Non-replicative transduction particles and transduction particle-based reporter systems
AssigneeGeneWeave Biosciences, Inc.
InventorsDiego Ariel Rey, Nikol de Forest, Heather Cox, Soni Shukla
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Abstract
Methods and systems are provided for packaging reporter nucleic acid molecules into non-replicative transduction particles for use as reporter molecules. The non-replicative transduction particles can be constructed from viruses and use viral transduction and replication systems. The reporter nucleic acid molecules include a reporter gene, such as a reporter molecule or selectable marker, for detecting target genes or cells. Methods and systems are provided for detection of cells and target nucleic acid molecules using the non-replicative transduction particles as reporter molecules.
Claims17
A bacterial cell packaging system for packaging a reporter nucleic acid molecule into a non-replicative transduction particle (NRTP) for introduction into a cell, the packaging system comprising:
- a host bacteria cell;
- a first nucleic acid construct inside the host bacteria cell, comprising of a bacteriophage genome having a first bacteriophage gene that contains a non-functional packaging initiation site sequence, wherein the non-functional packaging initiation site sequence prevents packaging of the bacteriophage genome into the NRTP;
- and
- a second nucleic acid construct inside the host bacteria cell and separate from the first nucleic acid construct, comprising of the reporter nucleic acid molecule having a reporter gene and a second bacteriophage gene that contains a functional packaging initiation site sequence for facilitating packaging of a replicon of the reporter nucleic acid molecule into the NRTP,
- wherein the functional packaging initiation site sequence on the second nucleic acid construct complements the non-functional packaging initiation site sequence in the bacteriophage genome on the first nucleic acid construct.
A bacterial cell packaging system for packaging a reporter plasmid into a non-replicative transduction particle (NRTP) for introduction into a cell, the packaging system comprising:
- a host bacteria cell;
- a first nucleic acid construct inside the host bacteria cell, comprising of a ϕ80α bacteriophage genome having a non-functional packaging initiation site sequence within a ϕ80α small terminase (terS) gene, wherein the non-functional packaging initiation site sequence prevents packaging of the ϕ80α bacteriophage genome into the NRTP;
- and
- a second nucleic acid construct inside the host bacteria cell and separate from the first nucleic acid construct, comprising of the reporter plasmid having a luxAB reporter gene operatively linked to a S. aureus clpB promoter comprising the sequence of SEQ ID NO:
- 12, a pT181 origin of replication comprising the sequence of SEQ ID NO:
- 5, and a ϕ80α small terminase (terS) gene comprising the sequence of SEQ ID NO:
- 9 that contains a functional packaging initiation site sequence that facilitates packaging of a replicon of the reporter plasmid into the NRTP,
- wherein the functional packaging initiation site sequence in the ϕ80α small terminase (terS) gene on the second nucleic acid construct complements the non-functional packaging initiation site sequence in the ϕ80α bacteriophage genome on the first nucleic acid construct.
A bacterial cell packaging system for packaging a reporter plasmid into a non-replicative transduction particle (NRTP) for introduction into a cell, the packaging system comprising:
- a host bacteria cell;
- a first nucleic acid construct inside the host bacteria cell, comprising of an Enterobacteriaceae bacteriophage P1 genome having a non-functional packaging initiation site sequence within a bacteriophage P1 pacA terminase gene, wherein the non-functional packaging initiation site sequence prevents packaging of the bacteriophage P1 genome into the NRTP;
- and
- a second nucleic acid construct inside the host bacteria cell and separate from the first nucleic acid construct, comprising of the reporter plasmid having a luxAB reporter gene operatively linked to an Enterobacteriaceae β-lactamase gene bla promoter, a pBBR1-derived origin of replication, and a bacteriophage P1 pacA terminase gene comprising the sequence of SEQ ID NO:
- 7 that contains a functional packaging initiation site sequence that facilitates packaging of a replicon of the reporter plasmid into the NRTP,
- wherein the functional packaging initiation site sequence in the bacteriophage P1 pacA terminase gene on the second nucleic acid construct complements the non-functional packaging initiation site sequence in the bacteriophage P1 genome on the first nucleic acid construct.
Dependencies are inferred from the claim text.