Abstract: Disclosed are methods, systems, components, and compositions for synthesis of sequence defined polymers. The methods, systems, components, and compositions may be utilized for incorporating novel substrates that include non-standard amino acid monomers and non-amino acid monomers into sequence defined polymers. As disclosed herein, the novel substrates may be utilized for acylation of tRNA via flexizyme catalyzed reactions. The tRNAs thus acylated with the novel substrates may be utilized in synthesis platforms for incorporating the novel substrates into a sequence defined polymer.

Abstract: Disclosed are compositions, methods, and kits for performing cell-free RNA transcription and/or cell-free protein synthesis (CFPS). The disclosed compositions, methods, and kits include or utilize components prepared from Vibrio species such as cellular extracts from Vibrio natriegens.

Abstract: Disclosed are compositions, methods, and kits for performing cell-free protein synthesis (CFPS). The disclosed compositions, methods, and kits include or utilize components prepared from plant plastids or extracts thereof. The compositions, methods, and kits may be used for in vitro protein synthesis and prototyping of genetic expression in plants and are suitable for automation.

Abstract: Disclosed are methods, devices, kits, components, and compositions for detecting a target molecule in a test sample using a cell-free protein synthesis (CFPS) reaction. The methods, devices, kits, components, and compositions may be utilized for detecting target molecules which may include small molecules and/or metabolites of small molecules. The components and compositions used in the disclosed methods, devices, and kits may be dried or lyophilized and may be present or immobilized on a paper substrate, for example, as a paper test article.

Abstract: Disclosed are methods, systems, components, and compositions for cell-free synthesis of proteins and glycoproteins. The methods, systems, components, and compositions may be utilized for incorporating non-standard amino acids (nsAAs) into cell-free synthesized proteins and glycosylating or otherwise modifying the cell-free synthesized proteins in vitro. The nsAAs of the cell-free synthesized protein may be modified via glycosylation or other modification.

Abstract: Disclosed are methods, systems, components, and compositions for cell-free synthesis of glycosylated proteins. The glycosylated proteins may be utilized in vaccines, including anti-bacterial vaccines. The glycosylated proteins may include a bacterial polysaccharide conjugated to a carrier, which may be utilized to generate an immune response in an immunized host against the polysaccharide conjugated to the carrier. The glycosylated proteins may be synthesized in cell-free glycoprotein synthesis (CFGpS) systems using prokaryote cell lysates that are enriched in components for glycoprotein synthesis such as oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs) including OSTs and LLOs associated with synthesis of bacterial O antigens.

Abstract: Disclosed are methods, systems, components, and compositions for cell-free synthesis of glycosylated proteins. The glycosylated proteins may be utilized in vaccines, including anti-bacterial vaccines. The glycosylated proteins may include a bacterial polysaccharide conjugated to a carrier, which may be utilized to generate an immune response in an immunized host against the polysaccharide conjugated to the carrier. The glycosylated proteins may be synthesized in cell-free glycoprotein synthesis (CFGpS) systems using prokaryote cell lysates that are enriched in components for glycoprotein synthesis such as oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs) including OSTs and LLOs associated with synthesis of bacterial O antigens.

Abstract: Disclosed are cell-free systems for metabolic engineering, methods for cell-free metabolic engineering, kits for preparing the disclosed systems, and kits for performing the disclosed methods. The disclosed systems, methods, and kits may be utilized to prepare a chemical product or natural product and to optimize conditions for preparing a chemical product or natural product. The disclosed systems, methods, and kits also may be utilized for combinatorial cell-free metabolism engineering.

Abstract: Disclosed are compositions, methods, and kits for performing cell-free RNA transcription and/or cell-free protein synthesis (CFPS). The disclosed compositions, methods, and kits include or utilize components prepared from a species of Clostridia such as cellular extracts from Clostridium autoethanogenum.

Abstract: An engineered ribosome that includes a tethered subunit arrangement is disclosed, in which the engineered ribosome supports translation of a sequence defined polymer. Methods for making and using the engineered ribosome are also disclosed, including a method for preparing a sequence defined polymer using the engineered ribosome and a method for preparing a sequence defined polymer using the engineered ribosome in a two-protein translation system. The engineered ribosomes may be utilized in methods for incorporating unnatural amino acids into a sequence defined polymer. Also disclosed are optimized polynucleotide sequences for use as tethers and Shine-Dalgarno/anti-Shine-Dalgarno sequences.

