Non-replicative transduction particles and transduction particle-based reporter systems
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.
1. 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.
2. The bacterial cell packaging system of claim 1 , wherein the reporter gene is luxAB.
3. The bacterial cell packaging system of claim 1 , wherein the reporter gene is operatively linked to a constitutive promoter.
4. The bacterial cell packaging system of claim 3 , wherein the constitutive promoter is a Staphylococcus aureus ( S. aureus ) clpB promoter.
5. The bacterial cell packaging system of claim 4 , wherein the S. aureus clpB promoter comprises the sequence of SEQ ID NO: 12.
6. The bacterial cell packaging system of claim 1 , wherein the bacteriophage genome is a ϕ80α genome.
7. The bacterial cell packaging system of claim 1 , wherein the first bacteriophage gene and the second bacteriophage gene is a ϕ80α small terminase (terS) gene.
8. The bacterial cell packaging system of claim 1 , wherein the second bacteriophage gene comprises the sequence of SEQ ID NO: 9.
9. The bacterial cell packaging system of claim 1 , wherein the bacteriophage genome is an Enterobacteriaceae bacteriophage P1 genome.
10. The bacterial cell packaging system of claim 1 , wherein the first bacteriophage gene and the second bacteriophage gene is a pacA terminase gene of Enterobacteriaceae bacteriophage P1.
11. The bacterial cell packaging system of claim 1 , wherein the second bacteriophage gene comprises the sequence of SEQ ID NO: 7.
12. The bacterial cell packaging system of claim 1 , wherein the reporter nucleic acid molecule is a plasmid comprising at least one origin of replication.
13. The bacterial cell packaging system of claim 12 , wherein the at least one origin of replication is a pT181 origin of replication.
14. The bacterial cell packaging system of claim 12 , wherein the at least one origin of replication comprises the sequence of SEQ ID NO: 5.
15. The bacterial cell packaging system of claim 1 , wherein the reporter nucleic acid molecule is a plasmid comprising the sequence of SEQ ID NO: 14.
16. 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.
17. 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.