Featured
-
-
Article
| Open AccessSynthetic intrinsically disordered protein fusion tags that enhance protein solubility
Insoluble protein expression continues to be a bottleneck for biotechnology. Here, Chilkoti and colleagues report a method for generating and identifying hypersoluble intrinsically disordered protein fusion tags to improve soluble protein expression and rescue protein function.
- Nicholas C. Tang
- , Jonathan C. Su
- & Ashutosh Chilkoti
-
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
-
Article
| Open AccessHydrophobic mismatch drives self-organization of designer proteins into synthetic membranes
The organization of membrane proteins is critical to cellular function. Here the authors explore how computational protein design, MD simulation, and cell-free systems can be combined to elucidate how membrane-protein hydrophobic mismatch affects protein folding and organization in synthetic lipid membranes.
- Justin A. Peruzzi
- , Jan Steinkühler
- & Neha P. Kamat
-
Article
| Open AccessA universal molecular control for DNA, mRNA and protein expression
Multi-omics analyses powerfully combine gene expression and translation, however no available controls can be used across these techniques. Here the authors develop pREF, a universal control construct designed for use in DNA, RNA and protein analyses.
- Helen M. Gunter
- , Scott E. Youlten
- & Tim R. Mercer
-
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
-
Article
| Open AccessdCas13-mediated translational repression for accurate gene silencing in mammalian cells
Current gene silencing tools can have drawbacks. Here the authors report CRISPRδ, an approach for translational silencing, harnessing catalytically inactive Cas13 proteins (dCas13): they also show that fusion of a translational repressor to dCas13 further improved the performance.
- Antonios Apostolopoulos
- , Naohiro Kawamoto
- & Shintaro Iwasaki
-
Article
| Open AccessCryo-EM structure of cell-free synthesized human histamine 2 receptor/Gs complex in nanodisc environment
The study describes the molecular structure of the human histamine 2 receptor in active conformation and in complex with Gs heterotrimer, synthesized in a cell-free system and co-translationally inserted into preformed nanodiscs.
- Zoe Köck
- , Kilian Schnelle
- & Frank Bernhard
-
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
-
Article
| Open AccessEngineering tumor-colonizing E. coli Nissle 1917 for detection and treatment of colorectal neoplasia
There is an unmet medical need for the detection and treatment of early adenomas to prevent their progression to malignant disease. Here the authors show that orally administered E. coli Nissle 1917 can selectively colonize adenomas in mouse models and in patients as a detection tool, as well as deliver immunotherapeutics for colorectal neoplasia treatment.
- Candice R. Gurbatri
- , Georgette A. Radford
- & Tal Danino
-
Article
| Open AccessCross-feeding promotes heterogeneity within yeast cell populations
Cellular heterogeneity in cell populations of isogenic origin is driven by diverse factors such as nutrient availability and interactions with neighbouring cells. Here, Hu et al. study plasmid retention dynamics in yeast populations, and show that the exchange of complementary metabolites between plasmid-carrying prototrophs and plasmid-free auxotrophs allows the latter to proliferate in selective environments.
- Kevin K. Y. Hu
- , Ankita Suri
- & Victoria S. Haritos
-
Article
| Open AccessRedox-enabled electronic interrogation and feedback control of hierarchical and networked biological systems
With redox-linked synthetic biology and electrobiofabrication, electronic information can be transmitted in a bidirectional manner between biology and electronics. Here the authors design an electrogenetic platform that allows real time electronic control of biological functions from proteins and gene circuits to cell consortia.
- Sally Wang
- , Chen-Yu Chen
- & William E. Bentley
-
Article
| Open AccessCell-free biosynthesis combined with deep learning accelerates de novo-development of antimicrobial peptides
Deep learning holds a great promise for the discovery and design of bioactive peptides, but experimental approaches to validate candidates in high throughput and at low cost are needed. Here, the authors combine deep learning and cell free biosynthesis for antimicrobial peptide (AMP) development and identify 30 functional AMPs, of which six with broad-spectrum activity against drug-resistant pathogens.
- Amir Pandi
- , David Adam
- & Tobias J. Erb
-
Article
| Open AccessCircular single-stranded DNA as switchable vector for gene expression in mammalian cells
Synthetic gene networks in mammalian cells are currently limited to either transcription factors or RNA regulators. Here, the authors develop a regulatory approach based on circular single-stranded DNA, which can be used as a conformationally switchable genetic vector in mammalian cells.