Abstract: Disclosed are cell-free systems for metabolic engineering, methods for cell-free metabolic engineering, kits for preparing the disclosed systems, and kits for performing the disclosed methods. The disclosed systems, methods, and kits may be utilized to prepare a chemical product and to optimize conditions for preparing a chemical product. The disclosed systems, methods, and kits also may be utilized for combinatorial cell-free metabolism engineering.

Abstract: Disclosed are methods, components, compositions, and kits for preparing and identifying engineered E. coli ribosomes. The E. coli ribosomes may be prepared and identified under a set of defined conditions, such as in the presences of antibiotics, in order to obtain an engineered ribosome that is resistant to the antibiotic.

Abstract: Disclosed are methods, systems, components, and compositions related to genomically recoded and engineered organisms. The disclosed genomically recoded and engineered organisms may be used to prepare extracts for use in platforms and methods for preparing sequence defined biopolymers in vitro. In particular, the methods, systems, components, and compositions relate to genomically recoded and engineered organisms comprising a strain deficient in release factor 1 (RF-1) or a genetic homolog thereof, wherein the genomically recoded organisms have been engineered to express a heterologous RNA polymerase that may be utilized to express a target protein from a transcription template comprising a promoter for the heterologous RNA polymerase, such as bacteriophage T7 RNA polymerase.

Abstract: Disclosed are methods, components, compositions, and kits for preparing and identifying engineered E. coli ribosomes. The E. coli ribosomes may be prepared and identified under a set of defined conditions, such as in the presences of antibiotics, in order to obtain an engineered ribosome that is resistant to the antibiotic.

Abstract: Disclosed are methods, systems, components, and compositions for cell-free synthesis of glycosylated carrier proteins. The glycosylated carrier proteins may be utilized in vaccines, including anti-bacterial vaccines. The glycosylated carrier proteins may include a bacterial polysaccharide conjugated to a carrier, which may be utilized to generate an immune response in an immunized host against the polysaccharide conjugated to the carrier. The glycosylated carrier proteins may be synthesized in cell-free glycoprotein synthesis (CFGpS) systems using prokaryote cell lysates that are enriched in components for glycoprotein synthesis such as oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs) including OSTs and LLOs associated with synthesis of bacterial O antigens.

Abstract: Disclosed are components and systems for cell-free glycoprotein synthesis (CFGpS). In particular, the components and systems include and utilize prokaryotic cell lysates from engineered prokaryotic cell strains that have been engineered to enable cell-free synthesis of glycoproteins.

Abstract: A platform for preparing a sequence defined biopolymer in vitro is disclosed. The platform includes a ribosome-depleted cellular extract ribosomal RNAs prepared by in vitro transcription and purified ribosomal proteins depleted of ribosomal RNAs. A method of synthesizing and assembling ribosomes in vitro for use in the platform is provided, as well as a method for preparing a sequence defined biopolymer in vitro using assembling ribosomes and the platform.

Abstract: Disclosed are methods, systems, components, and compositions for cell-free synthesis of glycosylated proteins. The glycosylated proteins may be utilized in vaccines, including anti-bacterial vaccines. The glycosylated proteins may include a bacterial polysaccharide conjugated to a carrier, which may be utilized to generate an immune response in an immunized host against the polysaccharide conjugated to the carrier. The glycosylated proteins may be synthesized in cell-free glycoprotein synthesis (CFGpS) systems using prokaryote cell lysates that are enriched in components for glycoprotein synthesis such as oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs) including OSTs and LLOs associated with synthesis of bacterial O antigens.

Abstract: Disclosed are methods, systems, components, and compositions related to genomically recoded and engineered organisms. The disclosed genomically recoded and engineered organisms may be used to prepare extracts for use in platforms and methods for preparing sequence defined biopolymers in vitro. In particular, the methods, systems, components, and compositions relate to genomically recoded and engineered organisms comprising a strain deficient in release factor 1 (RF-1) or a genetic homolog thereof, wherein the genomically recoded organisms have been engineered to express a heterologous RNA polymerase that may be utilized to express a target protein from a transcription template comprising a promoter for the heterologous RNA polymerase, such as bacteriophage T7 RNA polymerase.

Abstract: Disclosed are compositions, methods, and kits for enhanced synthesis of a biological macromolecule in vitro using cell-free protein synthesis. The compositions, methods, and kits include or utilize: a cell-free extract; a phosphate-free energy source; and a phosphate source, and typically do not include an exogenous nucleoside triphosphate or an exogenous nucleoside diphosphate.