NOVEL METHOD FOR TRANSDUCING PROTEIN-PROTEIN INTERACTIONS

Abstract

The present invention relates to a cell comprising a first nucleic acid sequence encoding a first polypeptide fused to the N-terminus of a first variant of a histidine kinase comprising a DHp domain and a CA domain, wherein said first variant does not comprise a transmembrane domain, a second nucleic acid sequence encoding a second polypeptide fused to the N-terminus of a second variant of said histidine kinase comprising a DHp domain and a CA domain, wherein said second variant does not comprise a transmembrane domain, and a third nucleic acid sequence encoding a response regulatory protein specifically phosphorylatable by said DHp domain of said first or said second variant. The present invention further relates to uses of the cell of the invention.

Claims

1. A cell, particularly a mammalian cell, more particularly a human cell, wherein said cell comprises a first nucleic acid sequence encoding a first polypeptide fused to the N-terminus of a first variant of a histidine kinase comprising a DHp domain and a CA domain, a second nucleic acid sequence encoding a second polypeptide fused to the N-terminus of a second variant of said histidine kinase comprising a DHp domain and a CA domain, and a third nucleic acid sequence encoding a response regulatory protein specifically phosphorylatable by said DHp domain of said first or said second variant.

2. The cell according to claim 1, wherein said first variant and/or said second variant does not comprise a transmembrane domain of said histidine kinase.

3. The cell according to claim 1 or 2, wherein said first variant does not comprise a functional transmitter domain and/or a functional sensor domain of said histidine kinase, and/or said second variant does not comprise a functional transmitter domain and/or a functional sensor domain of said histidine kinase.

4. The cell according to any one of the preceding claims, wherein said response regulatory protein comprises a receiver domain fused to an effector domain, wherein said receiver domain is phosphorylatable by said DHp domain of said first or said second variant, and said effector domain is activatable or inhibitable by the phosphorylated receiver domain.

5. The cell according to any one of the preceding claims, wherein said effector domain is a transcriptional activating domain, said cell comprises a fourth nucleic acid comprising a gene of interest under control of an inducible promoter recognizable by said transcriptional activating domain, wherein upon activation of said transcriptional activating domain the expression of said gene of interest is induced.

6. The cell according to claim 5, wherein said gene of interest encodes a protein of interest or an RNA of interest, wherein particularly said protein of interest is a luminescent protein.

7. The cell according to any one of the preceding claims, wherein said first and said second variant are variants of the EnvZ kinase, said response regulatory protein comprises or a is the OmpR response regulatory protein, or said first and said second variant are variants of the NarX kinase, said receiver domain comprises or a is the NarL response regulatory protein (SEQ ID 6), and said effector domain is or comprises a VP16 transcriptional activation domain (Vp48, SEQ ID 7).

8. The cell according to any one of the preceding claims, wherein said first or said second variant is or comprises a variant selected from EnvZ.sup.180to450 (SEQ ID 8), EnvZ.sup.223to450 (SEQ ID 9), NarX.sup.176to598 (SEQ ID 10) and NarX.sup.379to598 (SEQ ID 11).

9. The cell according to any one of claims 5 to 8, wherein said inducible promoter is selected from OmpR promoter (SEQ ID NO 1), and the NarL-RE (SEQ ID NO 2).

10. The cell according to any one of the preceding claims, wherein said first nucleic acid sequence and/or said second nucleic acid sequence and/or said third nucleic acid sequence is optimized towards the codon usage of said cell.

11. The cell according to any one of the preceding claims, wherein said first nucleic acid sequence and/or said second nucleic acid sequence and/or said third nucleic acid sequence is under transcriptional control of a constitutive promoter.

12. The cell according to claim 11, wherein said constitutive promoter is selected from CMV (SEQ ID NO 3), EF1α (SEQ ID NO 4), and EF1α-V1 (SEQ ID NO 5).

13. The cell according to any one of the preceding claims, wherein said first variant and said second variant are identical.

14. The cell according to claim any one of the preceding claims, wherein said histidine kinase belongs to the transphosphorylation family.

15. The cell according to any one of the preceding claims, wherein said first variant comprises a DHp domain that does not comprise a histidine residue accessible by said CA domain of first or said second variant of said histidine kinase, and/or said second variant comprises a CA domain that is not able to bind ATP.

16. The cell according to any one of the preceding claims, wherein said first variant is or comprises variant NarX.sup.379-598 (H399Q) (SEQ ID NO 12) or NarX.sup.176to598 (H399Q), and/or said second variant is or comprises variant NarX.sup.379-598 (N509A) (SEQ ID NO 13) or NarX.sup.176to598 (N509A).

17. The cell according to any one of the preceding claims, wherein specific binding of said first polypeptide and said second polypeptide is triggerable by a ligand specifically recognizable by said first and/or said second polypeptide.

18. The cell according to claim 17, wherein said first polypeptide is or comprises a receptor and said second polypeptide is or comprises a binding partner of said receptor, wherein binding of said receptor and said binding partner is triggerable by said ligand recognizable by said receptor.

19. The cell according to claim 18, wherein said receptor is a transmembrane receptor, and said binding partner is a cytosolic protein.

20. The cell according to any one of the preceding claims, wherein said first polypeptide consists of or comprises a G-protein coupled receptor and said second polypeptide consists of or comprises a cytosolic ligand of said G-protein coupled, particularly beta-arrestin, or said first polypeptide consists of or comprises a T cell receptor and said second polypeptide is or comprises ZAP-70.

21. The cell according to any one of claims 5 to 20, wherein said gene of interest encodes an immunoprotein, particularly a cytokine or an antibody, or a microRNA that affects the cell's function or integral state.

22. The cell according to any one of the preceding claims, wherein said cell is a mammalian cell, particularly a human cell.

23. A method for assessing a protein-protein interaction, wherein the method comprises the steps of: providing a cell according to any one of claims 1 to 22, said cell comprising, a first nucleic acid sequence encoding a first polypeptide fused to the N-terminus of a first variant of a histidine kinase comprising a DHp domain and a CA domain, a second nucleic acid sequence encoding a second polypeptide fused to the N-terminus of a second variant of said histidine kinase comprising a DHp domain and a CA domain, and a third nucleic acid sequence encoding a response regulatory protein specifically phosphorylatable by said DHp domain of said first or said second variant, and determining the activity of said response regulatory protein, wherein upon specific binding of said first polypeptide and said second polypeptide said first and second variant dimerize such that said CA domain of said first or second variant phosphorylates said DHp domain of said first or second variant, and the activity of said response regulatory protein is modulated, particularly activated or inhibited, by phosphorylation by said DHp domain of said first and/or said second variant.

24. A method for assessing the effect of a compound on a protein-protein interaction, wherein the method comprises the steps of providing a cell according to any one of claims 1 to 22, said cell comprising, a first nucleic acid sequence encoding a first polypeptide fused to the N-terminus of a first variant of a histidine kinase comprising a DHp domain and a CA domain, a second nucleic acid sequence encoding a second polypeptide fused to the N-terminus of a second variant of said histidine kinase comprising a DHp domain and a CA domain, and a third nucleic acid sequence encoding a response regulatory protein specifically phosphorylatable by said DHp domain of said first or said second variant, and contacting said cell with a compound, and determining the activity of said response regulatory protein, wherein upon specific binding of said first polypeptide and said second polypeptide said first and second variant dimerize such that said CA domain of said first or second variant phosphorylates said DHp domain of said first or second variant, and the activity of said response regulatory protein is modulated, particularly activated or inhibited, by phosphorylation by said DHp domain of said first and/or said second variant, and the effect of said compound on said specific binding of said first polypeptide and said second polypeptide is determined by said activity of said response regulator protein.

25. A method for eliciting a desired response in response to a stimulus, wherein the method comprises the steps of: providing a cell according to any one of claims 1 to 22, said cell comprising, a first nucleic acid sequence encoding a first polypeptide fused to the N-terminus of a first variant of a histidine kinase comprising a DHp domain and a CA domain, a second nucleic acid sequence encoding a second polypeptide fused to the N-terminus of a second variant of said histidine kinase comprising a DHp domain and a CA domain, wherein specific binding of said first and second polypeptide is triggered by said stimulus, and a third nucleic acid sequence encoding a response regulatory protein specifically phosphorylatable by said DHp domain of said first or said second variant, wherein upon specific binding of said first polypeptide and said second polypeptide said first and second variant dimerize such that said CA domain of said first or second variant phosphorylates said DHp domain of said first or second variant, and the activity of said response regulatory protein is modulated by phosphorylation by said DHp domain of said first and/or second variant exposing said cell to said stimulus, wherein said desired response is mediated by or is the activity of said response regulatory protein.

26. The method according to any one of claims 23 to 25, wherein said response regulatory protein comprises a receiver domain fused to an effector domain, wherein said receiver domain is phosphorylatable by said DHp domain of said first or said second variant, and said effector domain is activatable by the phosphorylated receiver domain; said effector domain is a transcriptional activating domain, said cell further comprises a fourth nucleic acid sequence encoding a gene of interest under control of an inducible promoter recognizable by said transcriptional activating domain, wherein upon activation of said transcriptional activating domain the expression of said gene of interest is induced,

27. The method according to claim 26, wherein the presence of the expression product of said gene of interest is determined as the activity of said response regulatory protein, or the expression product of said gene of interest is or mediates said desired response.

28. A vector, particularly suitable for transfecting or transducing a mammalian cell, particularly a human cell, comprising: a first nucleic acid sequence as recited in any one of claims 1 to 22, a second nucleic acid sequence as recited in any one of claims 1 to 22, a third nucleic acid sequence as recited in any one of claims 1 to 22, and optionally a fourth nucleic acid sequence as recited in any one of claims 5 to 22.

Description

DESCRIPTION OF THE FIGURES

[0123] FIG. 1 shows schematics of native and transplanted two-component signaling pathways. (A) The native pathway consists of a receptor histidine kinase (HK) protein, which in presence of its signal would autophosphorylate and phosphorylate its cognate response regulator (RR) at the level of one asparagine present in the receiver domain. The phosphorylated RR would bind the specific response element present in promoter regulated by the RR. (B) The transplanted TCS in mammalian host are expressed from genes with human-optimized codon sequence. The histidine kinase transplanted in mammalian cell are always active and autophosphorylate and phosphorylate the RR. The transplanted RR are augmented with VP48 trans activating domain. The phosphorylated RR will bind the RE present in the engineered promoter that drives the expression of the gene of interest, in this case the fluorescent reporter cerulean. DNB, DNA binding domain. VP48, VP48 transactivator domain; Pmin, minimal mammalian promoter.

[0124] FIG. 2 shows Cis versus Trans autophosphorylation of the HK. (A) Schematics representation of the cis-autophsophorylation (the upper lane) and of the trans-autophosphorylation (lower lane) in mammalian cells expressing WT HK (first column), DHp mutant (second column), CA mutant (third column) or DHp mutant and CA mutant (fourth column). (B) Quantitative data for mammalian cells expressing the reporter gene alone or in combination with wild type HK, DHp mutant (EnvZ H243V, NarX H399Q and DcuS H350L), CA mutant (EnvZ N347A, NarX N509A and DcuS N445A) or DHp mutant and CA mutant. The bar charts display Cerulean level normalized to the expression of the transfection control in norm. u. as mean±SD of independent biological triplicates.

[0125] FIG. 3 shows the activity of truncated HK. (A) Schematic representation of phosphotransfer occurring in between element of EnvZ/OmpR (the upper lane) and of NarX/NarL (lower lane) in mammalian cells expressing WT HK (first column), sensor domain truncated mutant (second column), or sensor and transmitter domain truncated mutant (third column). (B) Quantitative data for mammalian cells expressing reporter gene alone or in combination with wild type HK, sensor domain truncated mutant (EnvZ.sup.180to450 and NarX.sup.176to598), or sensor and transmitter domain truncated mutant (EnvZ.sup.223to450 and NarX.sup.379to598). The bar charts display Cerulean level normalized to the expression of the transfection control in norm. u. as mean±SD of independent biological triplicates.

[0126] FIG. 4 shows the design of the protein/protein interaction assay. (A) Schematic representation of the PPI assay to monitor the interaction between two proteins P1 and P2. The interaction between P1 and P2, spontaneously or induced by the compound, allow the dimerization of the short cytoplasmic domain of HK of the trans phosphorylation family mutated at the level of the CA domain fused to P1 with short cytoplasmic domain of HK of the trans phosphorylation family mutated at the level of the DHp domain fused to P2. The dimerization will trigger the phosphorylation of the RR which will bind its RE and induces the expression of the reporter gene. (B) Quantitative data for mammalian cells expressing HK mutant fused to SZ1 or SZ2 domain. (C) Quantitative data for mammalian cells expressing HK mutant fused to FKBP or FRB domain in absence (white bar) or in presence of A/C heterodimezer (black bar) which induce the dimerization of the FKBP and FRB domain. The bar charts display Cerulean level normalized to the expression of the transfection control in norm. u. as mean±SD of independent biological triplicates.