- Linlin Tang
- , Zhijin Tian
- & Jie Song
-
Article
| Open AccessEngineering tRNA abundances for synthetic cellular systems
Mature fields of engineering use physics-based models to design systems that work reliably the first time. Here the authors show how a similar approach can be used to design and build a cellular-scale system, protein synthesis, from scratch.
- Akshay J. Maheshwari
- , Jonathan Calles
- & Drew Endy
-
Article
| Open AccessA rapid cell-free expression and screening platform for antibody discovery
Antibody discovery is bottlenecked by the individual expression and evaluation of antigen specific hits. Here, the authors build an antibody screening workflow leveraging cell-free protein synthesis that enables expression and evaluation of hundreds of antibody fragments in less than 24 h.
- Andrew C. Hunt
- , Bastian Vögeli
- & Michael C. Jewett
-
Article
| Open AccessIsolation of full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of Escherichia coli
Discovery of full-length antibodies can be slow and labor intensive. Here, the authors describe a robust genetic assay for facile isolation of IgG antibodies from combinatorial libraries expressed in the cytoplasm of redox-engineered bacteria.
- Michael-Paul Robinson
- , Jinjoo Jung
- & Matthew P. DeLisa
-
Article
| Open AccessA Vaccinia-based system for directed evolution of GPCRs in mammalian cells
G protein-coupled receptors are a major class of drug targets. Here, the authors develop a method whereby their biophysical and functional properties can be altered through directed evolution in mammalian cells, leading to variants exhibiting features such as high stability and expression, or increased allosteric coupling.
- Christoph Klenk
- , Maria Scrivens
- & Andreas Plückthun
-
Article
| Open AccessEngineered Lactococcus lactis secreting Flt3L and OX40 ligand for in situ vaccination-based cancer immunotherapy
The probiotic Lactococcus lactis has been used for the delivery of therapeutic molecules. Here the authors engineer Lactococcus lactis to express a fusion protein of Flt3L and OX40 ligand, eliciting anti-tumor immune response in preclinical cancer models.
- Junmeng Zhu
- , Yaohua Ke
- & Baorui Liu
-
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
-
Article
| Open AccessA synthetic transcription platform for programmable gene expression in mammalian cells
Precise and scalable regulation of gene expression in mammalian cells is challenging. Here, the authors created a highly tunable CRISPR-based synthetic transcription system for programmable control of mammalian gene expression and cellular activity.
- William C. W. Chen
- , Leonid Gaidukov
- & Timothy K. Lu
-
Article
| Open AccessExpression strategies for the efficient synthesis of antimicrobial peptides in plastids
Antimicrobial peptides (AMPs) are promising next-generation antibiotics, but are difficult to produce due to the toxicity to bacterial hosts. Here, the authors report the utilization of transplastomic tobacco plants for AMPs production without cytotoxic effects via inducible expression systems and fusions to cleavable carrier protein.
- Matthijs P. Hoelscher
- , Joachim Forner
- & Ralph Bock
-
Article
| Open AccessA 33-residue peptide tag increases solubility and stability of Escherichia coli produced single-chain antibody fragments
Low solubility and stability of Escherichia coli produced single chain variable fragments (scFvs) restrict their applications. Here the authors report a 33-residue peptide tag which simultaneously increases the solubility and thermostability of multiple scFvs produced in Escherichia coli SHuffle strain.
- Yang Wang
- , Wenjie Yuan
- & Yong-Xiang Wang
-
Article
| Open AccessA microfluidic optimal experimental design platform for forward design of cell-free genetic networks
Characterization of cell-free genetic networks is inherently difficult. Here the authors use optimal experimental design and microfluidics to improve characterization, demonstrating modularity and predictability of parts in applied test cases.
- Bob van Sluijs
- , Roel J. M. Maas
- & Wilhelm T. S. Huck
-
Article
| Open AccessPlasmidMaker is a versatile, automated, and high throughput end-to-end platform for plasmid construction
Despite their broad utility, design and construction of plasmids remains laborious and time-consuming. Here the authors report a robust, versatile, and automated end-to-end platform that enables scarless construction of virtually any plasmid.
- Behnam Enghiad
- , Pu Xue
- & Huimin Zhao
-
Article
| Open AccessMassively parallel characterization of engineered transcript isoforms using direct RNA sequencing
Transcriptional terminators are generally viewed as hard endpoints for transcribing RNA polymerases. Here, the authors reimagine terminators as transcriptional valves with predictable read through. They engineer and characterize 1780 valves and use them for multiplexed gene regulation.
