	US 11,891,630 B2
	Bacteriophage engineering via semi-synthesis
Kirsty A. McFarland, Boston, MA (US); Miles T. Rogers, Boston, MA (US); Connor McBrine, Somerville, MA (US); and Jason W. Holder, Swampscott, MA (US)
Assigned to The Charles Stark Draper Laboratory, Inc.
Filed by The Charles Stark Draper Laboratory, Inc., Cambridge, MA (US)
Filed on Aug. 7, 2018, as Appl. No. 16/057,416.
Claims priority of provisional application 62/542,609, filed on Aug. 8, 2017.
Prior Publication US 2019/0048325 A1, Feb. 14, 2019
Int. Cl. C12N 7/02 (2006.01); C12N 15/66 (2006.01); C12N 15/10 (2006.01); C12N 15/63 (2006.01)

CPC C12N 7/02 (2013.01) [C12N 7/025 (2013.01); C12N 15/1031 (2013.01); C12N 15/63 (2013.01); C12N 15/66 (2013.01); C12N 2795/00041 (2013.01); C12N 2800/202 (2013.01)]

8 Claims

1. A method for generating a recombinant Klebsiella phage K11 bacteriophage genome comprising:

(a) generating a plurality of PCR fragments that are no more than 15 kilobases in length from a template comprising a first Klebsiella phage K11 bacteriophage DNA genome using polymerase chain reaction (PCR), wherein the plurality of PCR fragments collectively span the entire length of the first Klebsiella phage K11 bacteriophage DNA genome, wherein at least one end of each PCR fragment comprises a sequence that is homologous to an opposite end of another PCR fragment and wherein each PCR fragment is no more than 15 kilobases in length; and

(b) recombining in vitro the plurality of PCR fragments with a heterologous nucleic acid in the presence of a recombination system under conditions to produce a recombinant Klebsiella phage K11 bacteriophage genome, wherein the recombinant Klebsiella phage K11 bacteriophage genome comprises the nucleic acid sequence of SEQ ID NO: 23.