratulić
- & Ahmed H. Badran
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| Open AccessRapid prototyping and design of cybergenetic single-cell controllers
Practical implementation of genetic circuits is difficult due to low predictability and time-intensive troubleshooting. Here the authors present Cyberloop, which interfaces a computer with single cells to enable cell-in-the-loop testing and optimization of circuit designs before they are built.
- Sant Kumar
- , Marc Rullan
- & Mustafa Khammash