The present disclosure provides RNA polymerase variants for high efficiency transcription.

Some aspects of this disclosure provide methods for phage-assisted continuous evolution (PACE) of proteases. Some aspects of this invention provide methods for evaluating and selecting protease inhibitors based on the likelihood of the emergence of resistant proteases as determined by the protease PACE methods provided herein. Some aspects of this disclosure provide strategies, methods, and reagents for protease PACE, including fusion proteins for translating a desired protease activity into a selective advantage for phage particles encoding a protease exhibiting such an activity and improved mutagenesis-promoting expression constructs. Evolved proteases that recognize target cleavage sites which differ from their canonical cleavage site are also provided herein.

Methods and compositions for capping RNA in an in vitro transcription mixture are provided that include a thermostable RNA polymerase variant and a cap analog such that when a DNA template is added to the mixture, and the mixture is then incubated under conditions for in vitro transcription, capped RNA is produced.

This invention relates to nucleotide analogs and their derivatives (termed non-standard nucleotides) that, when incorporated into DNA and RNA, expand the number of nucleotides beyond the four found in standard DNA and RNA. The invention further relates to enzymatic processes that incorporate those non-standard nucleotide analogs into oligonucleotide products using the corresponding triphosphate derivatives. The RNA polymerases of the instant invention transcribe DNA containing nonstandard nucleotides to give RNA containing nonstandard nucleotides, where certain of those nucleotides have nucleobases that do not present electron density to the minor groove.

Provided herein are T7 RNA polymerase variants (T7 RNAP variants) having increased RNA polymerase activity and/or thermal stability, and methods of use thereof.

Provided herein, among other things, is a method for producing an RNA product that has reduced immunogenicity. In some embodiments, the method involves transcribing a template DNA with a thermostable RNA polymerase at a temperature of greater than 44° C.

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.

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.

The present invention provides methods and compositions for stable genetic modification of cultured mammalian cells. The genetic modifications can be used to produce cultured mammalian cells for therapeutic or diagnostic purposes.

The present invention relates to a novel variant RNA polymerase sigma factor 70 (σ.sup.70) polypeptide, a polynucleotide encoding the same, a microorganism containing the polypeptide, and a method for producing L-threonine by using the microorganism.