[0127] FIG. 5 shows the design of the protein/protein interaction assay (PPI) for GPCR. (A) Schematic representation of the TANGO assay to monitor the interaction between GPCR and beta-arrestin. The interaction between GPCR and beta-arrestin induced by the agonist allow the beta-arrestin-TEV protease fusion to localize at the level of the GPCR-tTA and trigger the release of the transcription factor tTA. (B) Schematic representation of the PPI assay to monitor the interaction between GPCR and beta-arrestin. The interaction of GPCR and beta-arrestin induced by the agonist, allows the dimerization of the short cytoplasmic domain of HK of the trans phosphorylation family mutated at the level of the CA domain fused to GPCR with short cytoplasmic domain of HK of the trans phosphorylation family mutated at the level of the DHp domain fused to beta-arrestin. The dimerization will trigger the phosphorylation of the RR which will bind its RE and induces the expression of the reporter gene. (C) Quantitative data for mammalian cells expressing the TANGO. (D) Quantitative data for mammalian cells expressing HK mutant fused to GPCR or beta-arrestin in absence (white bar) or in presence of procaterol (black bar). The bar charts display Cerulean level normalized to the expression of the transfection control in norm. u. as mean±SD of independent biological triplicates.

[0128] FIG. 6 shows the restoration of two-component signalling via forced dimerization of protein moieties fused at the C-terminus or at the N-terminus of NarX. Signalling levels in mammalian cells expressing the response regulator NarL and NarL-regulated mCerulean fluorescent protein reporter, alone or with different combinations of SynZip1 and SynZip2 fused at the C-terminus or at the N-terminus of NarX, as indicated in the chart. The bar charts display Cerulean level normalized to the expression of the transfection control in norm. u. as mean±SD of independent biological triplicates.

[0129] FIG. 7 shows the comparison of CMV and EF1α promoters. (a) iRFP fluorescence of HEK cells transfected with the plasmid expressing iRFP from the CMV promoter (white bar) or from the EF1α promoter (black bars). The reporter expression in DMEM without any ligand or in the presence of 1 μM of procaterol or 2 μM of epinephrine is shown as indicated. The bar charts display iRFP level normalized to the frequency of the transfection marker Citrine-positive cells (rel. u.) as mean±SD of independent biological triplicates. (b) Activity of the NarX/NarL expressed from CMV or EF1a promoters. Every transfection contains NarL-regulated mCerulean fluorescent protein reporter and plasmids expressing NarL and NarX from CMV, EF1α or EF1α-V1 promoters, as indicated in the chart. The bar chart displays Cerulean levels normalized to the expression of the transfection control in norm. u. as mean±SD of independent biological triplicates.

[0130] FIG. 8 shows the restoration of two-component signalling via forced dimerization fused to wild-type NarX or various NarX mutants. Signalling levels in mammalian cells expressing the response regulator NarL and NarL-regulated mCerulean fluorescent protein reporter, alone or with different combinations of SynZip1 and SynZip2 fused to wild-type NarX or various NarX mutants, as indicated in the chart. The bar charts display Cerulean level normalized to the expression of the transfection control in norm. u. as mean±SD of independent biological triplicates.

[0131] FIG. 9 shows the restoration of two-component signalling via forced dimerization. Signalling levels in mammalian cells expressing the response regulator NarL and NarL-regulated mCerulean fluorescent protein reporter, alone or with only one different variant of NarX mutant fused to SynZip1 or SynZip2, as indicated in the chart. The bar charts display Cerulean level normalized to the expression of the transfection control in norm. u. as mean±SD of independent biological triplicates.

[0132] FIG. 10 shows the transduction of cytoplasmic ligand concentration to gene expression. (a) Signalling levels in mammalian cells expressing the response regulator NarL and NarL-regulated mCerulean fluorescent protein reporter, alone or with only one different variant of NarX mutant fused to FKBP and FRB, as indicated in the chart. For each pair of bars, the bar on the left (white) represents reporter expression without the A/C ligand, and the bar on the right (black) represents reporter expression with the ligand (100 nM). The bar charts display Cerulean level normalized to the expression of the transfection control in norm. u. (b) Representative microscopy images of the same transfections whose images are shown in FIG. 3b, including the transfection control channel (Red). In all panels the top and the bottom row of images show, respectively, the expression of mCherry transfection reporter (red pseudocolor), and pathway-induced mCerulean protein output (cyan pseudocolor) in the same transfection with or without ligand. The bar charts display Cerulean level normalized to the expression of the transfection control in norm. u. as mean±SD of independent biological triplicates.

[0133] FIG. 11 shows the rewiring of the GPCR activity to the expression of a reporter gene. (a) Signalling levels in mammalian cells expressing the response regulator NarL and NarL-regulated mCerulean fluorescent protein reporter, and different combinations of the indicated protein domains and their fusions. (b) Signalling levels in mammalian cells expressing the TANGO assay components from CMV or EF1α-V1 promoter. For each pair of bars in panels a and b, the bar on the left (white) represents reporter expression without procaterol, and the bar on the right (black) represents reporter expression with procaterol (2 μM). The bar charts display Cerulean level normalized to the expression of the transfection control in norm. u. as mean±SD of independent biological triplicates. (c) Representative microscopy images of the same selected transfections whose images are shown in FIG. 4, here also showing the expression of the mCherry transfection control. In all panels the top and the bottom row of images show, respectively, the expression of mCherry transfection reporter (red pseudocolor), and pathway-induced mCerulean protein output (cyan pseudocolor) in the same transfection with or without ligand.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

[0134] The present invention provides a novel approach to transduce protein/protein interactions into gene expression in mammalian cells using components derived from the two-component system present in bacteria. The invention can be used to develop new screening assay for protein-protein interactions in general, and for GPCR signaling modulation in particular. The herein described system is characterized by superior dynamic range compared to the state of the art (e.g., the TANGO system from ThermoFischer), and it has great potential for high-throughput multiplexing thanks to an almost unlimited supply of the building blocks.

[0135] The present invention may provide the basis of a synthetic signal transduction module in cell-based biosensors and engineered therapeutic cells, with such properties as low background levels, high dynamic range, and reversibility. As the approach can be multiplexed, it will allow the creation of complex logic-based circuits which could provide novel capabilities to the modified cell.

[0136] Particularly, the present invention comprises 3 different features: [0137] the HK (histidine kinase) component (split into two different mutants) [0138] the RR component [0139] the genetic construct containing the chimeric promoter allowing the expression of the gene of interest.

[0140] The HK domains fused to the 2 interacting proteins are mutated to increase the dynamic range. Wild-type domains can also be used, but so far this resulted in lower dynamic range. Nevertheless, the use of the wild type domain can confer some advantages when the system is used to detect homodimerization. In addition, in case of homodimerization of a protein of interest, HK belonging to the cis family can be used. The advantage of this approach is the reduction in the number of genetic constructs. Nevertheless, in all cases the important feature of the invention is the fusion of the protein of interest to a size-reduced domain of the HK that does not dimerize on its own and therefore does not transduce transcriptional activity on its own, unless forced to dimerize with the help of the fused components.

[0141] The RR (response regulator protein) used in this experiment is fused with VP48 as transcriptional activating domain functional in mammalian cells. Other transcriptional activating domains could be fused to the RR, for example p65 (RelA) domain or Rta. In the present examples, the inventors used RR that binds directly to DNA, but other types of RR can be used depending of the desired readout. For example, some RR can bind to RNA and others are enzymes, catalyzing the production of a compound feeding into a secondary signal transduction chain, e.g., cyclic di-GMP.

[0142] The gene construct expressing the gene of interest (GOI) comprises 2 elements. The first part is a chimeric promoter. The inventors used a minimal promoter linked to an upstream sequence with a number of binding sites for the RR. The distance between the minimal promoter and the number of RE can be modified to tune the expression of the GOI, either up- or down.

[0143] The GOI used in the experiment is a fluorescent reporter Cerulean but this can be replaced with any other protein- or RNA-coding gene. For example, GOI could be a miRNA or guide RNA that itself can regulate endogenous genes.

[0144] The present invention differs from the aforementioned systems TANGO and ChaCha in that these systems release the transcription factor previously fused, respectively, to the GPCR or beta-arrestin, and this transcription factor accumulates over time. On the contrary, in the present invention, all the elements are still functional after one act of protein-protein interaction and therefore they exhibit multiple turnover. The gene of interest modulated by both the TANGO and the system of the invention is under the regulation of a chimeric promoter. In the ChaCha system, endogenous genes can also be modulated using appropriately designed gRNA. Another difference between this invention and the TANGO assay is that in this invention, one needs an additional component, namely, the RR in addition to the chimeric GPCR fusion and beta-arrestin fusion.

[0145] The size of the HK domains fused to the GPCR and to the beta-arrestin are only 223 aa. In the TANGO the size of the fused proteins is 240 aa and 341 aa, respectively. For the ChaCha they are 240 aa and 1900 aa, respectively, in addition to the requirement for gRNA expression. Due to this smaller size the system is easier to construct and it places less burden on the cells.

[0146] The system of the invention contains two signal amplification steps. The first step, unique to the invention, results from the reconstituted HK which phosphorylates multiple copies of the RR. The second level of amplification, also present in the TANGO and ChaCha system, is the catalytic nature of gene induction by the RR. Two levels of amplification in the present invention result in a 10-fold improvement in the dynamic range compared to the TANGO system.

[0147] The system of the invention is not desensitized in the course of time; the same protein can be re-activated after several cycles of presence/absence of ligand. On the contrary, the elements of the TANGO and ChaCha system can only be used once and therefore their system activity relies on the protein degradation and de novo protein synthesis, an inherently slow process. This characteristic lets the system of the invention switch more rapidly from On state to Off and vice versa.

[0148] Due to the fact that the present invention contains one extra level of amplification compared to the other approaches, it can detect low amounts of protein and weak protein-protein interactions. The advantage of this feature is that the expression of the components needed for the assay of the invention can be modulated in the cells from low to high. In this way, the level of expression of the system components can be set at a level that enables an appropriate ligand-inducible dynamic range. Therefore, the present approach reduces ligand-independent signalling that can occur due to protein overexpression in the earlier approaches.

[0149] Specifically, the assay of the invention shows higher dynamic range than the TANGO assay. This parameter would facilitate the automated analysis of the result generated by the present invention compared to the TANGO.

[0150] Another advantage of the system of the invention is that it can be multiplexed by employing different HK-RR pairs simultaneously. Due to the very large number of natural two-component systems, the potential to multiplex is very large. Each chimeric pair would be independent and induce a different output. Therefore, in the same experiment one will be able to test the effect of a compound on multiple protein-protein interactions or multiple GPCRs at once. Multiplexing is more difficult with other approaches because the number of well characterized TEV proteases is limited.

[0151] The system of the invention also enables reversibility due to the fact that the RR spontaneously dephosphorylates. In the absence of the interaction between protein pairs, the kinase is not active anymore and does not phosphorylate the RR. The unphosphorylated RR cannot induce the expression of the GOI. In the case of the TANGO and ChaCha, the reversibility of the system is difficult because it requires time-consuming degradation of the transcriptional activator released after the interaction between the GPCR and beta-arrestin.

[0152] Lastly, the assay of the invention is highly modular. The same pair of complementing HK fragments can be utilized to detect a protein-protein interaction in the cytoplasm and membrane-localized protein-protein interaction, as in the case of GPCR induction assay. Other assays require big adjustments for probing different types of protein-protein interaction, and assays such as TANGO are specific to GPCR pathways and have not been used to probe generic protein-protein interaction.

[0153] The present invention can have multiple applications.

[0154] 1) It can be used for the development of a new screening assay for the identification of compounds interacting with a GPCR. The resulting assay is expected to have better specificity and a higher dynamic range than the existing ones. It will also be easier to multiplex. The commercialization of the assays can be done by selling stable cell lines containing the GPCR fused to the HK, similar to what is currently done by DiscoverX and ThermoFischer (PathHunter and the TANGO).

[0155] 2) It can be used to screen for compounds modulating protein-protein interactions and therefore for drug discovery. As opposed to the yeast 2 hybrid, the screening assays could be done in mammalian cells, which is a more relevant system. In addition, with the present invention, the same assay can be used for proteins localized to different cell compartments (nucleus, cytoplasm or at the membrane).

