Resumen
[0001] The present invention relates to the design, preparation and use of cascade regulatory circuits for amplification of gene expression. The genetic cirucit is based on a plurality (e.g., two or more) of regulatory genes organized in a hierarchical order of expression in a genetic construct or constructs, which can be established in a cell, e.g., a gram-negative bacteria, by means of autoreplicative vectors or by chromosomal insertion. In one embodiment, the genetic construct(s) can be stabiliy maintained in the chromosome without selective pressure, and gene expression induced three orders of magnitude therefrom using economical biodegradable benzoate derivatives.
Reivindicaciones
1. A cascade genetic circuit, comprising: a) one or more nucleic acid constructs encoding the transcriptional regulator NahR or a mutant form thereof and the transcriptional regulator XylS or a mutant form thereof; wherein said encoded transcriptional regulators are arranged in a hierarchical order such that expression of the upstream located NahR transcriptional regulator or the mutant form thereof stimulates expression of the downstream located XylS transcriptional regulator or the mutant form thereof and; wherein the NahR transcriptional regulator or the mutant form thereof and the XylS transcriptional regulator or the mutant form thereof are responsive to the same inducer b) a final target promoter; wherein said final target promoter is responsive in a dose-dependent fashion to the XylS transcriptional regulator or the mutant form thereof. 2. The cascade genetic circuit of claim 1 further comprising a multiple cloning site downstream of said final target promoter. 3. The cascade genetic circuit of claim 1 or 2 wherein at least one of said one or more nucleic acid constructs is present as a chromosomal integration. 4. The cascade genetic circuit of any of claims 1 to 3 wherein at least one of said one or more nucleic acid constructs is present as an autoreplicative plasmid. 5. The cascade genetic circuit of any of claims 1 to 4 wherein said inducer is a benzoate derivative. 6. The cascade genetic circuit of claim 5 wherein said benzoate derivative is salicylate, anthranylate, 2-acetyl salicylate, 4-chloro salicylate, 5-chloro salicylate, 3,5-dichloro salicylate, 5-methoxy-salicylate, 3-methyl benzoate, 2- methoxy-benzoate, 3-methyl salicylate, 4-methyl salicylate, or 5-methyl salicylate. 7. The cascade genetic circuit of any of claims 1 to 6 wherein the expression of the XylS transcriptional regulator or the mutant form thereof is modulated by a nucleic acid molecule having the transcription modulating activity ofPsal. 8. The cascade genetic circuit of any of claims 1 to 7 wherein said final target promoter comprises a nucleic acid molecule having the promoter activity ofPm . 9. The cascade genetic circuit of any of claims 1 to 8 wherein the mutant form of the NahR transcriptional regulator is NahR3 or NahR4. 10. The cascade genetic circuit of any of claims 1 to 9 wherein the mutant form of the XylS transcriptional regulator is XylS2. 11. A cell comprising the cascade genetic circuit of any of the preceding claims. 12. The cell of claim 11 wherein said cell is a prokaryotic cell. 13. The cell of claim 12 wherein said prokaryotic cell is a gram-negative bacterial cell. 14. The cell of claim 13 wherein said gram-negative bacterial cell is aPseudomonas putida or anEscherichia coli cell. 15. A method of regulating the expression of a.nucleic acid sequence, comprising: providing the cell of any of claims 11 to 14; placing said nucleic acid sequence under control of said final target promoter; and inducing said cascade genetic circuit to stimulate expression of said nucleic acid sequence. 16. The method of claim 15 wherein said nucleic acid sequence encodes a member selected from the group consisting of enzymes, hormones, growth factors, apolipoproteins, therapeutic proteins, diagnostic molecules or proteins, anti-sense molecules, ribozymes, rRNAs, tRNAs, snRNAs, and portions or derivatives thereof. 17. The method of claim 16 wherein said encoded member is a diagnostic reporter molecule.
