Compositions comprising modified recombinant polymerizing enzymes are provided, along with nucleic acid molecules encoding the modified polymerizing enzymes. In some aspects, methods of using such polymerizing enzymes to synthesize a nucleic acid molecule or to sequence a nucleic acid template are provided.

The present invention provides methods for preparing optimized DNA sequences as templates for in vitro transcription of mRNA. These DNA sequences are optimized to avoid premature termination of transcription by RNA polymerase. The invention also provides methods for preparing optimized DNA sequences that include one or more termination signal at their 3 end to reduce or prevent non-templated runoff 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.

The invention relates to new methods for synthesising polynucleotide molecules according to a predefined nucleotide sequence. The invention also relates to methods for the assembly of synthetic polynucleotides following synthesis, as well as systems and kits for performing the synthesis and/or assembly methods.

Polynucleotide synthesis performed with a substrate independent polymerase such as terminal deoxynucleotidyl transferase (TdT) is regulated by controlling the oxidation state of a metal cofactor. The oxidation state of the metal cofactor is changed to +2, thus activating the polymerase, by applying a voltage with electrodes or by introducing a chemical redox reagent. Addressable polynucleotide synthesis creates polynucleotides with different arbitrary sequences through use of spatial control of cofactor oxidation states to add nucleotides only at selected locations on an array. Control of metal oxidation states is regulated by selective activation of a microelectrode array, controlled addition of redox reagents to specific locations on the array, or controlled activation of photocatalysts at specific locations on the array. Scavengers in solution prevent cofactors distant from the selected locations from catalyzing polymerase activity and thereby maintain the localized effect of polymerase activation.

The invention relates to an in vitro cell-free expression system incorporating a novel inorganic polyphosphate-based energy regeneration system. In certain embodiments, the invention includes a cell-free expression system where the cellular energy source, ATP, is regenerated from inorganic polyphosphate using a dual enzyme system. In this embodiment, this dual enzyme system may include thermostable Adenosyl Kinase, and/or Polyphosphate Kinase enzymes.

Expression vectors ideal for use in vaccinating individuals against disease based on vaccinia virus and other chordopoxviruses having high expression of recombinant genes and low expression of vector genes in target animals, and low expression of recombinant genes and high expression of vector genes in cells used for propagation.

A protein expression system for use in a prokaryotic host is provided, the expression system comprising: a) an expression cassette comprising a nucleic acid sequence encoding a protein of interest operably linked to a T7 RNA polymerase-dependent promoter; and b) an expression cassette comprising a nucleic acid sequence encoding T7 RNA polymerase operably linked to a host polymerase-dependent k phage promoter and a single perfect palindrome operator sequence; wherein the expression cassette for T7 RNA polymerase is located on the chromosome of a host cell.

The disclosure provides methods, compositions, systems, and kits for the concurrent detection and analysis of different structural and chemical forms of nucleic acids in a sample.

Disclosed herein are compositions, methods, cells, engineered microorganisms, and kits for increasing the production of proteins or polypeptides comprising one or more unnatural amino acids. Further provided are compositions, cells, engineered microorganisms, and kits for increasing the retention of unnatural nucleic acids encoding the unnatural amino acids in an engineered cell, or semi-synthetic organism.