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Cell-free protein synthesis can enable the combinatorial screening of many different components and concentrations. However, manual pipetting methods are unfit to handle many cell-free reactions. Here, we describe a microfluidic method that can generate hundreds of unique submicroliter scale reactions. The method is coupled with a high yield cell-free system that can be applied for broad protein screening assays.
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http://dx.doi.org/10.1007/978-1-0716-1998-8_7 | DOI Listing |
In this work, we present a streamlined one-pot cloning and protein expression platform that integrates mutagenesis, plasmid assembly, and functional protein testing in a single reaction. By combining Golden Gate cloning with cell-free transcription-translation, we demonstrate efficient generation and screening of genetic variants without the need for intermediate purification or bacterial amplification. Using fluorescent proteins, luciferase enzymes, antibiotic-converting enzymes, and the violacein biosynthetic pathway, we validate the versatility of this approach for single-and multi-site mutagenesis, combinatorial variant libraries, metabolic pathway programming, and whole-plasmid assembly.
View Article and Find Full Text PDFFront Immunol
September 2025
Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Chimeric Antigen Receptor (CAR)-engineered cell therapies excel against hematologic malignancies, however, their efficacy in solid tumors is hampered by toxicity, poor tumor infiltration, immunosuppressive microenvironments, limited persistence, and expansion difficulties. Recently, exosomes derived from CAR-immune cells (CAR-Exosomes) have emerged rapidly as an innovative therapeutic platform. CAR-Exosomes, utilizing nanoscale communication pathways, inherit their parental cells' tumor-targeting capabilities while offering distinct advantage.
View Article and Find Full Text PDFSci Transl Med
September 2025
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Antibody discovery is crucial for developing therapeutics and vaccines and for understanding adaptive immunity. However, the lack of approaches to synthesize antibodies with defined sequences in a high-throughput manner represents a major bottleneck in antibody discovery. Here, we present oPool display, a high-throughput cell-free platform that combined oligo pool synthesis and mRNA display to rapidly construct and characterize hundreds to thousands of natively paired antibodies in parallel.
View Article and Find Full Text PDFMicrobiology (Reading)
September 2025
Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada.
The opportunistic pathogens and are often found together causing persistent infections where they exhibit complex interactions that affect their virulence and resistance to treatment. We sought to clarify how interactions between these organisms affect their resistance to the antimicrobial metal silver (AgNO). As previous work showed that cell-free supernatant from enhances the resistance of we aimed to identify the exact factor(s) responsible for this increase.
View Article and Find Full Text PDFACS Synth Biol
September 2025
Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, United States.
Rapid and portable antigen detection is essential for managing infectious diseases and responding to toxic exposures, yet current methods face significant limitations. Highly sensitive platforms like the Enzyme-Linked Immunosorbent Assay (ELISA) are time- and cost-prohibitive for point-of-need detection, while portable options like lateral flow assays (LFAs) require systemic overhauls for new targets. Furthermore, the complex infrastructure, high production costs, and extended timelines for assay development constrain the manufacturing of traditional diagnostic platforms in low-resource settings.
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