Expression systems

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Phosphoregulation is a key mechanism of signal processing. Here the authors build a phosphoregulated relay system in mammalian cells for orthogonal signal transduction.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Tools for conditional induction of gene expression in C. elegans are limited compared to other organisms. Here the authors present a tetracycline-dependent ribozyme that allows conditional control of a gene of interest.

Classic monodirectional promoters are of limited use for multiple gene co-expression. Here the authors generate a library of 168 bidirectional promoters for the yeast K. phaffii (syn. P. pastoris) with diverse expression profiles to optimize metabolic pathway design.

Previous efforts to assemble Rubisco within a cyanobacterial carboxysome-derived protein shell in plant chloroplasts to concentrate CO₂ have been unsuccessful. Here, Long et al. produce carboxysomes in tobacco chloroplasts that encapsulate the introduced Rubisco and enable autotrophic growth at elevated CO₂.

The ability to produce homogeneous glycoproteins is expected to advance fundamental understanding in glycoscience, but current in vivo-based production systems have several limitations. Here, the authors develop an E. coli extract-based one-pot system for customized production of N-linked glycoproteins.

The ability to combine the production of multiple biologics into a single ‘on demand’ system could help in situations where resources are limited. Here the authors demonstrate a proof-of-concept approach for the co-production of three biologics, allowing singular, mixed and combination drug products.

For synthetic gene circuits to behave as designed, ligand-inducible promoters should display predictable ON/OFF characteristics. Here the authors design multi-input hybrid promoters to build transcriptional logic gates.

Binary expression systems enable researchers to deliver loss-of-function or gain-of-function transgenes with spatial-temporal resolution in vivo. Here, the authors present a programmable TALE-based system for multiplexed orthogonal activation of transgenes in Drosophila.

The contribution of metabolic pathways to protein secretion is largely unknown. Here, the authors find conserved metabolic patterns in yeast by examining genome-wide transcriptional responses in high protein secretion mutants and reveal critical factors that can be tuned for efficient protein secretion.

Despite the success of current vaccination against poliomyelitis, safe, cheap and effective vaccines remain sought for continuing eradication effort. Here the authors use plants to express stabilized virus-like particles of type 3 poliovirus that can induce a protective immune response in mice transgenic for the human poliovirus receptor.

The maximum titre of therapeutic viral vectors can be adversely affected by the encoded transgene. Here the authors repress transgene expression in producing cells by employing the tryptophan RNA-binding attenuation protein and show that it improves titre of RNA- and DNA-based viral vectors expressing toxic transgenes.

Optogenetics has emerged as a promising means to achieve gene expression control in bioprocess engineering, but current systems cannot respond to fluctuations in growth conditions. Here the authors overcome this limitation and develop an automated optogenetic feedback control system for precise and robust control of protein production in E. coli.

Hybridomas are widely used for antibody screening and production due to their genetic stability and rapid proliferation. Here the authors demonstrate the rapid reprogramming of antibody specificity in hybridomas using CRISPR-Cas9.

Recombination-based tools for generating targeted mutations in Mycobacterium tuberculosislack efficiency. Here the authors present a CRISPR interference approach that is able to efficiently repress the expression of target genes in mycobacteria, in a rapid and cost-effective manner.

Fluorescent proteins are widely used in molecular biology to visualize protein expression and localization. Here, Krizet al. describe an efficient and flexible modular plasmid-based eukaryotic expression cloning strategy for the homogeneous expression of several fluorescent proteins in one cell.