[0156] 3) Existing therapeutic cell-based agents often use signaling pathways that involve protein-protein interactions at the cytoplasmic side of the membrane. This includes CAR-T cells where the binding of the antigen to the extracellular antibody fragment recruits protein interaction partners; these interactions can then be rewired to result in therapeutic effects using the inventors' approach.

Examples

[0157] It is known that intracellular cytoplasmic domains of HK are capable of dimerizing and autophosphorylating.

[0158] The present invention is based on the question whether partial cytoplasmic domains have a reduced inherent capacity to signal. To this end, the inventors undertook stepwise truncation mutagenesis of the HKs to identify domains that fail to dimerize on their own (FIG. 1b). The first set of truncated mutants comprised the entire cytoplasmic domain of EnvZ and NarX (EnvZ.sup.180to450 and NarX.sup.176to598, respectively) and the second set featured deeper truncation, with the N-terminus about 20 amino acids (aa) upstream of the histidine (EnvZ.sup.223to450 and NarX.sup.379to598, respectively). The inventors found that the truncation mutants of EnvZ, coexpressed with the cognate response regulator OmpR in HEK293 cells, were able to signal constitutively and induce the expression of OmpR-regulated reporter mCerulean at a level comparable with the wild-type EnvZ (FIG. 1c). This result is consistent with the fact that a small EnvZ domain containing the histidine (“Domain A”, aa 223-289) is responsible for homodimerization. On the other hand, truncated mutants of NarX showed size-dependent reduction in basal signalling in the presence of the cognate RR NarL and NarL-inducible reporter, reaching background levels with the shortest mutant, NarX.sup.379to598 (FIG. 1d). A number of explanations were possible for this result, including (1) reduction in protein stability; (2) the decrease in the kinase activity and/or the increase in the phosphatase activity of the truncated mutant and (3) the inability of the mutant to dimerize on its own. Among these explanations, only the last one would support the eventual establishment of synthetic signalling. To check if forced dimerization would restore signalling, the inventors attempted to fuse the truncated NarX domain to a pair of proteins forming strong heterodimers in mammalian cells. The reasoning was if the dimerization alone was impaired, these same mutants, attached to proteins with strong affinity to each other, would dimerize and transduce the signal downstream.

SynZip1/SynZip2

[0159] The inventors used a known interaction pair SynZip1 and SynZip2, and fused them to the N- and the C-termini of the short NarX domain NarX.sup.379to598. It was observed (FIG. 6) that coexpression of SynZip1 and SynZip2 fusions to the N-terminus of the short NarX domain resulted in an increased reporter expression, although the expression was much lower than the signalling of the full-length NarX. On the other hand, coexpression of SynZip fusions to the C-terminus of NarX did not result in any dimerization-triggered increase reporter expression. Overall, the result hinted at the possibility of forced dimerization as a signalling mechanism via fusion to the N-terminus of the short cytoplasmic NarX domain, consistent with the position of the sensor domain relative to the cytoplasmic domain in the full-length HK. However, the quantitative behaviour was poor. In parallel to this study, the inventors optimized the promoters driving their constructs to make sure they do not respond to external stimulation and that the expression levels are balanced (FIG. 7).

[0160] For any synthetic signalling system, it is important to avoid non-specific changes to the signalling readout. In the system of the invention, the components are expressed from constitutive promoters. However, these promoters are in fact controlled by highly-expressed transcription factors such as Sp1, and there is always a risk that these factors are directly affected by external stimuli via unrelated endogenous pathways. This would result in the change of constitutive expression, and apparent change in the signalling readout that is unrelated to the studied effect and is artefactual. To eliminate these confounding factors, the inventors examined a number of constitutive promoters for their robustness under various stimulation conditions and compared the expression of iRFP from the CMV and EF1α promoters in the presence of different compounds often used to induce cell signalling (Epinephrine and procaterol). In the medium without any compound, the activity of both promoters is similar. However, in presence of epinephrine and procaterol the activity of the CMV promoter is induced by a factor of two, while the activity of the EF1α promoter is not affected (FIG. 7a).

[0161] The activity of the NarX/NarL system expressed from CMV, EF1α, and EF1α-V1 promoters was quantified to determine whether a too-high expression of the truncated HK cytoplasmic domain would increase the background level and respond to non-specific interactions. The EF1α-V1 promoter is around 5 times weaker than EF1α. The results demonstrate that comparable expression of the reporter gene is obtained with NarX expressed from any of the tested promoter (FIG. 7b, compare lanes 2 and 3 and lanes 5 and 6). However, when NarL is expressed from the weaker promoter, a reduction in the expression of the reporter gene was observed (Supplementary FIG. 2b). In the light of these results, the inventors used EF1α-V1 to drive NarX-derived constructs and EF1α to drive NarL expression.

[0162] The inventors further explored possibilities to optimize the effect. It is known that the HKs can be divided into two families with respect to the autophosphorylation mechanism. In the “cis”-family, the phosphoryl group is transferred to the histidine from an ATP molecule bound to a CA domain of the same monomer. In the “trans”-family, the phosphoryl group is transferred from an ATP bound to one monomer to the phosphorylatable histidine in the other monomer (FIG. 2a). For all HKs, phosphoryl group transfer can be stopped by either mutating the ATP binding site or the histidine. A heterodimer formed between an ATP binding site mutant and a histidine mutant would be incapable of signalling in the case of a “cis”-family HK, but in the case of a “trans”-family HK, it will in fact still be able to signal via unidirectional phosphate transfer from the histidine mutant monomer to the ATP-binding site mutant monomer (FIG. 2b). Therefore, the mutants complement each other for “trans”-family HKs.

[0163] The inventors hypothesized that dimerization between complementing mutants could result in a more efficient transduction due to reduced phosphatase activity of the mutant HK towards its cognate response regulator. Using protein alignment, the inventors identified the putative residues important for ATP binding in the CA domains of NarX.

[0164] Mutational analysis of the aa present in the CA domain of EnvZ had allowed to identify the asparagine in position 347 to be primordial for kinase activity of EnvZ. The CA domains of the histidine kinase belongs to the large family of the ATPase domain of HSP90 chaperone/DNA topoisomerase II/histidine kinase protein (Superfamily 55874, http://supfam.org/SUPERFAMILY/cgi-bin/scop.cgi?sunid=55874). To identify if the N347 of EnvZ is conserved in NarX, the inventors aligned EnvZ and NarX with proteins containing the ATPase domain. The alignment identified the Asn509 for NarX as a conserved residue that is potentially important for ATP binding.

[0165] Because the full-length HKs were shown to signal constitutively in the mammalian cells, the inventors set up the complementation assay in HEK293 cells by cotransfecting different combinations of codon-optimized mutants of the NarX together with the cognate downstream RR NarL and the reporter gene driven by NarL-responsive promoter. In this assay (FIG. 2c), it was shown that (i) the full-length wild-type NarX is able to signal as shown before; (ii) the mutants of either the aspartate in the CA domain important for ATP binding, or the histidine in the DHp domain, are unable to signal on their own, as expected; (iii) co-expression of the complementing mutants of NarX partially restores the signalling to the levels obtained with the wild-type NarX.

[0166] Next, the same mutations were introduced in the short cytoplasmic domain of NarX (FIG. 2d), and the resulting mutants were fused at the N-terminus to the SynZip1 and SynZip2 peptides as conducted earlier with the wild-type domain, creating, respectively, the constructs SynZip1::NarX.sup.379to598H399Q (labelled for brevity from now on as SynZip1::H.sup.mut) and SynZip2::NarX.sup.379to598N509A (labelled SynZip2::N.sup.mut) (FIG. 2e). The inventors also inverted the fusion pairs, generating SynZip2::NarX.sup.379t.sup.0598H399Q (SynZip2::H.sup.mut) and SynZip1::NarX.sup.379to598N509A (SynZip1::N.sup.mut). It was found that when these pairs of constructs were coexpressed in HEK293 cells together with the NarL RR in the presence of the NarL-responsive reporter, the signalling via the NarL was fully restored to the level obtained with full-length, wild-type NarX, and generating a much stronger signal compared to the wild-type short NarX fusions (FIG. 8). The restoration was the same for both pairs (FIG. 2f, bars 11 and 12). When one or both of the fused SynZip domains were missing, the signalling stopped (FIG. 2f, bars 4-8, FIG. 9), indicating that the dimerization of the fusion domains was necessary and sufficient to restore the signalling. Interestingly, the pair of complementing NarX mutants, both fused to SynZip1 (SynZip1::H.sup.mut and SynZip1::N.sup.mut) also resulted in elevated signalling activity (FIG. 2f, bar 9), consistent with the (weaker) effect observed under similar conditions with the wild type domain (FIG. 6, bar 4). This leads to the hypothesis that SynZip1 domain is capable of homodimerizing, albeit with a reduced affinity compared to SynZip1-SynZip2 interaction. Indeed, examination of the original literature suggests that SynZip1 exists as both a monomer and a dimer in size exclusion chromatography experiment, and it does so to a larger extend than SynZip2 alone. To evaluate the dose-response behaviour of the signalling intensity, the inventors characterized output levels for varying plasmid dosage of the NarX-derived constructs (FIG. 2g). The activity increases proportionally to the plasmid dosage, with the full-length wild-type NarX exhibiting the highest dosage sensitivity, likely reflecting the strongest dimerization constant, the SynZip1-SynZip2 pair showing a slightly reduced but comparable dimerization behaviour, and SynZip1 clearly showing inferior dimerization. For example, reducing the plasmid amount by about 16-fold compared to the initially-used conditions (6.25 ng instead of 100 ng) reduces SynZip1-SynZip1 signalling to background level, while still resulting in strong SynZip1-SynZip2 dimerization, consistent with expectation.

[0167] In summary, these experiments suggest that NarX-enabled signalling can be restored upon forced dimerization of the complementing, truncated mutant domains, and that this restoration is dose- and interaction strength dependent. Consistently with the current knowledge of two component signalling stoichiometry with about 1:30 HK:RR ratio in E. coli, NarX expression of about 10% compared to NarL fully activates the reporter output. This is because the promoter driving NarL, EF1α-V1 (see Methods), is about 5 times weaker than wild-type EF1α promoter driving NarL, and in addition plasmid dosage ratio of 1:2 (i.e., 50 ng of NarX-derived plasmid vs 100 ng of the RR-encoding plasmid) already saturates the response.

FK506/FKBP

[0168] To enable bona fide signalling, the dimerization of the NarX domains should be preferably controlled by an external stimulus. Inducible protein-protein interaction is a common signalling mechanism both in the cytoplasm and across the membrane. A well characterized ligand-induced heterodimerization takes place between the proteins FK506-binding protein 12 (FKBP) and FKBP12-rapamycin binding domain (FRB) mutant FRB.sup.T2098L in the presence of the small molecule A/C heterodimerize (a rapamycin analog Cl 6-(S)-7-methylindolerapamycin, known also as AP21967).

[0169] To find out if NarX domains are capable of transducing this interaction (FIG. 3a), the complementing histidine and asparagine NarX mutants described above were fused at their N-terminus to the FKBP (FK) and the FRB.sup.T2098L (FR) proteins, respectively, resulting in the fusions FK::NarX.sup.379to598H399Q (FK::H.sup.mut) and FR::NarX.sup.379to598N509A (FR::N.sup.mut), and the inverse pair FR::H.sup.mut and FK::N.sup.mut. First, it was confirmed that NC did not affect the wild-type NarX/NarL system, transfecting HEK cells with NarX or NarX.sup.379to598 in the absence or the presence of 100 nM of A/C (FIG. 3b, bars 2 and 3). Next, the inventors probed the ligand-induced signalling in a fashion similar to the one used for SynZip1-SynZip2 experiments, expressing different fusion variants and control constructs in HEK293 cells, in the presence of the response regulator NarL and NarL-activated reporter construct, this time with and without the ligand (FIG. 3b, FIG. 8). As expected, full length wild type NarX was constitutively active. In all the cases except with wild-type NarX, there was no signalling in the absence of the ligand. High level of the ligand was able to induce strong signalling (FIG. 3b, bars 11 and 12) only when (i) the NarX-derived domains contained complementary histidine and asparagine mutations and (ii) they were fused, respectively, to FKBP and FRB interaction partners. The inventors then proceeded to characterize the dose-response behaviour of this engineered signal transduction pathway using the pair FR::H.sup.mut and FK::N.sup.mut (FIG. 3c). The dose-response shows an expected Hill function dependency, from which the EC50 in the context of the assay was determined to be 1.3 nM as compared to the published values of 10 nM and 36 nM.

