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 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 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 system of claim 1 , wherein the reporter gene is luxAB.
3. The system of claim 1 , wherein the reporter gene is operatively linked to a constitutive promoter.
4. The system of claim 3 , wherein the constitutive promoter is a Staphylococcus aureus ( S. aureus ) clpB promoter.
5. The system of claim 4 , wherein the S. aureus clpB promoter comprises the sequence of SEQ ID NO: 12.
6. The system of claim 1 , wherein the functional packaging initiation site sequence comprises the sequence of SEQ ID NO: 9.
7. The system of claim 1 , wherein the reporter nucleic acid molecule is a plasmid comprising at least one origin of replication.
8. The system of claim 7 , wherein the at least one origin of replication is a pT181 origin of replication.
9. The system of claim 7 , wherein the at least one origin of replication comprises the sequence of SEQ ID NO: 5.
10. The system of claim 1 , wherein the reporter nucleic acid molecule is a plasmid comprising the sequence of SEQ ID NO: 14.
11. The system of claim 1 , wherein the functional packaging initiation site sequence is a φ80α small terminase (terS) gene.
12. The system of claim 1 , wherein the bacteriophage is φ80α.
13. The system of claim 1 , wherein the non-functional and the functional packaging initiation site sequences are small terminase (terS) genes.
14. 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 φ80α small terminase (terS) gene, wherein the non-functional φ80α terS gene 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 functional φ80α terS gene comprising the sequence of SEQ ID NO: 9, the functional φ80α terS gene facilitating packaging of a replicon of the reporter nucleic acid molecule into the NRTP,
wherein the functional φ80α terS gene on the second nucleic acid construct complements the non-functional φ80α terS gene in the φ80α bacteriophage genome on the first nucleic acid construct.