Cell-free TXTL Synthesis of Infectious Bacteriophage T4 in a Single Test Tube Reaction
Abstract. The bottom-up construction of biological entities from genetic information provides a broad range of opportunities to better understand fundamental p
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A Detailed Cell-Free Transcription-Translation-Based Assay to Decipher CRISPR Protospacer-Adjacent Motifs
The RNA-guided nucleases derived from the CRISPR-Cas systems in bacteria and archaea have found numerous applications in biotechnology, including genome editing, imaging, and gene regulation. However, the discovery of novel…
TXTL-based approach to synthetic cells
…reactions and liposomes for three proteins: the soluble reporter protein eGFP, the membrane proteins alpha-hemolysin (AH) from Staphylococcus aureus, and the mechanosensitive channel of large conductance (MscL) from E. coli….
Multiplex transcriptional characterizations across diverse bacterial species using cell-free systems
Abstract Cell-free expression systems enable rapid prototyping of genetic programs in vitro. However, current throughput of cell-free measurements is limited by the use of channel-limited fluorescent readouts. Here, we describe…
Cell-free transcription–translation: engineering biology from the nanometer to the millimeter scale (review)
Cell-free transcription–translation (TXTL) has become a highly versatile technology to construct, characterize and interrogate genetically programmed biomolecular systems implemented outside living organisms. By recapitulating gene expression in vitro, TXTL offers…
An enhanced assay to characterize anti-CRISPR proteins using a cell-free transcription-translation system
The characterization of CRISPR-Cas immune systems in bacteria was quickly followed by the discovery of anti-CRISPR proteins (Acrs) in bacteriophages. These proteins block different steps of CRISPR-based immunity and, as…
Quantitative modeling of transcription and translation of an all-E. coli cell-free system
…in vitro gene expression, using relevant kinetic constants and concentrations of molecular components, remains insufficiently examined. In this work, we present an ODE (Ordinary Differential Equation)-based model of the expression…
CRISPR-Cas13 Inhibitors Block RNA Editing in Bacteria and Mammalian Cells
…RNA targeting and editing by Cas13a in human cells. Strikingly, type VI-A anti-CRISPRs (AcrVIAs) also significantly muffle the single-nucleic-acid editing ability of the dCas13a RNA-editing system. Mechanistically, AcrVIA1, -4, -5,…
Synthesis of 2.3 mg per ml of Protein with an all Escherichia coli Cell-Free Transcription Translation System
…produce more ATP. As a result, the ATP regeneration is more efficient and cell-free protein synthesis lasts up to 10 h. Using a commercial E. coli strain, we show for…
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