[0170] The above results illustrate the ability of TCS-based components to mediate signal transduction in the cytoplasm. However, a bulk of signalling takes place across the membrane. Many transmembrane signalling pathways involve protein-protein interactions at the cytoplasmic surface of the lipid bilayer, including an important class of signalling pathways initiated by G-protein coupled receptors (GPCRs), a family of a few hundred proteins. A key step of GPCR signal transduction is the formation of a complex between the GPCR itself and the protein beta-arrestin, followed by various processes that include GPCR internalization, recycling, and signaling. This interaction was previously shown to be sufficient for rewiring GPCR signalling by specific proteolytic cleavage of a fused transcriptional activator. The inventors hypothesized that this interaction could also enable catalytic transmembrane signalling via the two-component pathway. To this end, the inventors fused truncated histidine mutant of NarX, NarX.sup.379to598H399Q, to a GPCR ADRB2-AVPR2: the procaterol-activated chimera of Adrenoceptor Beta 2 (ADRB2) and the cytoplasmic fragment of Arginine Vasopressin Receptor 2 (AVPR2). The inventors also fused the truncated asparagine mutant of NarX, NarX.sup.379to598N509A, to beta-arrestin 2 (FIG. 4a). Lastly, the inventors implemented these fusions in reverse to check if the effect was symmetric.

[0171] First, it was confirmed that procaterol did not affect the signalling via NarX/NarL system. HEK293 cells co-transfected with NarL and NarL-activated reporter with either NarX or NarX.sup.379to598, in the absence or presence of 100 nM procaterol, showed respectively fully induced and background reporter expression independent of the procaterol (FIG. 4b, bars 2 and 3). In the experiments (FIG. 4b, FIG. 11a) combining the complementary NarX mutants fused, respectively, to the GPCR receptor, beta-arrestin, or both, it was found that there was a certain amount of procaterol-independent signalling with beta-arrestin and the GPCR receptor alone (FIG. 4b, bars 9 and 10). The effect was more pronounced with the GPCR, suggesting that the receptors dimerize in ligand-independent fashion. Importantly, very strong procaterol-triggered signalling took place when the complementary NarX mutants were fused to the GPCR receptor and the beta-arrestin, respectively, with an induction dynamic range above 3 orders of magnitude (FIG. 4b, bars 11 and 12). The obtained dynamic range with the two-component based system was higher than the one obtained in a TANGO assay, a proteolysis-based assay for GPCR activation (FIG. 11b).

[0172] To determine whether the synthetic signalling cascade is able to recapitulate the effects of different known GPCR ligands, the inventors characterized the dose response of a system comprising the pair ADRB2-AVPR2::H.sup.mut and beta-arrestin::N.sup.mut in the presence of two agonists (procaterol, isoproterenol) and one partial agonist (clenbuterol). It was observed that the two full agonists, procaterol (FIG. 4c, blue line) and isoproterenol (FIG. 4c, red line), induce strong downstream gene expression in a dose-dependent manner and reach the same maximum response at saturating doses. In the presence of the partial agonist clenbuterol, the expression of the reporter gene is 3.5 times lower than in presence of the full agonists (FIG. 4c, compare green line with blue and red lines). In addition, single-cell flow cytometry data suggests monomodal, rather than bimodal, induction (FIG. 4d, TCS data). EC50 values of procaterol, isoproterenol and clenbuterol were determined from the dose-response curves to be 5 nM, 30 nM and 14 nM, respectively. These values are similar to the ones described in the literature and to the values that were determined by using the TANGO assay (FIG. 4c, dashed lines, secondary axes). Note that while the TANGO assay results in higher absolute reporter expression, the leakage is much higher compared to TCS-based mechanism and the single cell data is bimodal (FIG. 4d, TANGO data). The inventors also characterized the effect of the antagonist propranolol in the presence of the procaterol, and found that in the signalling cascade of the invention, the antagonist inhibited the effect of the procaterol in a dose-dependent fashion (FIG. 4e, blue line). The inventors determined the IC.sub.50 of the antagonist in the context of this assay to be 2 nM, which is similar to the value obtained by using the TANGO assay (FIG. 4e, dashed line). These results demonstrate that the system of the invention can faithfully transduce a variety of known effects of agonists and antagonists on the GPCR activity and it can be used to extract quantitative data on interaction parameters.

SUMMARY

[0173] Implementing non-native signalling modalities in cells, in particular mammalian cells, is highly desirable for rational control of cell behaviour and ultimately, engineering novel cellular functions for basic research, biotechnology and medicine. Two-component signalling is evolutionary extremely divergent from vertebrate signalling, and to the best of the inventors' knowledge, not a single instance of histidine to aspartate phosphoryl transfer has been described in vertebrate cells. The native mechanism of TCS signal transduction in prokaryotes relies on ligand-induced conformation change of the HK dimer in the membrane, but direct implementation of this mechanism in mammalian cells has been elusive. Instead, here the inventors pursued a different strategy to achieve essentially the same end result by controlling the signalling via switching between dissociated and associated states of the HK cytoplasmic domains. In the cases the inventors show here, the switching was accomplished by ligand-induced dimerization of proteins fused, respectively, to histidine and asparagine mutant of a truncated cytoplasmic domain of an HK NarX. A similar qualitative effect is observed when wild-type truncated domain is used instead of the mutants. However, the quantitative behaviour is inferior and more importantly, the resulting effect does not distinguish between ligand-induced dimerization of the two interaction partners and homo-dimerization of one of the partners, as was the case with SynZip1 and GPCR. One reason for the reduced dynamic range could be the stronger phosphatase activity of the wild-type domain, compared to the histidine mutant.

[0174] The approach retains many of the features of the original prokaryotic signalling. It is an amplifying, multiple turnover process with a single NarX dimer capable of phosphorylating multiple copies of the response regulator NarL, which in turn can induce multiple transcription initiation events. The inventors speculate that the two-step amplification resulted in a greatly improved dynamic range compared to the proteolytic-cleavage based approach. Further, upon stimulus withdrawal, the signalling will cease due to spontaneous dephosphorylation of the RR; this can be facilitated by judicious employment of the wild-type HK domains that retain their full phosphatase activity if quick signalling quiescence is required. Given the rich variety of TCS pathways, multiplexing of the synthetic signalling pathways is feasible by using the methods described above. Together, the result point toward a novel modality for sensing and signal transduction in mammalian cells, both in the cytoplasm and across the membrane.

Methods

[0175] Standard molecular cloning techniques were employed as available to the skilled person.

Plasmid Construction.

[0176] Plasmids were constructed using standard cloning techniques. All restriction enzymes used in this work were purchased from New England Biolabs (NEB). Q5 High-Fidelity DNA Polymerase (NEB) was used for fragment amplification. Single-strand oligonucleotides were synthesized by Sigma-Aldrich. Digestion products or PCR fragments were purified using GenElute Gel Extraction Kit or Gen Elute PCR Clean Up Kit (Sigma-Aldrich). Ligations were performed using T4 DNA Ligase (NEB) by temperature cycle ligation with 140 cycles between 30 s at 10° C. and 30 s at 30° C. Gibson assembly was done as described below. 5 μl of the ligation product or the Gibson assembly product were transformed into chemically competent E. Coli DH5a or E. coli TOP10 that were plated on LB Agar with Ampicillin at 100 μg/ml. The resulting clones where screened directly by colony-PCR (Dream Taq Green PCR Master Mix, Thermo Scientific). The inventors expanded single clones in LB Broth Miller Difco (BD) supplemented with ampicillin and purified their plasmid DNA using GenElute Plasmid Miniprep Kit (Sigma-Aldrich). All the resulting plasmids were sequence-verified by Microsynth using Sanger sequencing method. The DNA for mammalian transfection was obtained from 100 ml of liquid culture using the Promega PureYield™ Plasmid Midiprep System (A2495). The recovered DNA was further purified using the Norgen Endotoxin Removal Kit Mini (Cat. #27700) or Midi (Cat. #52200). A short cloning procedure for each construct used within this work is described below.

Gibson Assembly Protocol

[0177] The Gibson assembly was performed in 10 μl final volume by mixing vectors (0.018 pmol) and inserts (0.09 pmol) in 1× Gibson assembly buffer (0.1 M Tris-HCl, pH 7.5, 0.01 M MgCl.sub.2, 0.2 mM dGTP, 0.2 mM dATP, 0.2 mM dTTP, 0.2 mM dCTP, 0.01 M DTT, 5% (w/v) PEG-8000, 1 mM NAD), 0.04 units of T5 exonuclease (NEB), 0.25 units of Phusion DNA polymerase (NEB) and 40 units of Taq DNA ligase (NEB). Negative controls for Gibson assemblies included vectors alone. The Gibson assembly was realized at 50° C. for 1 h.

Recombinant DNA Cloning Protocols

[0178] OmpR_RE-cerulean (pMZ1): The mCerulean coding sequence from EF1α-cerulean (pKH24) was digested with NotI and SmaI and cloned into the plasmid OmpR_RE-amCyan (pJH008) digested with AfeI and PspOMI CMV-envZN347A (pMZ37): The 5′ and the 3′ fragments of envZ was PCR amplified with PR3687/PR3708 and PR3707/PR3709 from the plasmid CMV-envZ (pJH001). The primers were designed to introduce a mutation exchanging the codon encoding for the asparagine (N) at the 347th position to codon encoding for an alanine (A). Both PCR products and the plasmid CMV-envZ (pJH001.sup.14) digested with XhoI and PvuII were assembled using Gibson mix.

[0179] CMV-envZ.sup.223to450 (pMZ123): The 3′ fragments of envZ was PCR amplified with PR4345/PR4346 from the plasmid CMV-envZ (pJH1). The primers were designed to amplify the sequence from the 20th codon upstream of the codon encoding for the phosphorylable histidine at the position 243 till the end of the gene, and to insert ATG sequence in front of this amplified sequence. The PCR products and the plasmid CMV-envZ (pJH001) digested with XhoI and AgeI were assembled using Gibson mix.

[0180] CMV-narX N509A (pMZ160): The 5′ and the 3′ fragments of narX were PCR amplified with PR4122/PR4541 and PR4346/PR4542 from the plasmid CMV-narX (pJH002). The primers were designed to introduce a mutation exchanging the codon encoding for the asparagine (N) at the 509th position to codon encoding for an alanine (A). Both PCR products and the plasmid CMV-envZ (pJH001) digested with XhoI and AgeI were assembled using Gibson mix.

[0181] CMV-narX.sup.379to598 (pMZ163): The 3′ fragment of narX was PCR amplified with PR4345/PR4546 from the plasmid CMV-narX (pJH002.sup.14). The primers were designed to amplify the sequence from the 20th codon upstream the codon encoding for the phosphorylable histidine at the position 399 till the end of the gene, and to insert ATG sequence in front of this amplified sequence. The PCR products and the plasmid CMV-envZ (pJH001) digested with XhoI and AgeI were assembled using Gibson mix.

[0182] EF1α-V1-envZ-mCherry (pMZ194): The EF1α-V1, as shortened version of EF1α, was PCR amplified with PR4733/PR4734 from the plasmid pRA114 (Altamura et al, manuscript in preparation). The promoter and the plasmid EnvZ-GGGGS-mCherry (pEM017.sup.14) digested with PspOMI and AgeI were assembled using Gibson mix.

[0183] CMV-SynZip1::narX.sup.379to598 (pMZ200): the inventors performed de novo synthesis of gBlock sequence encoding for SynZip1 and G4S linker (gBlock264) via IDT. The coding sequence of NarX.sup.379to598 was PCR amplified with PR4346/PR4747 from the plasmid CMV-narX.sup.176to598 (JH010.sup.14). The gBlock, the PCR product and the plasmid CMV-envZ (pJH001) digested with AgeI and XhoI were assembled using Gibson mix.

[0184] CMV-narX.sup.379to598::SynZip1 (pMZ202): The inventors performed a de novo synthesis of gBlock sequence encoding for G4S linker and SynZip1 (gBlock265) via IDT. The coding sequence of NarX.sup.379to598 was PCR amplified with PR4122/PR4747 from the plasmid CMV-narX.sup.379to598 (pMZ163). The gBlock, the PCR product and the plasmid CMV-envZ (pJH001) digested with AgeI and XhoI were assembled using Gibson mix.

[0185] CMV-SynZip2::narX.sup.379to598 (pMZ206): The inventors performed a de novo synthesis of gBlock sequence encoding for SynZip2 and G4S linker (gBlock269) via IDT. The coding sequence of NarX.sup.379to598 was PCR amplified with PR4346/PR4747 from the plasmid CMV-narX.sup.176to598 (JH010). The gBlock, the PCR product and the plasmid CMV-envZ (pJH001) digested with AgeI and XhoI were assembled using Gibson mix.

