M13 bacteriophage with a high cysteine content and genetically engineerable hydrogels

A method of making a genetically engineered bacteriophage genome construct, the method comprising:

• (a) contacting a bacteriophage genome with restriction enzymes to produce a cleaved linear bacteriophage genome;
• (b) contacting a heterologous nucleic acid with a second set of restriction enzymes to produce a cleaved heterologous nucleic acid, wherein the heterologous nucleic acid encodes a peptide having two or more cysteine residues;
• and
• (c) ligating the cleaved linear bacteriophage genome with the cleaved heterologous nucleic acid in the presence of a ligase enzyme to produce a genetically engineered bacteriophage genome construct.

A method of making a genetically engineered bacteriophage genome construct, the method comprising:

• (a) contacting a bacteriophage genome with a DNA polymerase enzyme in a polymerase chain reaction (PCR) to produce an extended bacteriophage genome;
• and
• (b) ligating the extended bacteriophage genome with a heterologous nucleic acid in the presence of a ligase enzyme to produce a genetically engineered bacteriophage genome construct, wherein the heterologous nucleic acid encodes a peptide having two or more cysteine residues;
• (c) optionally, prior to step (a), contacting a bacteriophage genome with restriction enzymes to produce a cleaved linear bacteriophage genome is performed.

A method of making a genetically engineered bacteriophage genome construct comprising:

• (a) contacting a bacteriophage with DNA polymerase enzyme in a polymerase chain reaction (PCR), wherein the enzyme amplifies a part of the bacteriophage;
• (b-1) contacting a heterologous nucleic acid, the heterologous nucleic acid encoding a peptide having two or more cysteine residues, with DNA polymerase enzyme in a different PCR, wherein the enzyme amplifies the heterologous nucleic acid, or
• (b-2) contacting two complementary heterologous nucleic acids, wherein one of the heterologous nucleic acids encodes a peptide having two or more cysteine residues, with DNA polymerase enzyme in a different PCR, wherein the enzyme amplifies and anneals the two complementary nucleic acids;
• and
• (c) ligating the amplified bacteriophage with the amplified heterologous nucleic acid in the presence of an exonuclease, a DNA polymerase, and a DNA ligase to produce a genetically engineered bacteriophage genome construct.

A genetically engineered bacteriophage comprising:

• (i) a first plurality of peptides expressed at a surface of the bacteriophage, wherein each peptide comprises two or more cysteine residues and a pVIII major coat protein;
• and
• (ii-1) a second plurality of fusion peptides expressed at a surface of the bacteriophage, wherein the second plurality of fusion peptides comprises a pVIII major coat protein or a pIII, pVI, pVII, or pIX minor coat protein fused to a distinct moiety designed for a specific purpose;
• or
• (ii-2) a second plurality of peptides, wherein the second plurality of peptides comprises peptides that are expressed and/or secreted by a bacterial host cell.

A method of making a genetically engineered bacteriophage, the method comprising expressing

• (i) a first plurality of peptides expressed at a surface of the bacteriophage, wherein each peptide comprises two or more cysteine residues and a pVIII major coat protein;
• and
• (ii-1) a second plurality of peptides expressed at a surface of the bacteriophage, wherein the second plurality of peptides comprises a pVIII major coat protein or a pIII, pVI, pVII, or pIX minor coat protein fused to a distinct moiety designed for a specific purpose;
• or
• (ii-2) a second plurality of peptides comprising peptides that are expressed and/or secreted by a bacterial host cell when the bacteriophage genome construct is propagated therein.

A method of making a genetically engineered bacteriophage genome construct, the method comprising:

• (a) contacting a bacteriophage genome with restriction enzymes to produce a cleaved linear bacteriophage genome;
• (b) contacting a first heterologous nucleic acid with a second set of restriction enzymes to produce a first cleaved heterologous nucleic acid, wherein the first heterologous nucleic acid encodes a peptide having two or more cysteine residues and a pVIII major coat protein;
• (c) contacting a second heterologous nucleic acid with a third set of restriction enzymes to produce a second heterologous nucleic acid, wherein the second heterologous nucleic acid is selected from the group consisting of (i) and (ii), wherein the second heterologous nucleic acid;
• (i) encodes a pVIII major coat protein or a pIII, pVI, pVII, or pIX minor coat protein fused to a distinct moiety designed for a specific purpose;
• and
• (ii) encodes a plurality of peptides expressed and/or secreted by a bacterial host cell when the bacteriophage genome construct is propagated therein;
• and
• (d) ligating the cleaved linear bacteriophage genome with the first and second cleaved heterologous nucleic acids in the presence of a ligase enzyme to produce a genetically engineered bacteriophage genome construct.

A method of making a genetically engineered bacteriophage genome construct, the method comprising:

• (a) contacting a bacteriophage genome with a DNA polymerase enzyme in a polymerase chain reaction (PCR) to produce an extended bacteriophage genome;
• and
• (b) ligating the extended bacteriophage genome with a first and second heterologous nucleic acid in the presence of a ligase enzyme to produce a genetically engineered bacteriophage genome construct;
• (c) optionally, prior to step (a), contacting a bacteriophage genome with restriction enzymes to produce a cleaved linear bacteriophage genome;
• wherein the first heterologous nucleic acid encodes a peptide having two or more cysteine residues and a pVIII major coat protein, and
• wherein the second heterologous nucleic acid (i) encodes a pVIII major coat protein or a pIII, pVI, pVII, or pIX minor coat protein fused to a distinct moiety designed for a specific purpose or (ii) encodes a plurality of peptides expressed and/or secreted by a bacterial host cell when the bacteriophage genome construct is propagated therein.