Cell-free protein synthesis (CFPS) is one approach to address the increasing demand for complex recombinant proteins in various applications, especially in the pharmaceutical sector. CFPS offers a var...

Nuclear biomolecular condensates are functional sub-compartments within the cell nucleus. Here, the authors develop a synthetic DNA protonucleus that enables RNA transcription and condensation into di...

Climate change made 213 historical heatwaves reported over 2000–2023 more likely and more intense, to which each of the 180 carbon majors (fossil fuel and cement producers) substantially contributed.

The programmability of synthetic cells, comprising lipid vesicles that are capable of imitating the structure and function of living cells, facilitates their application as drug delivery devices. Now,...

tRNAs are essential for translating genetic information into proteins. Here, the authors develop a method to synthesize all 21 essential tRNAs from a single DNA in vitro, enabling protein production a...

Synthetic cells encapsulating cell-free protein synthesis machinery are currently limited to laboratory use due to preservation challenges. They are typically produced and used immediately. We present...

Plasmids remain by far the most common medium for delivering engineered DNA to microorganisms. However, the reliance on natural plasmid replication mechanisms limits their tunability, compatibility, a...

The study of “resource allocator” bacteriophage T7 RNA polymerase (T7RNAP) has garnered significant interest, particularly for optimizing transcriptional systems in microbial cell factories (MCFs). Mo...

The One-Pot PURE (Protein synthesis Using Recombinant Elements) system simplifies the preparation of traditional PURE systems by coculturing and purifying 36 essential proteins for gene expression in ...

Cell-free systems are emerging as powerful platforms for synthetic biology with widespread applications in both fundamental research, such as artificial cell construction, and practical uses like recombinant protein production. Among these, cell-free protein synthesis (CFPS) plays a crucial role in gene expression for various downstream applications. However, the development of CFPS systems based on certain chassis, such as Bacillus subtilis, still remains limited due to their low in vitro productivity. Here, we report the development of a highly productive CFPS system derived from an engineered B. subtilis 164T7P strain, which contains a genomic integration of the T7 RNA polymerase gene. This modification allows the preparation of cell extracts that inherently contain T7 RNA polymerase, enabling T7 promoter-based transcription without the supplementation of purified T7 RNA polymerase in CFPS reactions. Through systematic optimization of cell extract preparation and key reaction parameters, we achieved the synthesis of 286 ± 16.7 μg/mL of sfGFP in batch reactions, with yields increasing to over 1100 μg/mL in a semicontinuous format that can replenish substrates and remove inhibitory byproducts. We further demonstrated the system’s versatility by using it for two synthetic biology applications: prototyping ribosome binding site (RBS) elements and synthesizing pulcherriminic acid─a bioactive cyclodipeptide. The system successfully characterized RBS performance, with in vitro and in vivo rankings correlating with predicted strengths, and expressed two active biosynthetic enzymes (cyclodipeptide synthase─YvmC and cytochrome P450 enzyme─CypX), leading to the production of pulcherriminic acid. Overall, our B. subtilis-based CFPS system offers a robust platform for high-yield protein synthesis, in vitro prototyping of gene regulatory elements, and natural product biosynthesis, highlighting its broad potential for synthetic biology and biotechnology applications.

An in vitro system is capable of sustainably regenerating 20 aminoacyl-tRNA synthetases toward artificial cells.

A biohybrid system is presented for mimicking infections in both cellular and tissue contexts. Droplet interface bilayers (DIBs) are spatio-temporally controlled to enable antibiotic addition, withdr...

This perspective offers a cross-disciplinary roadmap toward synthetic life that does not shy away from technical and non-technical challenges, ethical concerns, and provocative implications, embracing...

Cell-free gene expression (CFE) systems are often constrained by numerous biochemical components required to maintain biocatalytic efficiency. Here, the authors propose a droplet-AI combined approach ...