[0186] CMV-narX.sup.379to598::SynZip1 (pMZ208): The inventors performed a de novo synthesis of gBlock sequence encoding for G4S linker and SynZip1 (gBlock270) via IDT. The coding sequence of NarX.sup.379to598 was PCR amplified with PR4122/PR4748 from the plasmid CMV-narX.sup.379to598 (pMZ163). The gBlock, the PCR product and the plasmid CMV-envZ (pJH001) digested with AgeI and XhoI were assembled using Gibson mix.

[0187] CMV-FRB T2098L::CBRC (pMZ211): The 5′ of FRB and the 3′ fragments of FRB with CBRC were PCR amplified with PR4122/PR4541 and PR4346/PR4542 from the plasmid FRB::CBRC.sup.27. The primers were designed to introduce a mutation exchanging the codon encoding for the threonine (T) at Both098.sup.th position (relative to the full protein Serine/Threonine-protein kinase TOR1) to codon encoding for a leucine (L). Both PCR products and the plasmid FRB::CBRC digested with BamHI and AgeI were assembled using Gibson mix.

[0188] CMV-FKBP::narX.sup.379to598 (pMZ214): The sequence encoding for FKBP was PCR amplified with PR4766/PR4767 from the plasmid CBRN::FKBP.sup.27. The coding sequence of NarX.sup.379to598 was PCR amplified with PR4346/PR4771 from the plasmid CMV-narX.sup.176to598 (pJH010). The primers were designed to insert (G4S)2 linker between the amplified fragment. Both PCR products and the plasmid CMV-envZ (pJH001) digested with AgeI and XhoI were assembled using Gibson mix.

[0189] CMV-FRB T2098L::narX.sup.379to598 (pMZ215): The sequence encoding for FRB T2098L was PCR amplified with PR4769/PR4770 from the plasmid CMV-FRB::CBRC (pMZ211). The coding sequence of NarX.sup.379to598 was PCR amplified with PR4346/PR4771 from the plasmid CMV-narX.sup.176to598 (pJH010). The primers were designed to insert (G4S)2 linker between the amplified fragment. Both PCR products and the plasmid CMV-envZ (pJH001) digested with AgeI and XhoI were assembled using Gibson mix.

[0190] NarL_RE-cerulean (pMZ219): The minimal response element NarL_RE was formed out by annealing the primers PR4892 and PR4893. The annealed product and the plasmid OmpR_RE-cerulean (pMZ1) digested with AscI and NdeI were assembled using Gibson mix.

[0191] EF1α-V1-SynZip1::narX.sup.379to598 (pMZ221): The sequence encoding for SynZip1, G4S linker and NarX.sup.379to383 was PCR amplified with PR3687/PR4971 from the plasmid CMV-SynZip1:: NarX.sup.379to598 (pMZ200). The PCR product and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.

[0192] EF1α-V1-SynZip2::narX.sup.379to598 (pMZ222): The sequence encoding for SynZip2, G4S linker and NarX.sup.379to383 was PCR amplified with PR3687/PR4971 from the plasmid CMV-SynZip2::narX.sup.379to598 (pMZ206). The PCR product and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.

[0193] EF1α-V1-SynZip1::narX.sup.379to598 H399Q (pMZ223): The sequence encoding for SynZip1 and G4S linker was PCR amplified with PR4971/PR4973 from the plasmid CMV-SynZip1::narX.sup.379to598 (pMZ200). The sequence encoding for NarX.sup.379to598 H399Q was PCR amplified with PR3687/PR4972 from the plasmid CMV-narX H399Q (pEM014). Both PCR products and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.

[0194] EF1α-V1-SynZip2::narX.sup.3′ H399Q (pMZ224): The sequence encoding for SynZip2 and G4S linker was PCR amplified with PR4971/PR4973 from the plasmid CMV-SynZip2::narX.sup.379to598 (pMZ206). The sequence encoding for NarX.sup.379to598 H399Q was PCR amplified with PR3687/PR4972 from the plasmid CMV-narX H399Q (pEM014). Both PCR products and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.

[0195] EF1α-V1-SynZip1::narX.sup.379to598 N509A (pMZ225): The sequence encoding for SynZip1 was PCR amplified with PR4971/PR4973 from the plasmid CMV-SynZip1::narX.sup.379to598 (pMZ200). The sequence encoding for NarX.sup.379to598 N509A was PCR amplified with PR3687/PR4972 from the plasmid CMV-narX N509A (pMZ160). Both PCR products and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.

[0196] EF1α-V1-SynZip2::narX.sup.379to598 N509A (pMZ226): The sequence encoding for SynZip2 was PCR amplified with PR4971/PR4973 from the plasmid CMV-SynZip2::narX.sup.379to598 (pMZ206). The sequence encoding for NarX.sup.379to598 N509A was PCR amplified with PR3687/PR4972 from CMV-narX N509A (pMZ160). Both PCR products and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.

[0197] EF1α-V1-FKBP::narX.sup.379to598 H399Q (pMZ229): The sequence encoding for FKBP and (G4S)2 linker was PCR amplified with PR4974/PR4973 from the plasmid CMV-FKBP:: NarX.sup.379to598 (pMZ214). The sequence encoding for NarX.sup.379to598 H399Q was PCR amplified with PR3687/PR4972 from the plasmid CMV-narX H399Q (pEM014). Both PCR products and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.

[0198] EF1α-V1-FRB T2098L::narX.sup.379to598 H399Q (pMZ230): The sequence encoding for FRB T2098L and (G4S)2 linker was PCR amplified with PR4975/PR4973 from the plasmid CMV-FRB T2098L::narX.sup.379to598 (pMZ215). The sequence encoding for NarX.sup.379to598 H399Q was PCR amplified with PR3687/PR4972 from the plasmid CMV-narX H399Q (pEM014). Both PCR products and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.

[0199] EF1α-V1-FKBP::narX.sup.379to598 N509A (pMZ231): The sequence encoding for FKBP and (G4S)2 linker was PCR amplified with PR4974/PR4973 from the plasmid CMV-FKBP::narX.sup.379to598 (pMZ214). The sequence encoding for NarX.sup.379to598 N509A was PCR amplified with PR3687/PR4972 from the plasmid CMV-narX N509A (pMZ160). Both PCR products and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.

[0200] EF1α-V1-FRB T2098L::narX.sup.379to598 N509A (pMZ232): The sequence encoding for FRB T2098L and (G4S)2 linker was PCR amplified with PR4975/PR4973 from the plasmid CMV-FRB T2098L::narX.sup.379to598 (pMZ215). The sequence encoding for NarX.sup.379to598 N509A was PCR amplified with PR3687/PR4972 from the plasmid CMV-narX N509A (pMZ160). Both PCR products and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.

[0201] EF1α-V1-narX (pMZ239): The sequence encoding for NarX was PCR amplified with PR3687/PR4979 from the plasmid CMV-narX (pJH002.sup.14). The PCR product and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix

[0202] EF1α-V1-narX.sup.379to598 (pMZ241): The 3′ fragment of narX was PCR amplified with PR4977/PR3687 from the plasmid CMV-narX (pJH002). The primers were designed to amplify the sequence from the 20th codon upstream the codon encoding for the phosphorylable histidine at the position 399 till the end of the gene and to insert the ATG sequence in front of this amplified sequence. The PCR product and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.

[0203] EF1α-V1-narX H399Q (pMZ242): The sequence encoding for NarX was PCR amplified with PR3687/PR4979 from the plasmid CMV-narX H399Q (pEM014.sup.14). The PCR product and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.

[0204] EF1α-V1-narX.sup.379to598 H399Q (pMZ244): The 3′ fragments of narX was PCR amplified with PR4977/PR3687 from the plasmid CMV-narX H399Q (pEM014). The primers were designed to amplify the sequence from the 20th codon upstream of the codon encoding for the phosphorylable histidine at the position 399 till the end of the gene and to insert the ATG sequence in front of this amplified sequence. The PCR product and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.

[0205] EF1α-V1-narX N509A (pMZ245): The sequence encoding for NarX was PCR amplified with PR3687/PR4979 from the plasmid CMV-narX N509A (pMZ160). The PCR product and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.

[0206] EF1α-V1-narX.sup.379to598 N509A (pMZ247): The 3′ fragment of narX was PCR amplified with PR4977/PR3687 from the plasmid CMV-narX N509A (pMZ160). The primers were designed to amplify the sequence from 20 codon upstream the codon encoding for the phosphorylable histidine at the position 399 to the end of the gene and to insert the ATG sequence in front of this amplified sequence. The PCR product and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.

[0207] EF1α-narL (pMZ248): The EF1α promoter was PCR amplified with PR4732/PR4978 from the plasmid pRA58 (Altamura et al, manuscript in preparation). The PCR product and the plasmid the plasmid CMV-narL (pJH004) digested with PspOMI and AgeI were assembled using Gibson mix

[0208] EF1α-V1-narL (pMZ249): The EF1α-V1 promoter was PCR amplified with PR4734/PR4978 from the plasmid pRA114 (Altamura et al, manuscript in preparation). The PCR product and the plasmid the plasmid CMV-narL (pJH004) digested with PspOMI and AgeI were assembled using Gibson mix.

[0209] EF1α-V1-ARRB2::narX.sup.379to598 (pMZ250): The sequence encoding for ARRB2 was PCR amplified with PR4980/PR4981 from the plasmid CMV-ARRB2::TEV protease (pBH302). The sequence encoding for NarX.sup.379to598 was PCR amplified with PR3687/PR4982 from the plasmid CMV-narX (pJH2). The primers were designed to insert G4S linker between the amplified fragment. Both PCR products and EF1α-V1-envZ-mCherry the plasmid (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.

[0210] EF1α-V1-ARRB2::narX.sup.379to598 H399Q (pMZ251): The sequence encoding for ARRB2 was PCR amplified with PR4980/PR4981 from the plasmid CMV-ARRB2::TEV protease (pBH302). The sequence encoding for NarX.sup.379to598 H399Q was PCR amplified with PR3687/PR4982 from the plasmid CMV-narX H399Q (pEM014). The primers were designed to insert G4S linker between the amplified fragment. Both PCR products and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.

[0211] EF1α-V1-ARRB2::narX.sup.3′N509A (pMZ252): The sequence encoding for ARRB2 was PCR amplified with PR4980/PR4981 from the plasmid CMV-ARRB2::TEV protease (pBH302). The sequence encoding for NarX.sup.379to598 N509A was PCR amplified with PR3687/PR4982 from the plasmid CMV-narX N509A (pMZ160). The primers were designed to insert G4S linker between the amplified fragment. Both PCR products and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.

[0212] EF1α-V1-ADRB2.sup.1to371::AVPR2.sup.343to371::narX.sup.379to598 H399Q (pMZ257): The sequence encoding for ADRB2.sup.1to341::AVPR2.sup.343to371 was PCR amplified with PR4983/PR4985 from the plasmid CMV-ADRB2.sup.1to341::AVPR2.sup.343to371::tTA (pBH312). The sequence encoding for NarX.sup.379to598 H399Q was PCR amplified with PR3687/PR4982 from the plasmid CMV-narX H399Q (pEM014). The primers were designed to insert G4S linker between the amplified fragment. Both PCR products and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.

[0213] EF1α-V1-ADRB2.sup.1to341::AVPR2.sup.343to371::narX.sup.379to598 N509A (pMZ258): The sequence encoding for ADRB2.sup.1to341::AVPR2.sup.343to371 was PCR amplified with PR4983/PR4985 from the plasmid CMV-ADRB2.sup.1to341::AVPR2.sup.343to371::tTA (pBH312). The sequence encoding for NarX.sup.379to598 N509A was PCR amplified with PR3687/PR4982 from the plasmid CMV-narX N509A (pMZ160). The primers were designed to insert G4S linker between the amplified fragment. Both PCR products and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.

[0214] tTA_RE-cerulean (pMZ290): The promoter regulated by tTA was PCR amplified with PR5226/PR5227 from the plasmid tTA_RE-mCherry (pIM003.sup.12). The PCR product and the plasmid DcuR_RE-cerulean (pMZ259) digested with AscI and AgeI were assembled using Gibson mix.

[0215] EF1α-V1-ARRB2::TEV protease (pMZ291): The sequence encoding for ARRB2.sup.283to409 and for the TEV protease was PCR amplified with PR5228/PR5229 from the plasmid CMV-ARRB2::TEV protease (pBH302). The sequence of the bGH poly(A) signal was PCR amplified with PR5230/PR5231 from the plasmid EF1α-V1-ARRB2::narX.sup.379to598 (pMZ250). Both PCR products and the plasmid EF1α-V1-ARRB2::narX.sup.379to598 (pMZ250) digested with BsaI and AvrII were assembled using Gibson mix.