- Matthew J. Tarnowski
- & Thomas E. Gorochowski
-
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
-
Article
| Open AccessMassively parallel interrogation of protein fragment secretability using SECRiFY reveals features influencing secretory system transit
The exact protein features that control passage through the eukaryotic secretory system remain largely unknown. Here the authors report SECRiFY which they use to evaluate the secretory potential of polypeptides on a proteome-wide scale in yeast, revealing a role for flexibility and intrinsic disorder.
- Morgane Boone
- , Pathmanaban Ramasamy
- & Nico Callewaert
-
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
-
Article
| Open AccessSingle-component near-infrared optogenetic systems for gene transcription regulation
Current near-IR optogenetic systems to regulate transcription consist of a number of large protein components. Here the authors report a smaller single-component near-IR system, iLight, developed from a bacterial phytochrome that they use to control gene transcription in bacterial and mammalian cells.
- Andrii A. Kaberniuk
- , Mikhail Baloban
- & Vladislav V. Verkhusha
-
Article
| Open AccessScalable recombinase-based gene expression cascades
There are few robust circuit architectures for sequential gene perturbations. Here, the authors use a modular recombinase-based design that sequentially edits loci, synchronizes cells, and deletes itself.
- Tackhoon Kim
- , Benjamin Weinberg
- & Timothy K. Lu
-
Article
| Open AccessImproving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles
Cell-free gene expression systems are an attractive platform for biomanufacturing and synthetic biology. Here the authors characterize native membrane vesicles in E. coli extracts for improved glycoengineering.
- Jasmine M. Hershewe
- , Katherine F. Warfel
- & Michael C. Jewett
-
Article
| Open AccessRobust and flexible platform for directed evolution of yeast genetic switches
Eukaryotic genetic switches are more complex than prokaryotic ones, complicating their design. Here the authors present a workflow for parallel screening, selection and evolution of yeast genetic switches.
- Masahiro Tominaga
- , Kenta Nozaki
- & Akihiko Kondo
-
Article
| Open AccessIn vivo and in vitro reconstitution of unique key steps in cystobactamid antibiotic biosynthesis
The biosynthetic pathway leading to the production of cystobactamids, a group of myxobacteria-derived topoisomerase inhibitors with potent anti-Gram-negative activity, remains unclear. Here, the authors report in vivo and in vitro evidence for unique steps in cystobactamid biosynthesis.
- Sebastian Groß
- , Bastien Schnell
- & Rolf Müller
-
Article
| Open AccessSARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface
Antibodies targeting the spike protein of coronaviruses are potential candidates for therapeutic development. Here, Bertoglio et al. use phage display to select anti-SARS-CoV-2 spike antibodies from the human naïve universal antibody gene libraries HAL9/10 that block interaction with ACE2 receptor to inhibit infection.
- Federico Bertoglio
- , Doris Meier
- & Michael Hust
-
Article
| Open AccessA glucose meter interface for point-of-care gene circuit-based diagnostics
Getting synthetic biology circuit-based sensors into field applications is still a challenge. Here the authors combine a circuit sensor with a glucose meter for small analyte and nucleic acid detection.
- Evan Amalfitano
- , Margot Karlikow
- & Keith Pardee
-
Article
| Open AccessModelling genetic mosaicism of neurodevelopmental disorders in vivo by a Cre-amplifying fluorescent reporter
Genetic mosaicism is frequently present in monogenic diseases of the central nervous system. Here the authors design a dual-colour reporter system that can be used to tune the degree of mosaicism in mouse models.
- Francesco Trovato
- , Riccardo Parra
- & Gian Michele Ratto
-
Article
| Open AccessThe auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice
Auxin-inducible degron systems can be leaky and require high doses of auxin. Here the authors establish AID2 which uses an OsTIR1 mutant and the ligand 5-Ph-IAA to overcome these problems and establish AID-mediated target depletion in mice.
- Aisha Yesbolatova
- , Yuichiro Saito
- & Masato T. Kanemaki
-
Article
| Open AccessPhosphoregulated orthogonal signal transduction in mammalian cells
Phosphoregulation is a key mechanism of signal processing. Here the authors build a phosphoregulated relay system in mammalian cells for orthogonal signal transduction.
