Recombination systems for high-throughput chromosomal engineering of bacteria
AssigneeUniversity of Massachusetts
InventorsKenan C. Murphy, Christopher Sassetti
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Abstract
In some aspects, the disclosure relates methods and compositions for genetic engineering of cells (e.g., bacterial cells, etc.). The disclosure is based, in part, on a combination of genetic recombination systems (e.g., a “targeting oligonucleotide” and a “payload plasmid) that enable high-throughput chromosomal engineering and do not involve the preparation of double-stranded DNA (dsDNA) recombination substrates. In some aspects, the disclosure provides engineered bacterial cells comprising a targeting oligonucleotide and a payload plasmid.
Claims19
A method for genetic modification of a bacterium, the method comprising contacting a bacterium with:
- (i) a single-stranded targeting oligonucleotide comprising an attP attachment site flanked by homology arms that are homologous to a target nucleic acid sequence of the bacterium;
- and,
- (ii) a non-replicating payload plasmid comprising an attB attachment site and a nucleic acid sequence encoding a selectable marker,
- wherein a single strand annealing protein (SSAP) and/or an integrase protein is expressed in the bacterium.
A bacterial cell comprising:
- (i) a single-stranded targeting oligonucleotide comprising an attP attachment site flanked by homology arms that are homologous to a target nucleic acid sequence of the bacterial cell;
- and,
- (ii) a non-replicating payload plasmid comprising an attB attachment site and a nucleic acid sequence encoding a selectable marker,
- wherein a single strand annealing protein (SSAP) and/or an integrase protein is expressed in the bacterial cell.
Dependencies are inferred from the claim text.