Abstract: Provided herein is a stabilized DNase I polypeptide containing a non-standard amino acid that maintains enzymatic activity even under harsh conditions, such as reducing environments. The stabilized DNase I polypeptide has enzymatic activity in reducing environments that is higher than a corresponding DNase I polypeptide without the non-standard amino acids under the same conditions. Also provided herein are polynucleotides encoding the stabilized DNase I polypeptide, cells for expressing and/or producing the stabilized DNase I polypeptide, and methods of use of the stabilized DNase I polypeptide.

Abstract: Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Biosensors are produced based on a ligand-binding domain (LBD) using a method that, in principle, can be applied for any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. The power of this method is illustrated by developing biosensors for digoxin and progesterone. Addition of ligand to cells expressing a biosensor activates transcription in yeast, mammalian cells and plants, with a dynamic range of up to about 100-fold or more. The biosensors are used to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules.

Abstract: Provided herein is a stabilized DNase I polypeptide containing a non-standard amino acid that maintains enzymatic activity even under harsh conditions, such as reducing environments. The stabilized DNase I polypeptide has enzymatic activity in reducing environments that is higher than a corresponding DNase I polypeptide without the non-standard amino acids under the same conditions. Also provided herein are polynucleotides encoding the stabilized DNase I polypeptide, cells for expressing and/or producing the stabilized DNase I polypeptide, and methods of use of the stabilized DNase I polypeptide.

Abstract: Polypeptides that fold into biologies are stabilized by diselenide bonds between selenocysteine amino acids. Methods to produce such polypeptides in genomically recoded organisms (GRO) can be scaled up for industrial production. Since diselenides have the same geometric bond angles and torsions as disulfides, as well as very similar bond lengths, they can be substituted into polypeptides without disrupting the three dimensional structure of the polypeptides. Diselenides render the polypeptides resistant to reduction when they are exposed to blood serum or to reducing components of blood serum or to reducing components components within cells.

Abstract: Provided herein are pharmaceutical compositions for treating human lung diseases comprising a stabilized DNase I polypeptide containing a non-standard amino acid, a functional fragment thereof, or a variant thereof that maintains enzymatic activity even under harsh conditions, such as reducing environments. The present disclosure also relates to methods of using the composition for the treatment of human of lung diseases or for the disruption of biofilms.

Abstract: Provided herein is a stabilized DNase I polypeptide containing a non-standard amino acid that maintains enzymatic activity even under harsh conditions, such as reducing environments. The stabilized DNase I polypeptide has enzymatic activity in reducing environments that is higher than a corresponding DNase I polypeptide without the non-standard amino acids under the same conditions. Also provided herein are polynucleotides encoding the stabilized DNase I polypeptide, cells for expressing and/or producing the stabilized DNase I polypeptide, and methods of use of the stabilized DNase I polypeptide.

Abstract: A method of making a polypeptide including at least one covalent bond between a pair of reactive side chains of corresponding amino acids, wherein the covalent bond is insensitive to reduction is provided including genetically modifying a genomically recoded organism to express a corresponding synthetase, tRNA or synthetase/tRNA pair for translating mRNA encoding the corresponding amino acids having the reactive side chains into the polypeptide and to express the polypeptide including the at least one pair of the reactive side chains wherein the reactive side chains are oriented near one another when the expressed polypeptide is in a folded configuration, wherein the reactive side chains react to form the covalent bond that is insensitive to reduction.

Abstract: The disclosure provides a biosensor having a ligand binding domain (LBD) or its variant, wherein the stability of the LBD or its variant is conditioned on the presence of specific small molecule ligands, and wherein the LBD or its variant is fused to a reporter protein. The disclosure also provides a method of screening for small molecules that modulate protein stability.

Abstract: Polypeptides that fold into biologies are stabilized by diselenide bonds between selenocysteine amino acids. Methods to produce such polypeptides in genomically recoded organisms (GRO) can be scaled up for industrial production. Since diselenides have the same geometric bond angles and torsions as disulfides, as well as very similar bond lengths, they can be substituted into polypeptides without disrupting the three dimensional structure of the polypeptides. Diselenides render the polypeptides resistant to reduction when they are exposed to blood serum or to reducing components of blood serum or to reducing components components within cells.