[0216] EF1α::iRFP (pCS184): The iRFP coding sequence from CMV-iRFP (pCS12) was PCR amplified with PR2258/PR2259. The PCR product and the plasmid EF1a::citrine (pRA001, Altamura et al, manuscript in preparation) digested with BmtI and XbaI were assembled using ligation mix.

[0217] EF1α-V1-ADRB2.sup.1to341::AVPR2.sup.343to371::tTA (pBH292): The sequence encoding for ADRB2.sup.254to341 AVPR2.sup.343to371 and tTA was PCR amplified with PR5232/PR5233 from the plasmid CMV-ADRB2.sup.1to341::AVPR2.sup.343to371::tTA (pBH312). The PCR products and the the plasmid EF1α-V1-ADRB2.sup.1to341::AVPR2.sup.343to371::narX.sup.379to598 H399Q (pMZ257) digested with BglII and XhoI were assembled using Gibson mix.

[0218] CMV-ARRB2::TEV protease (pBH302): the inventors performed de novo synthesis of gBlock sequence encoding for Beta-arrestin-2 fused with the TEV protease with 2 gBlock (gBlock112 and gBlock 113) via IDT. The gBlock and the plasmid pZsYellow1-N1 (Clontech 632445) digested with NotI and EcoRI were assembled using Gibson mix.

[0219] CMV-OPRK1.sup.1to345::AVPR2.sup.343to371::tTA (pBH309): Via IDT, the inventors performed de novo synthesis of gBlock (gBlock114) sequence encoding for KOR-1 and of gBlock (gBlock115) sequence encoding for V2R fused to tTA. The sequence encoding for KOR-1.sup.1to345 was PCR amplified with PR2442/PR2443 from gBlock114. The PCR product, gBlock115 and the plasmid pZsYellow1-N1 (Clontech 632445) digested with XhoI and MfeI were assembled using Gibson mix.

[0220] CMV-ADRB2.sup.1to341::AVPR2.sup.343to371::tTA (pBH312): Via IDT, the inventors performed de novo synthesis of gBlock (gBlock118) sequence encoding for Beta-2 adrenergic receptor. The sequence encoding for ADRB2.sup.1to341 was PCR amplified with PR2442/PR2444 from gBlock118. The PCR product and the plasmid CMV-OPRK1.sup.1to345::AVPR2.sup.343to371::tTA (pBH309) digested with XhoI and BasHII were assembled using Gibson mix.

[0221] CMV-narX.sup.176to598 (pJH010): The 3′ fragment of narX was PCR amplified with PR1021/PR1023 from CMV-narX (pJH002). The primers were designed to amplify the sequence of NarX from the codon encoding the alanine at the position 176 to the end of the gene and to insert the ATG sequence in front of this amplified sequence. The PCR products and CMV-narX (pJH002) were digested with XhoI and AgeI. The two digested products are then ligated together.

[0222] The following plasmids were reported previously: CMV-envZ (pJH001), CMV-narX (pJH002), CMV-ompR (pJH003), CMV-narL (pJH004), OmpR_RE-AmCyan (pJH008), CMV-envZ_cyt (pJH009), CMV-envZ H243V (pEM013), CMV-narX H399Q (pEM014) and EnvZ-GGGGS-mCherry (pEM017) (Hansen, J. et al. Proc Natl Acad Sci USA 111, 15705-15710 (2014)). CBRN::FKBP and FRB::CBRC (Schramm, A. et al. Int J Mol Sci 19 (2018)). Ef1α-mCerulean (pKH024), Ef1α-citrine (pKH025) Ef1α-mCherry (pKH026) and Junk-DNA (pBH265) (Prochazka, et al., Nat Commun 5, 4729 (2014)). pTRE Bidirectional mCherry-pA (pIM003) (Angelici, B., et al., Cell Rep 16, 2525-2537 (2016)). The plasmid CMV-iRFP (pCS12) was obtained from Addgene (plasmid 31857 (Filonov, G. S. et al. Angewandte Chemie International Edition 51, 1448-1451 (2012))).

Cell Culture

[0223] The experiments in this work are performed on HEK293 purchased from Life technology (Cat #11631-017). Cell were cultured at 37° C., 5% CO.sub.2 in DMEM (Gibco, Life Technologies; Cat #41966-052), supplemented with 10% FBS (Sigma-Aldrich; Cat #F9665) and with 1% Penicillin/Streptogamine Solution (Sigma-Aldrich, Cat #P4333). Splitting was performed every 3-4 days using 0.25% Trypsin-EDTA (Gibco, Life technologies; Cat #25200-072). Cultures were propagated for at most two months before being replaced by fresh cell stock.

Transfections

[0224] All transfections were performed using Lipofectamine 2000 Transfection Reagent (Life Technologies; Cat #11668027). All transfections were performed in 24-well plates (Thermo Scientific Nunc; NC-142475) and 400 ng of DNA was transfected. The cells were seeded 24 h before transfection at a density per well of 50 000 in 500 μl of DMEM. The plasmids for each sample were mixed as indicated in Supplementary Tables 3-18 and completed with the volume of Opti-MEM I Reduced Serum (Gibco, Life technologies Cat #31985-962) to have final volume of 50 μl. 1.5 μl of lipofectamine 2000 was diluted in 50 μl Opti-MEM I per sample to have a final amount of 3.75:1 μl Reagent/μg DNA ratio. After an incubation at least of 5 minutes the diluted Lipofectamine was added to the mixed DNA sample. The resulting mixture was briefly mixed by gentle vortexing and incubated 20 minutes at room temperature before being added to the cells. 4 hours after the DNA was added to the cells the medium was removed and replaced with 500 μl of fresh medium. When required 5 μl of the chemical tested were added to the medium. The different stock solution at 100× of the desired final concentration were prepared as indicated below:

[0225] A/C Heterodimerizer (Clontech; Cat #635057) stock solution was prepared in ethanol (Honeywell; Cat #02860): 250 μM, 50 μM, 20 μM, 8 μM, 3.2 μM, 1.28 μM, 512 nM, 205 nM, 81.9 nM, 32.8 nM, 13.1 nM, 5.24 nM, 1.04 nM.

[0226] Procaterol (Sigma; Cat #P9180-10MG) stock solution was prepared in DMSO (Sigma; Cat #D4540, BCBT0803): 1 mM, 286 μM, 81.6 μM, 23.3 μM, 10 μM, 6.6 μM, 1.9 μM, 544 nM, 155 nM, 44.4 nM and 12.7 nM

[0227] Isoproterenol (Sigma; Cat #16504) stock solution was prepared in DMSO (Sigma; Cat #D4540): 1 mM, 286 μM, 81.6 μM, 23.3 μM, 6.6 μM, 1.9 μM, 544 nM, 155 nM, 44.4 nM and 12.7 nM

[0228] Clenbuterol (Sigma; Cat #C5423) stock solution was prepared in DMSO (Sigma; Cat #D4540): 1 mM, 286 μM, 81.6 μM, 23.3 μM, 6.6 μM, 1.9 μM, 544 nM, 155 nM, 44.4 nM and 12.7 nM

[0229] Propranolol (Sigma; Cat #P0884) stock solution was prepared in water (Invitrogen; Cat #10977-035): 1 mM, 286 μM, 81.6 μM, 23.3 μM, 6.6 μM, 1.9 μM, 544 nM, 155 nM, 44.4 nM and 12.7 nM

Microscopy

[0230] Microscopy images were taken from 48 h after transfection. The inventors used a Nikon Eclipse Ti microscope equipped with a mechanized stage and temperature control chamber held at 37° C. during the image acquisition. The excitation light was generated by a Nikon IntensiLight C-HGFI mercury lamp and filtered through a set of optimized Semrock filter cubes. The resulting images were collected by an Hamamatsu, ORCA R2 camera using a 10× objective. Each Semrock cube is assembled from an excitation filter, a dichroic mirror and an emission filter. In order to minimize the crosstalk between the different fluorescent proteins the inventors used the following setup: Cerulean: CFP HC (HC 438/24, BS 458, HC 483/32), mCherry: TxRed HC (HC 624/40, BS 593, HC 562/40). The images were acquired with an exposure of 40 ms for Cerulean and mCherry.

[0231] The acquired images were processed by ImageJ software performing uniform contrast-enhancement to improve visualization.

Flow Cytometry

[0232] The cells were prepared for FACS analysis 48 h after transfection by removing the medium and incubating the cells with 200 μl StemPro™ Accutase™ Cell Dissociation Reagent (Gibco, cat #A11105-01) at 37° C. for 5 minutes. After incubation, the plates was transferred on ice. To avoid potential cell damage the samples were prepared in successive batches so that no single sample was kept on ice for more than 1 h. The prepared samples were measured using a BD LSR Fortessa II Cell Analyzer with a combination of excitation and emission that minimizes the crosstalk between different fluorescent reporters. Cerulean was measured with a 445 nm laser and a 473/10 nm emission filter, mCherry with a 561 nm excitation laser coupled to a 600 nm longpass filter and 610/20 emission filter. The Cerulean and the mCherry were measured, respectively, at PMT voltage of 330 and 310 in all the experiments. SPHERO RainBow Calibration particles (Spherotech; Cat #RCP-30-5A, BD) were used to ensure constant device performance.

Data Analysis

[0233] General flow cytometry data analysis for bar charts was performed using FlowJo software. In this work, the fluorescence values in the bar charts, shown as normalized expression units (Cerulean, norm. u.) are calculated as follows. Live cells are gated based on their forward and side scatters readouts. From this population single cell are gated based on their forward scatters area and forward scatters height. Within this gate, cells positive in a Cerulean are gated based on a negative control such that 99.9% of cells in this control sample fall outside of the selected gate. For each Cerulean positive cell, the mean value of the fluorescent intensity is calculated and multiplied by the frequency of the positive cells. This value is used as a measure for the total reporter signal in a sample and can be defined as Total Intensity (TI). The TI of the Cerulean is normalized by the TI of mCherry-positive cells (constitutive transfection control). The relative formula is therefore: Reporter intensity in norm. u.=[mean(Reporter in Reporter+ cells)*Frequency (Reporter+ cells)]/[mean (Transfection Marker in Transfection Marker+ cells)*Frequency (Transfection Marker+ cells)].

Sequences

[0234] The nucleic and amino acid sequences provided herewith are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file named 95083_315_29_seqlist, created Apr. 15, 2021, about 42 KB, which is incorporated by reference herein. In case that the sequences given below vary from the Sequence Listingsubmitted herewith in text format, the below sequences shall prevail.