- Leo Scheller
- , Marc Schmollack
- & Martin Fussenegger
-
Article
| Open AccessA redox-based electrogenetic CRISPR system to connect with and control biological information networks
Redox-responsive transcriptional regulators can enable user-specified electronic control over biological functions. Here the authors demonstrate electronic control of CRISPRa and CRISPRi using redox signalling.
- Narendranath Bhokisham
- , Eric VanArsdale
- & William E. Bentley
-
Article
| Open AccessModel-driven generation of artificial yeast promoters
A small set of promoters is used for most genetic construct design in S. cerevisiae. Here, the authors develop a predictive model of promoter activity trained on a data set of over one million sequences and use it to design large sets of high-activity promoters.
- Benjamin J. Kotopka
- & Christina D. Smolke
-
Article
| Open AccessA fully orthogonal system for protein synthesis in bacterial cells
Ribosome engineering is an emerging powerful approach for synthetic protein synthesis. Here the authors invert the Ribo-T system, using the engineered ribosome to translate the proteome while the native ribosome translates specific mRNA.
- Nikolay A. Aleksashin
- , Teresa Szal
- & Alexander S. Mankin
-
Article
| Open AccessA genome-wide approach for identification and characterisation of metabolite-inducible systems
Inducible gene expression tools have important applications as genetically encoded biosensors. Here the authors conduct a genome-wide approach to identify and utilise functional sensors.
- Erik K. R. Hanko
- , Ana C. Paiva
- & Naglis Malys
-
Article
| Open AccessAnti-BCMA chimeric antigen receptors with fully human heavy-chain-only antigen recognition domains
Chimeric antigen receptors (CAR) use antibody variable regions to activate anti-tumor immunity. Here the authors show that a mouse IgH/IgL variable region used in a clinical CAR induces host immune responses to possibly reduce therapy efficacy, but an IgH-only CAR T design achieves similar CAR T activity but is potentially less immunogenic.
- Norris Lam
- , Nathan D. Trinklein
- & James N. Kochenderfer
-
Article
| Open AccessA short translational ramp determines the efficiency of protein synthesis
Several factors contribute to the efficiency of protein expression. Here the authors show that the identity of amino acids encoded by codons at position 3–5 significantly impact translation efficiency and protein expression levels.
- Manasvi Verma
- , Junhong Choi
- & Sergej Djuranovic
-
Article
| Open AccessPolyunsaturated fatty acid production by Yarrowia lipolytica employing designed myxobacterial PUFA synthases
Sustainable sources are needed to meet the demand for long-chain polyunsaturated fatty acids. Here the authors construct an artificial biosynthetic gene cluster in Y. lipolytica capable of producing a high concentration of PUFAs.
- Katja Gemperlein
- , Demian Dietrich
- & Rolf Müller
-
Article
| Open AccessProgrammable mutually exclusive alternative splicing for generating RNA and protein diversity
Alternative splicing expands the genetic coding capacity and proteomic diversity of the cell. Here the authors create a synthetic biology platform for regulating four programmable exons in modular transcription factors.
- Melina Mathur
- , Cameron M. Kim
- & Christina D. Smolke
-
Article
| Open AccessDecoupling tRNA promoter and processing activities enables specific Pol-II Cas9 guide RNA expression
The utility of CRISPR-based technologies could be enhanced with the ability to control the spatial and temporal expression of gRNAs. Here the authors design a tRNA scaffold for highly specific gRNA production from a Pol II promoter.
- David J. H. F. Knapp
- , Yale S. Michaels
- & Tudor A. Fulga
-
Article
| Open AccessArtificial photosynthetic cell producing energy for protein synthesis
Artificial cells need to be supplied with ATP as they lack internal systems of energy generation. Here the authors reconstruct ATP synthase and bacteriorhodopsins for light-driven ATP generation, powering transcription and translation.
- Samuel Berhanu
- , Takuya Ueda
- & Yutetsu Kuruma
-
Article
| Open AccessPrecisely controlling endogenous protein dosage in hPSCs and derivatives to model FOXG1 syndrome
Altered dosage of developmental regulators such as transcription factors can result in disorders, such as FOXG1 syndrome. Here, the authors demonstrate the utility of SMASh technology for modulating protein dosage by modeling FOXG1 syndrome using human pluripotent stem cell-derived neurons and neural organoids.
- Wenliang Zhu
- , Boya Zhang
- & Baoyang Hu