CHIMERIC RECEPTOR FOR USE IN WHOLE-CELL SENSORS FOR DETECTING ANALYTES OF INTEREST
20200096507 ยท 2020-03-26
Inventors
Cpc classification
G01N33/6872
PHYSICS
C07K2319/80
CHEMISTRY; METALLURGY
G01N33/554
PHYSICS
G01N33/566
PHYSICS
C07K2317/80
CHEMISTRY; METALLURGY
G01N33/5308
PHYSICS
International classification
G01N33/566
PHYSICS
G01N33/53
PHYSICS
Abstract
The present invention relates to chimeric receptors that can be used in whole-cell sensors for detecting analytes of interest. The inventors showed that the DNA binding domains and downstream gene expression can be activated via dimerization of an artificial dimerization composed of a single chain variable domain. They demonstrated for the first time that an artificial bacterial receptor using an antibody-like domain can be activated and produce a transcriptional output upon ligand-binding. In particular, the present invention relates to a chimeric receptor polypeptide comprising: i) a first DNA binding domain, ii) at least one binding domain selected from the group consisting of heavy chain variable domain, camelid VHHs, or antibody mimetics having specificity for an analyte, and iii) a linker between the DNA binding domain and the binding domain.
Claims
1. A chimeric receptor polypeptide comprising i) a first DNA binding domain, ii) at least one binding domain having specificity for an analyte selected from the group consisting of a heavy chain variable domain, a camelid, and an antibody mimetic, and iii) a linker between the first DNA binding domain and the at least one binding domain.
2. The chimeric receptor polypeptide of claim 1 wherein the first DNA binding domain comprises an amino acid sequence having at least 70% identity with SEQ ID NO:1 or SEQ ID NO: 2 .
3. The chimeric receptor polypeptide of claim 1 wherein the first DNA binding domain comprises an amino acid sequence having at least 70% identity with SEQ ID NO: 13.
4. The chimeric receptor polypeptide of claim 1 wherein the linker is a transmembrane domain comprising an amino acid sequence having at least 50% identity with SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
5. The chimeric receptor polypeptide of claim 1 wherein the antibody mimetic is selected from the group consisting of a monobody, a DARPin and an alphaRep.
6. The chimeric receptor polypeptide of claim 1 which further comprises a spacer inserted between the linker and the at least one binding domain.
7. The chimeric receptor polypeptide of claim 6 wherein the spacer is or comprises the amino acid sequence DTRLPMS (SEQ ID NO:7) or GGGSG (SEQ ID NO:12).
8. The chimeric receptor polypeptide of claim 1 wherein the first DNA binding domain comprises an amino acid sequence having at least 70% of identity with SED ID NO:8 fused to one binding domain.
9. A nucleic acid encoding for the chimeric receptor of claim 1.
10. An expression cassette comprising the nucleic acid molecule of claim 9 operably linked to control sequences allowing expression in a prokaryotic cell.
11. A prokaryotic cell genetically engineered with: the nucleic acid molecule of claim 9, or an expression cassette comprising the nucleic acid.
12. The prokaryotic cell of claim 11 which is a gram-positive or a gram-negative bacteria.
13. The prokaryotic cell of claim 11 which further comprises a nucleic acid molecule encoding a detection protein operatively linked to a CadBA promoter.
14. A method for assaying for at least one analyte, comprising i) providing at least one set of prokaryotic cells of claim 11, each comprising a chimeric receptor capable of binding to said at least one analyte coupled to one detection protein; b) contacting said set of prokaryotic cells with a sample suspected of containing said at least one analyte for a time sufficient for binding of the chimeric receptors to the at least one analyte, and for expression of the detection protein; and c) detecting an expression level of the detection protein wherein the expression level is correlated with the amount of the at least one analyte present in the sample.
15. A cell-free system comprising the chimeric receptor polypeptide of claim 1.
16. The cell free- system of claim 15 wherein the chimeric receptor polypeptide is embedded partially or completely in a solid support.
17. The cell-free system of claim 16 wherein the solid support is a porous substrate.
18. The cell-free system of claim 16 wherein the solid support is paper.
19. The cell-free system of claim 16 wherein the solid support comprises several spatially distinct reaction regions where the cell-free system is confined.
20. The cell-free system of claim 15 comprising a reporter gene.
21. The cell-free system of claim 20 wherein the reporter gene encodes for a fluorescent protein.
22. The cell-free system of claim 16 which is lyophilized on the solid support.
23. A method of detecting an analyte, comprising: (i) providing the cell-free system of claim 20; (ii) contacting the cell-free system with a sample to be tested for said analyte; and (iii) detecting a signal from the reporter gene, wherein detection of said signal indicates the presence of said analyte.