Abstract: Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Biosensors are produced based on a ligand-binding domain (LBD) using a method that, in principle, can be applied for any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. The power of this method is illustrated by developing biosensors for digoxin and progesterone. Addition of ligand to cells expressing a biosensor activates transcription in yeast, mammalian cells and plants, with a dynamic range of up to about 100-fold or more. The biosensors are used to improve the biotrans-formation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules.

Abstract: Novel recombinant cells and recombinant organisms persistently expressing nonstandard amino acids (NSAAs) are provided. Methods of making novel recombinant cells and recombinant organisms dependent on persistently expressing NSAAs for survival are also provided. These methods may be used to make safe recombinant cells and recombinant organisms and/or to provide a selective pressure to maintain one or more reassigned codon functions in recombinant cells and recombinant organisms.

Abstract: Disclosed is a biosensor engineered to conditionally respond to the presence of specific small molecules, the biosensors including conditionally stable ligand-binding domains (LBDs) which respond to the presence of specific small molecules, wherein readout of binding is provided by reporter genes or transcription factors (TFs) fused to the LBDs.

Abstract: Recombinant cells and recombinant organisms persistently expressing nonstandard amino acids (NSAAs) are provided. Methods of making recombinant cells and recombinant organisms dependent on persistently expressing NSAAs for survival are also provided. These methods may be used to make safe recombinant cells and recombinant organisms and/or to provide a selective pressure to maintain one or more reassigned codon functions in recombinant cells and recombinant organisms.

Abstract: A method of making a polypeptide including at least one covalent bond between a pair of reactive side chains of corresponding amino acids, wherein the covalent bond is insensitive to reduction is provided including genetically modifying a genomically recoded organism to express a corresponding synthetase, tRNA or synthetase/tRNA pair for translating mRNA encoding the corresponding amino acids having the reactive side chains into the polypeptide and to express the polypeptide including the at least one pair of the reactive side chains wherein the reactive side chains are oriented near one another when the expressed polypeptide is in a folded configuration, wherein the reactive side chains react to form the covalent bond that is insensitive to reduction.

Abstract: Disclosed is a biosensor engineered to conditionally respond to the presence of specific small molecules, the biosensors including conditionally stable ligand-binding domains (LBDs) which respond to the presence of specific small molecules, wherein readout of binding is provided by reporter genes or transcription factors (TFs) fused to the LBDs.

Abstract: Novel recombinant cells and recombinant organisms persistently expressing nonstandard amino acids (NSAAs) are provided. Methods of making novel recombinant cells and recombinant organisms dependent on persistently expressing NSAAs for survival are also provided. These methods may be used to make safe recombinant cells and recombinant organisms and/or to provide a selective pressure to maintain one or more reassigned codon functions in recombinant cells and recombinant organisms.

Abstract: Disclosed is a biosensor engineered to conditionally respond to the presence of specific small molecules, the biosensors including conditionally stable ligand-binding domains (LBDs) which respond to the presence of specific small molecules, wherein readout of binding is provided by reporter genes or transcription factors (TFs) fused to the LBDs.

Abstract: An invention is provided for incorporating web services into JSP tag libraries for use in JSP pages. As a result, web page authors can develop JSP pages, which utilize web services from WSDL enabled servers, without an in-depth knowledge of WSDL. In general, a web services based document is received, which defines a set of web service operations. Each web service operation is translated into a custom action, and the custom actions are grouped into a tag library. Generally, the web services based document can be a WSDL document, and the custom actions can be JSP custom actions. Also, the tag library can be a JSP custom tag library. Particular custom actions from the custom actions that are grouped in the JSP custom tag library can be incorporated in a JSP page. Once the JSP page is created, content for the JSP page can be obtained from a web service using the JSP custom tag library. The JSP page can then generate a document based on the content.

Abstract: An invention is provided for incorporating web services into JSP tag libraries for use in JSP pages. As a result, web page authors can develop JSP pages, which utilize web services from WSDL enabled servers, without an in-depth knowledge of WSDL. In general, a web services based document is received, which defines a set of web service operations. Each web service operation is translated into a custom action, and the custom actions are grouped into a tag library. Generally, the web services based document can be a WSDL document, and the custom actions can be JSP custom actions. Also, the tag library can be a JSP custom tag library. Particular custom actions from the custom actions that are grouped in the JSP custom tag library can be incorporated in a JSP page. Once the JSP page is created, content for the JSP page can be obtained from a web service using the JSP custom tag library. The JSP page can then generate a document based on the content.