Abstract: Disclosed are engineered polynucleotides, engineered ribosomes comprising the engineered polynucleotides, engineered cells and systems comprising the engineered polynucleotides and ribosomes, and methods of making and using the engineered polynucleotides, engineered ribosomes, engineered cells and systems. The engineered polynucleotides, engineered ribosomes, and engineered cells may be utilized to prepare sequence defined polymers and to select for mutant ribosomes that are capable of incorporating non-canonical amino acids into a polymer.

Abstract: Disclosed are compositions, methods, and kits for performing cell-free protein synthesis (CFPS) and for expressing proteins in cells. Particularly disclosed are vectors comprising Golden Gate sites for cloning, methods for preparing such vectors, and the use thereof for performing CFPS and for expressing proteins in cells such as in naturally occurring or recombinant species of Clostridia, including Clostridium autoethanogenum.

Abstract: Disclosed are protocols for preparing cell-free extracts preparation protocols that are enriched in membrane vesicles and use of the disclosed extract in cell-free glycoprotein synthesis methods and platforms for increasing glycoprotein yields.

Abstract: Disclosed are components, systems, and methods for glycoprotein protein synthesis in vitro and in vivo. In particular, the disclosed components, systems, and methods relate to modular platforms for producing glycoproteins. The components, systems, and methods disclosed herein may be used in synthesizing glycoproteins and recombinant glycoproteins in cell-free protein synthesis (CFPS) and in modified cells.

Abstract: Disclosed are methods and systems for detecting fluoride as a target molecule in a test sample which utilize a cell-protein synthesis (CFPS) reaction and an engineered fluoride-sensing riboswitch. The components used in the disclosed methods and systems may be dried or lyophilized and may be present or immobilized on a paper substrate.

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 methods, devices, kits, components, and compositions employ one or more transcription templates that encode and conditionally express one or more exogenous RNA polymerases in the presence of the target molecule. The expressed RNA polymerases in turn induce expression of one or more reporter molecules from transcription templates comprising promoters for the RNA polymerases, thereby amplifying an output signal that is generated in the presence of a detected target molecule.

Abstract: Methods and kits are provided for calibrating a cell-free protein synthesis reaction for optimal activity. The method includes the steps of providing an extract competent for cell-free protein synthesis (CFPS); performing cell-free protein synthesis with the extract; measuring a first reaction end-point where in vitro protein synthesis plateaus; measuring a second reaction end-point where Energy Charge of the extract declines to a level in a range from about 0.40 to about 0.80 of Energy Charge of a control extract; and adjusting Energy Charge of the extract to a level in a range from about 0.80 to about 1.0 of the control extract.

Abstract: Cell-free protein synthesis systems and methods of using the same for producing in vitro protein materials in high yield are disclosed. The cell-free protein synthesis platform includes (a) a Saccharomyces cerevisiae cellular extract prepared from mid-exponential to late-exponential batch cultures in the range from about 6 OD600 to about 18 OD600 or fed-batch cultures harvested in mid-exponential to late-exponential phase; (b) a reaction buffer; and (c) a translation template or (c?) a transcription template from which a translation template can be prepared in situ with an RNA polymerase. A method of performing high-throughput protein synthesis in vitro is also provided that utilizes a combined transcription/translation reaction with the cell-free protein synthesis platform from Saccharomyces cerevisiae, an RNA polymerase and a transcription template prepared from a source DNA using an amplification procedure.

Abstract: Methods and kits are provided for calibrating a cell-free protein synthesis reaction for optimal activity. The method includes the steps of providing an extract competent for cell-free protein synthesis (CFPS); performing cell-free protein synthesis with the extract; measuring a first reaction end-point where in vitro protein synthesis plateaus; measuring a second reaction end-point where Energy Charge of the extract declines to a level in a range from about 0.40 to about 0.80 of Energy Charge of a control extract; and adjusting Energy Charge of the extract to a level in a range from about 0.80 to about 1.0 of the control extract.

Abstract: Cell-free protein synthesis systems and methods of using the same for producing in vitro protein materials in high yield are disclosed. The cell-free protein synthesis platform includes (a) a Saccharomyces cerevisiae cellular extract prepared from mid-exponential to late-exponential batch cultures in the range from about 6 OD600 to about 18 OD600 or fed-batch cultures harvested in mid-exponential to late-exponential phase; (b) a reaction buffer; and (c) a translation template or (c?) a transcription template from which a translation template can be prepared in situ with an RNA polymerase. A method of performing high-throughput protein synthesis in vitro is also provided that utilizes a combined transcription/translation reaction with the cell-free protein synthesis platform from Saccharomyces cerevisiae, an RNA polymerase and a transcription template prepared from a source DNA using an amplification procedure.

Abstract: This invention relates to a method of immobilizing biocatalysts including protein and cells by co-precipitation with silicate or organosilicate matrices through the action of an organic template molecule. The organic template molecule is in general a polyamine such as polyethylenimine (PEI), or polypeptide compound bearing at least two or three basic residues selected from the group consisting of lysine, arginine, histidine, proline, hydroxyproline, N-methylhistidine, ornithine, taurine, ?-hydroxylysine, and ?-hydroxy-?-N,N,N trimethyllysine. The invention is also directed to a silica biocomposite comprising co-precipitates of active biocatalysts, silica or organosilicates, and an N-containing organic template molecule. Such silica biocomposites are useful in biocatalysis, and other applications requiring an immobilized biocatalyst. Preferred biocatalysts for this invention are enzymes and whole cells.

Abstract: Methods for making in vitro assembled ribosomal subunits and in vitro assembled ribosomes are provided. Methods of transcribing synthetic rRNA and including the synthetic RNA in a synthetic ribosome are provided. Single vessel methods of transcribing synthetic rRNA, forming a synthetic ribosome that includes the synthetic RNA, and allowing the synthetic ribosome to translate a protein are also provided. Methods of screening for novel, synthetic ribosomal subunits and/or ribosomes are provided. Synthetic replicons and methods of making synthetic replicons are also provided.

Abstract: This invention relates to a method of immobilizing biocatalysts including protein and cells by co-precipitation with silicate or organosilicate matrices through the action of an organic template molecule. The organic template molecule is in general a polyamine such as polyethylenimine (PEI), or polypeptide compound bearing at least two or three basic residues selected from the group consisting of lysine, arginine, histidine, proline, hydroxyproline, N-methylhistidine, ornithine, taurine, ?-hydroxylysine, and ?-hydroxy-?-N,N,N trimethyllysine. The invention is also directed to a silica biocomposite comprising co-precipitates of active biocatalysts, silica or organosilicates, and an N-containing organic template molecule. Such silica biocomposites are useful in biocatalysis, and other applications requiring an immobilized biocatalyst. Preferred biocatalysts for this invention are enzymes and whole cells.

Abstract: This invention relates to a method of immobilizing biocatalysts including protein and cells by co-precipitation with silicate or organosilicate matrices through the action of an organic template molecule. The organic template molecule is in general a polyamine such as polyethylenimine (PEI), or polypeptide compound bearing at least two or three basic residues selected from the group consisting of lysine, arginine, histidine, proline, hydroxyproline, N-methylhistidine, ornithine, taurine, ?-hydroxylysine, and ?-hydroxy-?-N,N,N trimethyllysine. The invention is also directed to a silica biocomposite comprising co-precipitates of active biocatalysts, silica or organosilicates, and an N-containing organic template molecule. Such silica biocomposites are useful in biocatalysis, and other applications requiring an immobilized biocatalyst. Preferred biocatalysts for this invention are enzymes and whole cells.