The disclosure provides methods of making a protein having a desired non-standard amino acid incorporated at its N-terminus in a cell and methods of screening for an amino acyl tRNA synthetase variant that preferentially selects a non-standard amino acid against its standard amino acid counterpart or undesired non-standard amino acids for incorporation into a protein in a cell.

A red blood cell (RBC) having an agent linked thereto, wherein the agent is linked to at least one endogenous, non-engineered membrane protein of the RBC by a sortase-mediated reaction, preferably by a sortase-mediated glycine conjugation and/or a sortase-mediated lysine side chain ε-amino group conjugation, which may occurring at least on glycine (n) and/or lysine ε-amino group at internal sites of the extracellular domain of at least one endogenous, non-engineered membrane protein, preferably n being 1 or 2, as well as the use of the RBC for delivering drugs and probes.

A red blood cell (RBC) having an agent linked thereto, wherein the agent is linked to at least one endogenous, non-engineered membrane protein of the RBC by a sortase-mediated reaction, preferably by a sortase-mediated glycine conjugation and/or a sortase-mediated lysine side chain ε-amino group conjugation, which may occurring at least on glycine (n) and/or lysine ε-amino group at internal sites of the extracellular domain of at least one endogenous, non-engineered membrane protein, preferably n being 1 or 2, as well as the use of the RBC for delivering drugs and probes.

Engineered FnCas9 variants are provided that have an enhanced kinetic activity and a broader PAM recognition. The protein engineering methodology introduced specific mutations that stabilized interaction between Cas9 enzyme and target DNA. The enhanced kinetic activity increases NHEJ-mediated editing, owing to more efficient DSB generation potential than WT FnCas9, and the broadened PAM specificity increases the target range of FnCas9 variants. Thus, the scope and accessibility of CRISPR-Cas9 system targets are widened, along with generating robust and highly specific engineered FnCas9 variants.

The present invention provides a Mitrecin A polypeptide useful in prevention and treatment of one or more bacteria. Also provided is a method to kill or prevent growth of one or more bacteria comprising contacting the one or more bacteria with a Mitrecin A polypeptide. The target bacteria can be selected from the group consisting of a Gram-positive bacterium, a Gram-negative bacterium, or both. In one embodiment, the present invention is drawn to a polynucleotide encoding a Mitrecin A polypeptide, a vector comprising the polynucleotide, a host cell comprising the polynucleotide, or a composition comprising the Mitrecin A polypeptide, the polynucleotide, the vector, or the host cell.

The present invention provides a Mitrecin A polypeptide useful in prevention and treatment of one or more bacteria. Also provided is a method to kill or prevent growth of one or more bacteria comprising contacting the one or more bacteria with a Mitrecin A polypeptide. The target bacteria can be selected from the group consisting of a Gram-positive bacterium, a Gram-negative bacterium, or both. In one embodiment, the present invention is drawn to a polynucleotide encoding a Mitrecin A polypeptide, a vector comprising the polynucleotide, a host cell comprising the polynucleotide, or a composition comprising the Mitrecin A polypeptide, the polynucleotide, the vector, or the host cell.

The present disclosure relates to a gene recombinant vector of a collagenase, comprising a collagenase gene, wherein an amino acid sequence of a collagenase encoded by the collagenase gene is shown in SEQ ID NO. 1; moreover, a genetically engineered strain of the collagenase and a preparation method of the collagenase are also disclosed; and the collagenase prepared according to the invention is capable of degrading a bone collagen, and improving a yield of a low-molecular-weight bone collagen peptide.

This invention concerns a method for preparing a bacterial supernatant comprising culturing a cell of Pseudomonas environmental strain PF-11; and recovering the supernatant. This invention also concerns a method for reducing the amount of a biofilm on a surface, reducing adhesion of at least one organism to a surface, or reducing microfouling or macrofouling on a surface comprising contacting the surface with a supernatant, supernatant fraction, modified supernatant or modified supernatant fraction of Pseudomonas strain PF-11; or a composition comprising a supernatant, supernatant fraction, modified supernatant or modified supernatant fraction of Pseudomonas strain PF-11, and one or more acceptable carriers. This invention also concerns a method for killing or reducing the growth of a fungus or bacterial cell, or killing or inhibiting the development of an insect or marine copepod, comprising contacting the fungus, bacteria, insect or marine copepod with a supernatant, supernatant fraction, modified supernatant or modified supernatant fraction of a Pseudomonas strain PF-11 culture; or a composition comprising a supernatant, supernatant fraction, modified supernatant or modified supernatant fraction of a Pseudomonas strain PF-11 culture, and one or more acceptable carriers. This invention also concerns a substantially pure culture of Pseudomonas strain PF-11. This invention also concerns a culture that is enriched in Pseudomonas strain PF-11. This invention also provides a method of identifying whether a bacteria is capable of producing one or more extracellular proteases capable of digesting a high molecular weight substrate.

This invention concerns a method for preparing a bacterial supernatant comprising culturing a cell of Pseudomonas environmental strain PF-11; and recovering the supernatant. This invention also concerns a method for reducing the amount of a biofilm on a surface, reducing adhesion of at least one organism to a surface, or reducing microfouling or macrofouling on a surface comprising contacting the surface with a supernatant, supernatant fraction, modified supernatant or modified supernatant fraction of Pseudomonas strain PF-11; or a composition comprising a supernatant, supernatant fraction, modified supernatant or modified supernatant fraction of Pseudomonas strain PF-11, and one or more acceptable carriers. This invention also concerns a method for killing or reducing the growth of a fungus or bacterial cell, or killing or inhibiting the development of an insect or marine copepod, comprising contacting the fungus, bacteria, insect or marine copepod with a supernatant, supernatant fraction, modified supernatant or modified supernatant fraction of a Pseudomonas strain PF-11 culture; or a composition comprising a supernatant, supernatant fraction, modified supernatant or modified supernatant fraction of a Pseudomonas strain PF-11 culture, and one or more acceptable carriers. This invention also concerns a substantially pure culture of Pseudomonas strain PF-11. This invention also concerns a culture that is enriched in Pseudomonas strain PF-11. This invention also provides a method of identifying whether a bacteria is capable of producing one or more extracellular proteases capable of digesting a high molecular weight substrate.

The present invention relates to a modified polypeptide comprising a non-cytotoxic protease, a translocation domain, a destructive protease cleavage site and a Targeting Moiety that binds to a Binding Site on a nerve cell, wherein after cleavage of the destructive cleavage site the polypeptide has reduced potency. The destructive cleavage site is recognized and cleaved by a protease present at or in an off-site target cell, and, in one embodiment, the polypeptide is a modified clostridial neurotoxin. The present invention also relates to the use of said polypeptides for treating a range of conditions, and to nucleic acids encoding said polypeptides.