Recombinant phage and methods
This disclosure provided methods of cloning a phage genome. Also provided are methods of making a recombinant phage genome. In some embodiments the phage genome is engineered to comprise a heterologous nucleic acid sequence, for example a sequence comprising an open reading frame. In some embodiments the phage genome is cloned in a yeast artificial chromosome. Recombinant phage genomes and recombinant phage are also provided. In some embodiments the methods are high throughput methods such as methods of making a plurality of recombinant phage genomes or recombinant phage. Collections of recombinant phage genomes and recombinant phage are also provided.
1. A method of making a recombinant phage genome, comprising:
(a) selecting a starting phage genome and a eukaryotic vector;
(b) selecting a stitching oligonucleotide or oligonucleotide duplex for recombination with the starting phage genome and the eukaryotic vector comprising:
(i) detecting an imperfect oligo repeat at an end of the starting phage genome, and selecting a stitching oligonucleotide comprising a sequence from the ends of the starting phage genome and a sequence flanking the double strand break in the eukaryotic vector;
(ii) detecting a perfect oligo repeat at an end of the starting phage genome and selecting a oligonucleotide duplex comprising a sequence homologous to the starting phage genome and a sequence homologous to the eukaryotic vector;
(c) introducing the starting phage genome and the eukaryotic vector of step (a), and the stitching oligonucleotide or the oligonucleotide duplex of step (b) into a plurality of eukaryotic vector host cells that are not phage host cells;
(d) selecting a vector host cell comprising a recombinant vector comprising an intact starting phage genome as a result of insertion of the starting phage genome into the vector; and
(e) inserting a heterologous nucleic acid sequence comprising an open reading frame encoding a screenable marker into the intact starting phage genome to provide a recombinant intact phage genome, wherein the screenable marker is operatively linked to a regulatory control sequence.
2. The method of claim 1 , wherein c) comprises co-transforming the starting phage genome and the vector into a plurality of vector host cells, under conditions that allow insertion of the starting phage genome into the vector.
3. The method of claim 1 , wherein c) comprises transforming the starting phage genome into a plurality of vector host cells comprising the vector, under conditions that allow insertion of the starting phage genome into the vector.
4. The method of claim 1 , wherein the vector is a yeast artificial chromosome (YAC).
5. The method of claim 1 , wherein the heterologous nucleic acid sequence is inserted into the intact starting phage genome in vivo.
6. The method of claim 1 , wherein the heterologous nucleic acid sequence is inserted into the intact starting phage genome in vitro.
7. The method of claim 1 , further comprising f) transforming the recombinant intact phage genome into competent phage host cells.
8. The method of claim 7 , further comprising g) producing and recovering recombinant phage particles comprising the recombinant intact phage genome from said competent phage host cells.