TABLE-US-00001 TABLE 1 Primer Sequences Primer name Sequence PR1021 GCTGCTGCTCTCGAGTCATTATCATTCGTGAGTGTC (SEQ ID NO 14) PR1023 GCTGCTACCGGTCGCCACCATGGCCCGCTTGCTCCAGCCGTGG (SEQ ID NO 15) PR1964 CGCGCCTGATTACAAAACTTTAAAAAGTGCTGTAGCGCCGGCTGATTACAAAACTTTAAA AAGTGCTGTCCA (SEQ ID NO 16) PR1965 TATGGACAGCACTTTTTAAAGTTTTGTAATCAGCCGGCGCTACAGCACTTTTTAAAGTTTT GTAATCAGG (SEQ ID NO 17) PR2196 TAAGCGGAATTCATCTTGGCTGAGGAATCTT (SEQ ID NO 18) PR2197 GCGAATTCTAGACTACTTGTACAGCTCGTCC (SEQ ID NO 19) PR2258 AATGTGAAGCTAGCGCCACCATGGCTGAAGGATCCGTCG (SEQ ID NO 20) PR2259 AATGTAATCTAGATCACTCTTCCATCACGCCGATC (SEQ ID NO 21) PR2442 GCTAGCGCTACCGGACTCAGAT (SEQ ID NO 22) PR2443 TGGGTGGGGTGCGTCCGCGCGCACAGAAGTCCCGGAAACACCG (SEQ ID NO 23) PR2444 TGGGTGGGGTGCGTCCGCGCGCACACAGAAGCTCCTGGAAGGCAA (SEQ ID NO 24) PR3687 GGCACAGTCGAGGCTGATTTTC (SEQ ID NO 25) PR3707 TGGCAGCGGGCGTCAAGCAG (SEQ ID NO 26) PR3708 ATGGTGGTGGCAGCGGCGAGGTATGGCAACG (SEQ ID NO 27) PR3709 CTCGCCGCTGCCACCACCATGTTGGCGACG (SEQ ID NO 28) PR4122 GAAATTAATACGACTCACTATAGGGGAC (SEQ ID NO 29) PR4345 GAAATTAATACGACTCACTATAGGGGACCGGTCGCCACCATGGCAGCGGGCGTCAAG (SEQ ID NO 30) PR4346 CACAGTCGAGGCTGATTTTC (SEQ ID NO 31) PR4541 GTTTGAGAGCTGCCGAGAGTGCTTCTCTCGCG (SEQ ID NO 32) PR4542 AGCACTCTCGGCAGCTCTCAAACATAGCCAGG (SEQ ID NO 33) PR4543 CTTCCAGTGCAGCTTCAATCAGATTTCCCAGTGTG (SEQ ID NO 34) PR4544 TCTGATTGAAGCTGCACTGGAAGCTCTGGGAC (SEQ ID NO 35) PR4546 GAAATTAATACGACTCACTATAGGGGACCGGTCGCCACCATGCAAGAGCGGCAGCAGC AG (SEQ ID NO 36) PR4732 GACGGCCAGTCTTAAGCTCGGGCCCGCTCCGGTGCCCGTCAG (SEQ ID NO 37) PR4733 GAAGTCGTCGCATGGTGGCGACCGGTTCACGACACCTGAAATGGAAG (SEQ ID NO 38) PR4734 GACGGCCAGTCTTAAGCTCGGGCCCTGGGCGGGATTCGTCTTG (SEQ ID NO 39) PR4747 CAAGAGCGGCAGCAGCAG (SEQ ID NO 40) PR4748 TTCGTGAGTGTCACCCTGC (SEQ ID NO 41) PR4766 GAAATTAATACGACTCACTATAGGGGACCGGTCGCCACCATGGGCGTGCAGGTGGAG (SEQ ID NO 42) PR4767 CGCCACCGCCTGAACCGCCTCCACCTTCCAGTTTTAGAAGCTCCACATC (SEQ ID NO 43) PR4769 GAAATTAATACGACTCACTATAGGGGACCGGTCGCCACCATGGTAGCCATCCTCTGG (SEQ ID NO 44) PR4770 CGCCACCGCCTGAACCGCCTCCACCTGATATCCGTCTGAACACGTG (SEQ ID NO 45) PR4771 AGGCGGTTCAGGCGGTGGCGGGTCGCAAGAGCGGCAGCAGCAG (SEQ ID NO 46) PR4892 CGCGCCTACCCCTATAGGGGTATAGCGCCGGCTACCCCTATAGGGGTATCCA (SEQ ID NO 47) PR4893 TATGGATACCCCTATAGGGGTAGCCGGCGCTATACCCCTATAGGGGTAGG (SEQ ID NO 48) PR4971 TTCTTCCATTTCAGGTGTCGTGAACCGGTCGCCACCATGGGCTC (SEQ ID NO 49) PR4972 CATCGTCATGGAAGAGAGGGCGACTATTGC (SEQ ID NO 50) PR4973 GCAATAGTCGCCCTCTCTTCCATGACGATG (SEQ ID NO 51) PR4974 TTCTTCCATTTCAGGTGTCGTGAACCGGTCGCCACCATGGGCGT (SEQ ID NO 52) PR4975 TTCTTCCATTTCAGGTGTCGTGAACCGGTCGCCACCATGGTAGC (SEQ ID NO 53) PR4977 TTCTTCCATTTCAGGTGTCGTGAACCGGTCGCCACCATGCAAGAGCGGCAGCAGCAG (SEQ ID NO 54) PR4978 CCTGATTGGACATGGTGGCGACCGGTTCACGACACCTGA (SEQ ID NO 55) PR4979 TTCTTCCATTTCAGGTGTCGTGAACCGGTCGCCACCATGCTT (SEQ ID NO 56) PR4980 TCCATTTCAGGTGTCGTGAACCGGTCGCCACCATGGGGGAGAAACCCGGGAC (SEQ ID NO 57) PR4981 CTCTTGCGAGCCACCGCCACCGCAGAGTTGATCATCATAGTCGTC (SEQ ID NO 58) PR4982 GGTGGCGGTGGCTCGCAAGAGCGGCAGCAGCAG (SEQ ID NO 59) PR4983 TCCATTTCAGGTGTCGTGAACCGGTCGCCACCATGGGGCAACCCGGGAAC (SEQ ID NO 60) PR4985 CTCTTGCGAGCCACCGCCACCCGATGAAGTGTCCTTGGCC (SEQ ID NO 61) PR5226 ATTACGCCAAGCTACGGGGGCACCTCGACATACTCGAG (SEQ ID NO 62) PR5227 CCTTGCTCACCATGGTGGCGAGGTACCGAGCTCGAAATCTC (SEQ ID NO 63) PR5228 ACCGGGAGAAGCGGGGTCTCG (SEQ ID NO 64) PR5229 CAGTCGAGGCTGATTTTCTCGCTCAAGCGTAATCTGGAAC (SEQ ID NO 65) PR5230 GTTCCAGATTACGCTTGAGCGAGAAAATCAGCCTCGACTG (SEQ ID NO 66) PR5231 CCGGGAGCTTTTTGCAAAAGC (SEQ ID NO 67) PR5232 GGACGGGGCATGGACTCCGCAG (SEQ ID NO 68) PR5233 GCACAGTCGAGGCTGATTTTCTCGAGTCATTACTACCCACCGTACTCGTCAATTCC (SEQ ID NO 69)

TABLE-US-00002 TABLE 2 List of synthetic DNA used for plasmid constructs Synthetic DNA name Sequence gBlock112 agatctcgagctcaagcttcGAATTCGCCACCatgggggagaaacccgggaccagggtcttcaagaagtcgagc cctaactgcaagctcaccgtgtacttgggcaagcgggacttcgtagatcacctggacaaagtggaccctgtagatggcgtgg tgcttgtggaccctgactacctgaaggaccgcaaagtgtttgtgaccctcacctgcgccttccgctatggccgtgaagacctgg atgtgctgggcttgtccttccgcaaagacctgttcatcgccacctaccaggccttccccccggtgcccaacccaccccggccc cccacccgcctgcaggaccggctgctgaggaagctgggccagcatgcccaccccttcttcttcaccataccccagaatcttc catgctccgtcacactgcagccaggcccagaggatacaggaaaggcctgcggcgtagactttgagattcgagccttctgtg ctaaatcactagaagagaaaagccacaaaaggaactctgtgcggctggtgatccgaaaggtgcagttcgccccggagaa acccggcccccagccttcagccgaaaccacacgccacttcctcatgtctgaccggtccctgcacctcgaggcttccctggac aaggagctgtactaccatggggagcccctcaatgtaaatgtccacgtcaccaacaactccaccaagaccgtcaagaagat caaagtctctgtgagacagtacgccgacatctgcctcttcagcaccgcccagtacaagtgtcctgtggctcaactcgaacaa gatgaccaggtatctcccagctccacattctgtaaggtgtacaccataaccccactgctcagcgacaaccgggagaagcgg ggtctcgccctggatgggaaactcaagcacgaggacaccaacctggcttccagcaccatcgtgaaggagggtgccaaca aggaggtgctgggaatcctggtgtcct (SEQ ID NO 70) gBlock113 gctgggaatcctggtgtcctacagggtcaaggtgaagctggtggtgtctcgaggcggggatgtctctgtggagctgccttttgtt cttatgcaccccaagccccacgaccacatccccctccccagaccccagtcagccgctccggagacagatgtccctgtgga caccaacctcattgaatttgataccaactatgccacagatgatgacattgtgtttgaggactttgcccggcttcggctgaaggg gatgaAGGATGACGACTATGATGATCAACTCTGCGGATCCagcttgtttaagggaccacgtgattac aacccgatatcgagcaccatttgtcatttgacgaatgaatctgatgggcacacaacatcgttgtatggtattggatttggtccctt catcattacaaacaagcacttgtttagaagaaataatggaacactgttggtccaatcactacatggtgtattcaaggtcaaga acaccacgactttgcaacaacacctcattgatgggagggacatgataattattcgcatgcctaaggatttcccaccatttcctc aaaagctgaaatttagagagccacaaagggaagagcgcatatgtcttgtgacaaccaacttccaaactaagagcatgtcta gcatggtgtcagacactagttgcacattcccttcatctgatggcatattctggaagcattggattcaaaccaaggatgggcagt gtggcagtccattagtatcaactagagatgggttcattgttggtatacactcagcatcgaatttcaccaacacaaacaattatttc acaagcgtgccgaaaaacttcatggaattgttgacaaatcaggaggcgcagcagtgggttagtggttggcgattaaatgctg actcagtattgtgggggggccataaagttttcatgagcaaacctgaagagccttttcagccagttaaggaagcgactcAAC TCATGAATGAATTGGTGTACTCGCAATACCCATACGATGTTCCAGATTACGCTTGAgc GGCCGCgactctagatcataatcag (SEQ ID NO 71) gBlock114 GcgctaccggactcagatctcgagGCCACCatggactccccgatccagatcttccgcggggagccgggccctacctg cgccccgagcgcctgcctgccccccaacagcagcgcctggtttcccggctgggccgagcccgacagcaacggcagcgc cggctcggaggacgcgcagctggagcccgcgcacatctccccggccatcccggtcatcatcacggcggtctactccgtagt gttcgtcgtgggcttggtgggcaactcgctggtcatgttcgtgatcatccgatacacaaagatgaagacagcaaccaacattt acatatttaacctggctttggcagatgctttagttactacaaccatgccctttcagagtacggtctacttgatgaattcctggcctttt ggggatgtgctgtgcaagatagtaatttccattgattactacaacatgttcaccagcatcttcaccttgaccatgatgagcgtgg accgctacattgccgtgtgccaccccgtgaaggctttggacttccgcacacccttgaaggcaaagatcatcaatatctgcatct ggctgctgtcgtcatctgttggcatctctgcaatagtccttggaggcaccaaagtcagggaagacgtcgatgtcattgagtgct ccttgcagttcccagatgatgactactcctggtgggacctcttcatgaagatctgcgtcttcatctttgccttcgtgatccctgtcctc atcatcatcgtctgctacaccctgatgatcctgcgtctcaagagcgtccggctcctttctggctcccgagagaaagatcgcaac ctgcgtaggatcaccagactggtcctggtggtggtggcagtcttcgtcgtctgctggactcccattcacatattcatcctggtgga ggctctggggagcacctcccacagcacagctgctctctccagctattacttctgcatcgccttaggctataccaacagtagcct gaatcccattctctacgcctttcttgatgaaaacttcaagcggtgtttccgggacttctgTtttccactgaagatgaggatggagc ggcagagcactagcagagtccgaaatacagttcaggaCcctgcttacctgagggacatcgatgggatgaataaaccagt atgacaattgttgttgttaacttgtttattgc (SEQ ID NO 72) gBlock115 AGCGGTGTTTCCGGGACTTCTGTGCGCGCggacgcaccccacccagcctgggtccccaagatgagtc ctgcaccaccgccagctcctcccTGGCCAAGGACACTTCATCGgGATCCGAGAATCTGTACTTT CAGCTGagattagataaaagtaaagtgattaacagcgcattagagctgcttaatgaggtcggaatcgaaggtttaacaa cccgtaaactcgcccagaagctaggtgtagagcagcctacattgtattggcatgtaaaaaataagcgggctttgctcgacgc cttagccattgagatgttagataggcaccatactcacttttgccctttagaaggggaaagctggcaagattttttacgtaataacg ctaaaagttttagatgtgctttactaagtcatcgcgatggagcaaaagtacatttaggtacacggcctacagaaaaacagtat gaaactctcgaaaatcaattagcctttttatgccaacaaggtttttcactagagaatgcattatatgcactcagcgctgtggggc attttactttaggttgcgtattggaagatcaagagcatcaagtcgctaaagaagaaagggaaacacctactactgatagtatg ccgccattattacgacaagctatcgaattatttgatcaccaaggtgcagagccagccttcttattcggccttgaattgatcatatg cggattagaaaaacaacttaaatgtgaaagtgggtccgcgtacagccgGgcgcgtacgaaaaacaattacgggtctacc atcgagggcctgctcgatctcccggacgacgacgcccccgaagaggcggggctggcggctccgcgcctgtcctttctcccc gcgggacacacgcgcagactgtcgacggcccccccgaccgatgtcagcctgggggacgagctccacttagacggcgag gacgtggcgatggcgcatgccgacgcgctagacgatttcgatctggacatgttgggggacggggattccccgggtccggg atttaccccccacgactccgccccctacggcgctctggatatggccgacttcgagtttgagcagatgtttaccgatgcccTTG GAATTGACGAGTACGGTGGGTAGcaattgttgttgttaacttgtttattgc (SEQ ID NO 73) gBlock118 gctagcgctaccggactcagatctcgagGCCACCatggggcaacccgggaacggcagcgccttcttgctggcaccca atagaagccatgcgccggaccacgacgtcacgcagcaaagggacgaggtgtgggtggtgggcatgggcatcgtcatgtc tctcatcgtcctggccatcgtgtttggcaatgtgctggtcatcacagccattgccaagttcgagcgtctgcagacggtcaccaac tacttcatcacttcactggcctgtgctgatctggtcatgggcctggcagtggtgccctttggggccgcccatattcttatgaaaatg tggacttttggcaacttctggtgcgagttttggacttccattgatgtgctgtgcgtcacggccagcattgagaccctgtgcgtgatc gcagtggatcgctactttgccattacttcacctttcaagtaccagagcctgctgaccaagaataaggcccgggtgatcattctg atggtgtggattgtgtcaggccttacctccttcttgcccattcagatgcactggtaccgggccacccaccaggaagccatcaac tgctatgccaatgagacctgctgtgacttcttcacgaaccaagcctatgccattgcctcttccatcgtgtccttctacgttcccctg gtgatcatggtcttcgtctactccagggtctttcaggaggccaaaaggcagctccagaagattgacaaatctgagggccgctt ccatgtccagaaccttagccaggtggagcaggatgggcggacggggcatggactccgcagatcttccaagttctgcttgaa ggagcacaaagccctcaagacgttaggcatcatcatgggcactttcaccctctgctggctgcccttcttcatcgttaacattgtg catgtgatccaggataacctcatccgtaaggaagtttacatcctcctaaattggataggctatgtcaattctggtttcaatcccctt atctactgccggagcccagatttcaggattgccttccaggagcttctgtgTctgcgcaggtcttctttgaaggcctatgggaatg gctactccagcaacggcaacacaggggagcagagtggatatcacgtggaacaggagaaagaaaataaactgctgtgtg aagacctcccaggcacggaagactttgtgggccatcaaggtactgtgcctagcgataacattgattcacaagggaggaatt gtagtacaaatgactcactgctgtaacaattgttgttgttaacttgtttattgc (SEQ ID NO 74) gBlock143 ATCTTGGCTGAGGAATCTTCTAACAATTTAGAGCTTAAAAACGCCCACGAGGCGGAG AACGAAATATCCAGAGAGACGTTAGAAACGTTCAAAAACGTTCGCTAGCGCCACCAT GGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTG GACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATG CCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTG CCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCCTGATGTGCTTCGCCCGCTA CCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACG TCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAG GTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTT CAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACA ACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCC GCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACAC CCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGT CCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTC GTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAG (SEQ ID NO 75) gBlock264 gaaattaatacgactcactataggggaccggtcgccaccATGGGCTCGAGCAACCTGGTTGCGCAGCT CGAAAACGAAGTTGCGTCTCTGGAAAATGAGAACGAAACCCTGAAGAAAAAGAACCT GCACAAAAAAGACCTGATCGCGTACCTGGAGAAAGAAATCGCGAATCTGCGTAAGA AAATCGAAGAAGGCGGTGGCGGGTCGCAAGAGCGGCAGCAGCAGCT (SEQ ID NO 76) gBlock265 TGCAGGGTGACACTCACGAAGGCGGTGGCGGGTCGAACCTGGTTGCGCAGCTCGA AAACGAAGTTGCGTCTCTGGAAAATGAGAACGAAACCCTGAAGAAAAAGAACCTGCA CAAAAAAGACCTGATCGCGTACCTGGAGAAAGAAATCGCGAATCTGCGTAAGAAAAT CGAAGAATGATAATGACTCGAGAAAATCAGCCTCGACTGTG (SEQ ID NO 77) gBlock269 gaaattaatacgactcactataggggaccggtcgccaccATGGGCTCGAGCGCGCGTAACGCGTATCT GCGTAAGAAAATCGCACGTCTGAAAAAAGACAACCTGCAGCTGGAACGTGATGAAC AGAACCTGGAAAAAATCATCGCGAACCTGCGTGACGAAATCGCGCGTCTCGAAAAC GAAGTTGCGTCTCACGAACAGGGCGGTGGCGGGTCGCAAGAGCGGCAGCAGCAGC T (SEQ ID NO 78) gBlock270 TGCAGGGTGACACTCACGAAGGCGGTGGCGGGTCGGCGCGTAACGCGTATCTGCG TAAGAAAATCGCACGTCTGAAAAAAGACAACCTGCAGCTGGAACGTGATGAACAGAA CCTGGAAAAAATCATCGCGAACCTGCGTGACGAAATCGCGCGTCTCGAAAACGAAG TTGCGTCTCACGAACAGTGATAATGACTCGAGAAAATCAGCCTCGACTGTG (SEQ ID NO 79)

