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 bacterial cell, the bacterial cell packaging system comprising a host cell, comprising:
a bacteriophage genome comprising a first terminase gene comprising a disrupted packaging initiation site sequence, wherein in the absence of the disruption the first terminase gene encodes a first terminase protein and wherein the first terminase protein recognizes the first-packaging initiation site sequence, wherein the disruption prevents recognition of the packaging initiation site sequence by the first terminase protein, and wherein the disruption further renders non-functional the first terminase protein; and
a reporter nucleic acid molecule comprising a reporter gene, a first terminase gene comprising a non-disrupted packaging initiation site sequence and encoding a functional first terminase protein, and a second terminase gene encoding a second terminase protein, wherein the non-disrupted packaging initiation site sequence is configured to facilitate packaging of a replicon of the reporter nucleic acid molecule into the NRTP.
2. The bacterial cell packaging system of claim 1 , wherein the bacteriophage genome comprises a plurality of disrupted terminase genes, wherein in the absence of the disruptions, each of the plurality of disrupted terminase genes encodes a terminase protein.
3. The bacterial cell packaging system of claim 2 , wherein each of the plurality of disrupted genes on the bacteriophage genome is complemented by a functional, non-disrupted gene encoded by the reporter nucleic acid molecule.
4. The bacterial cell packaging system of claim 1 , wherein the reporter nucleic acid molecule comprises a sequence selected from SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:6.
5. The bacterial cell packaging system of claim 1 wherein the disruption of the first terminase gene is by an insertion, replacement, deletion, or mutation that disrupts the packaging initiation site.
6. The bacterial cell packaging system of claim 5 wherein the disruption of the first terminase gene is by an insertion of a gene encoding a detectable marker, a gene encoding a selectable marker or both a gene encoding a detectable marker and a gene encoding a selectable marker.
7. The bacterial cell packaging system of claim 1 , wherein both the non-disrupted first terminase gene and the second terminase gene on the reporter nucleic acid molecule are operably linked to a conditional promoter.
8. The bacterial cell packaging system of claim 7 , wherein the conditional promoter comprises the sequence of SEQ ID NO: 9.
9. The bacterial cell packaging system of claim 7 , wherein the conditional promoter is a native promoter of a terminase gene of the bacteriophage genome.
10. The bacterial cell packaging system of claim 7 , wherein expression of the non-disrupted first terminase gene or the second terminase gene is inhibited in the absence of activation of the lytic cycle of the bacteriophage, and wherein expression of the non-disrupted first terminase gene or the second terminase gene is activated upon activation of the lytic cycle of the bacteriophage.
11. The bacterial cell packaging system of claim 1 , wherein the reporter gene encodes a detectable marker or a selectable marker.
12. The bacterial cell packaging system of claim 1 , wherein the bacteriophage genome comprises SEQ ID NO: 12.
13. The bacterial cell packaging system of claim 1 , wherein the reporter nucleic acid molecule comprises an origin of replication.
14. The bacterial cell packaging system of claim 1 , wherein the replicon of the reporter nucleic acid molecule comprises a concatamer that is packaged into the non-replicative transduction particle.
15. The bacterial cell packaging system of claim 1 , wherein the replicon comprises the sequence of SEQ ID NO:2.
16. The bacterial cell packaging system of claim 1 , wherein the reporter nucleic acid molecule is operatively linked to a promoter.
17. The bacterial cell packaging system of claim 16 , wherein the promoter is selected for contributing to reactivity of a reporter molecule expressed from the reporter nucleic acid molecule in the bacterial cell.
18. A method for packaging a reporter nucleic acid molecule into a non-replicative transduction particle, comprising:
incubating the bacterial cell packaging system of claim 1 at a temperature that induces a lytic phase of the bacteriophage genome to produce non-replicative transduction particles packaged with the reporter nucleic acid molecule; and
collecting the non-replicative transduction particle comprising the reporter nucleic acid molecule.
19. The method of claim 18 , wherein the non-replicative transduction particle does not contain a replicated bacteriophage genome.
20. The method of claim 18 , wherein the non-replicative transduction particle comprises a portion of the bacteriophage genome due to recombination with the reporter nucleic acid molecule, and wherein the portion of the bacteriophage genome comprises the reporter gene.
21. The bacterial cell packaging system of claim 1 , wherein the first terminase gene is selected from group consisting of a pacA gene of bacteriophage P1, a terS gene of bacteriophage φ11 or φ80a, and a terA gene of bacteriophage φEfl1; and the second terminase gene is selected from the group consisting of a pacB gene of bacteriophage P1, a terL gene of bacteriophage φ11 or φ80a, and a terB gene of bacteriophage φEfl1.