[0235] The sequences of the synthetic promoters are indicated in the following table (underlinded sequences indicates the RR DNA binding sites, italic letters indicate the TATA Box. Start codon is shown in bold)

TABLE-US-00003 OmpR RE ATTTACATTTTGAAACATCTATAGCGCCGGCATTTACATTTTGAAACATC (SEQ ID 1) TATCCATATGCTCTAGAGGGTATATAATGGGGGCCACTAGTCTACTACC AGAGCTCATCGCTAGCGCTACCGGTCGCCACCATG NarL RE TACCCCTATAGGGGTATAGCGCCGGCTACCCCTATAGGGGTATCCATA (SEQ ID 2) TGCTCTAGAGGGTATATAATGGGGGCCACTAGTCTACTACCAGAGCTCA TCGCTAGCGCTACCGGTCGCCACCATG

[0236] The sequence of the promoter CMV, EF1α, and EF1α-V1

TABLE-US-00004 >CMV promoter (SEQ ID 3) gcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataa cttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgc caatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaa gtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggca gtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggg gatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactc cgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaact >EF1α (SEQ ID 4) GCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGG GGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGG AAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATAT AAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG GTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCG TGCCTTGAATTACTTCCACGCCCCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCG GGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGT GCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCAC CTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCT GCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACAC TGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCAC ATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGT CTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGC CCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCG CTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGG GCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCAT GTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTG GAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACT GAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATT TGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTT TTTTCTTCCATTTCAGGTGTCGTGA >EF1α-V1 (SEQ ID 5) ttaagctcgggcccTGGGCGGGATTCGTCTTGGGCGGGATCCTTGTCCACGTGATCGGGGGA GGGACTTTCCCGCTGGAGTGACTCATCTAGCCCACGTGATCTTCATGCCACGTGATCGA TATGGGGACTTTCCTGACTCCCACGTGATCGCACCCCCACGTGATCCCGTAAGGGACT TTCCCTACTTTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTA CTGGCTCCGCCTTTTTCCCGTGCTCAGGGGAGAACCGTATATAAGTGCAGTAGTCGCC GTGAACGTTCTTTTTCGCAACGTTAACTAGCACAGAACACAGGTAAGTGCCGTGTGTGG TTCCCGCGGGCGGCGACGGGGCCCGTGCCCACGTGATCAGGAGTTGGGCGGGATGTT ATGAGTGACTCACGCCATCCACGTGATCTCAGACGGGACTTTCCATATTAAGTGACTCA GGATAAGGGACTTTCCCTACGGCCACGTGATCTCTTTTTGGGCGGGATGAGATTGGGA CTTTCCTGTCCTGGGACTTTCCTACAGTTCAAACTCGACCACGTGATCTTATGACTGACG GGCGGGTGAGTCACCCACGGTGGCATGGGGGACTTTCCTTTAGGCGTTCATGTGACTC CACGGACAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAa The sequence of narL: >NarL (SEQ ID 6) MSNQEPATILLIDDHPMLRTGVKQLISMAPDITVVGEASNGEQGIELAESLDPDLILLD LNMPGMNGLETLDKLREKSLSGRIVVFSVSNHEEDVVTALKRGADGYLLKDMEPED LLKALHQAAAGEMVLSEALTPVLAASLRANRATTERDVNQLTPRERDILKLIAQGLP NKMIARRLDITESTVKVHVKHMLKKMKLKSRVEAAVWVHQERIF The sequence of VP48 >VP48 (SEQ ID 7) GPADALDDFDLDMLPADALDDFDLDMLPADALDDFDLDMLPG The aa sequence of EnvZ.sup.180to450, EnvZ.sup.223to450, NarX.sup.176to598 and NarX.sup.379to598 are indicated below the phosphorylatable histidine is underlined and italic, the asparagine important for the ATP binding domain is bold and underlined >EnvZ.sup.180to450 (SEQ ID 8) MRIQNRPLVDLEHAALQVGKGIIPPPLREYGASEVRSVTRAFNHMAAGVKQLADDRTLLMA GVS DLRTPLTRIRLATEMMSEQDGYLAESINKDIEECNAIIEQFIDYLRTGQEMPMEMADLN AVLGEVIAAESGYEREIETALYPGSIEVKMHPLSIKRAVANMVVNAARYGNGWIKVSSGTEP NRAWFQVEDDGPGIAPEQRKHLFQPFVRGDSARTISGTGLGLAIVQRIVDNHNGMLELGTS ERGGLSIRAWLPVPVTRAQGTTKEG >EnvZ.sup.223to450 (SEQ ID 9) MAAGVKQLADDRTLLMAGVS DLRTPLTRIRLATEMMSEQDGYLAESINKDIEECNAIIEQFI DYLRTGQEMPMEMADLNAVLGEVIAAESGYEREIETALYPGSIEVKMHPLSIKRAVANMVVN AARYGNGWIKVSSGTEPNRAWFQVEDDGPGIAPEQRKHLFQPFVRGDSARTISGTGLGLAI VQRIVDNHNGMLELGTSERGGLSIRAWLPVPVTRAQGTTKEG >NarX.sup.176to598 (SEQ ID 10) MARLLQPWRQLLAMASAVSHRDFTQRANISGRNEMAMLGTALNNMSAELAESYAVLEQRV QEKTAGLEHKNQILSFLWQANRRLHSRAPLCERLSPVLNGLQNLTLLRDIELRVYDTDDEEN HQEFTCQPDMTCDDKGCQLCPRGVLPVGDRGTTLKWRLADSHTQYGILLATLPQGRHLSH DQQQLVDTLVEQLTATLALDRHQERQQQLIVMEERATIAREL DSIAQSLSCMKMQVSCLQ MQGDALPESSRELLSQIRNELNASWAQLRELLTTFRLQLTEPGLRPALEASCEEYSAKFGFP VKLDYQLPPRLVPSHQAIHLLQIAREALSNALKHSQASEVVVTVAQNDNQVKLTVQDNGCG VPENAIRSNHYGMIIMRDRAQSLRGDCRVRRRESGGTEVVVTFIPEKTFTDVQGDTHE >NarX.sup.379to598 (SEQ ID 11) MQERQQQLIVMEERATIAREL DSIAQSLSCMKMQVSCLQMQGDALPESSRELLSQIRNEL NASWAQLRELLTTFRLQLTEPGLRPALEASCEEYSAKFGFPVKLDYQLPPRLVPSHQAIHLL QIAREALSNALKHSQASEVVVTVAQNDNQVKLTVQDNGCGVPENAIRSNHYGMIIMRDRAQ SLRGDCRVRRRESGGTEVVVTFIPEKTFTDVQGDTHE The aa of the mutated version of NarX379to598 the mutated aa is indicated in bold and underlined >NarX.sup.379to598 (H399Q) (SEQ ID 12) MQERQQQLIVMEERATIARELQDSIAQSLSCMKMQVSCLQMQGDALPESSRELLSQIRNEL NASWAQLRELLTTFRLQLTEPGLRPALEASCEEYSAKFGFPVKLDYQLPPRLVPSHQAIHLL QIAREALSNALKHSQASEVVVTVAQNDNQVKLTVQDNGCGVPENAIRSNHYGMIIMRDRAQ SLRGDCRVRRRESGGTEVVVTFIPEKTFTDVQGDTHE >NarX.sup.379to598 (N509A) (SEQ ID 13) MQERQQQLIVMEERATIARELHDSIAQSLSCMKMQVSCLQMQGDALPESSRELLSQIRNEL NASWAQLRELLTTFRLQLTEPGLRPALEASCEEYSAKFGFPVKLDYQLPPRLVPSHQAIHLL QIAREALSAALKHSQASEVVVTVAQNDNQVKLTVQDNGCGVPENAIRSNHYGMIIMRDRAQ SLRGDCRVRRRESGGTEVVVTFIPEKTFTDVQGDTHE