24. The prokaryotic cell of claim 12, wherein the gram-negative bacteria is an Escherichia coli.
Description
FIGURES
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EXAMPLE 1
Materials and Methods
Bacterial Strains, Plasmids, and Materials
[0064] CadC and LexA transcriptional factor DNA binding domain, Leucine zipper GCN4, V.sub.HHcafe and V.sub.HHRNase were synthesized by Integrated DNA Technologies (IDT). Different DNA components such as GFP reporter, CadBA promoter, pLexA promoter and CadC variants were constructed into BioBrick standard vector pSB4K5 by Gibson assembly methods. The resulting constructs were transformed into E.coli strain NEB10beta (New England Biolabs, NEB) and determined their performance. In all constructs, the expression level of CadC variants are under the control of pLac-1 promoter, which can be induced by IPTG. The CadC variants control the expression of reporter GFP through pCadBA promoter.
Design and Construction of CadC Variants
[0065] The sequence details of each construct are listed in supplementary information. The construct CadC operation unit with CadC-wtTM-GCN4 was built first as template for further replacement by other CadC variants. Three pairs of primers (see primer details) were used to amplify three gene blocks with 40bp overlap regions. These components were further assembled by Gibson assembly methods.
Generating PCR Amplified Blocks for CadC Operation Unit
[0066] The PCR amplification was carried out in a 40 l reaction mixture consisting of 0.110 ng of template DNA fragment, 1 l of each forward/reversed primer (20 M), and 20 L of Q5 hot start high-fidelity 2X master mix (NEB). After 30 seconds of initial denaturation at 98 C., 35 cycles were conducted with the PCR procedures of 10 seconds at 98 C., 30 seconds at corresponding annealing temperature (different with each primer combination, calculated with NEB Tm calculator: http://tmcalculator.neb.com/#!/), and elongation (2 kb/min) at 72 C., with a final extension at 72 C. for 10 minutes. The PCR product was verified by gel electrophoresis, then purified by PCR clean up kit and determined the DNA concentration by Nanodrop spectrophotometer.
Gibson Assembly and Electrotransformation
[0067] The DNA templates from E.coli in the purified PCR product was further digested with 1 l DpnI (20 units/l, NEB) in 40 l Cut Smart reaction buffer (NEB) at 37 C. for 1 hours. The resulting product was applied to Gibson assembly reaction directly. In each Gibson assembly reaction, 100 ng of vector DNA fragment and 3 -5 folds of insert fragments was incubated with 10 l of 2X Gibson assembly master mix (NEB) in a final volume of 20 L at 50 C. for 60 minutes. To prevent the DNA ligase activity in the reaction mix affecting the following electroporation efficiency, the reaction mix was further heat inactivated at 80 C. for 15 minutes. 1 l of Gibson assembly products was added into 40 l NEB10beta electro-competent cells and then transferred into the Biorad 0.1 cm gap Micropulser electroporation cuvettes. Right after electroporation with Biorad Micropulser electroporator and program EC1, 1 mL of prewarmed (37 C.) SOC medium was added into the transformants immediately. The cell culture was further incubated in 37 C. incubator with vigorous shaking for one more hour for rescuing the cells. The transformant was then plated on the selection plate with antibioitics (ex. kanamycin) and incubated at 37 C. overnight. The constructs were further verified by Sanger sequencing.
In Vivo Characterization
[0068] Pick a single colony of CadC operating unit variant into 3 mL of LB/kanamycin medium and incubate in 37 C. overnight with vigorous shaking. The overnight culture is further diluted 100 fold into 3 mL of LB/kanamycin medium and incubated in 37 C. for 4 hours to reach to exponential phase (O.D=0.40.6). The culture in exponential phase was further diluted 50 fold into mediums containing inducer 1 mM IPTG (for experiment: Activation of CadC transcriptional factor by periplasmic domain oligomerization) or 1 mM IPTG/1 mM caffeine (for experiment: activation of CadC transcriptional factor by analyte induced binding domain oligomerization), and then incubated at 37 C. overnight with vigorous shaking. The resulting cell culture was further analyzed with Attune NxT flow cytometer.
[0069] Designing a synthetic biosensor using split DNA-Binding domains. DNA binding of transcriptional regulators is generally dependent on dimerization of the DBD through the LBD. Deletion of the LBD/dimerization domain lead to an inactive, monomeric DBDs which function can be restored via dimerization driven by fusion proteins of interest. In order to engineer our split-DBD system, we used the DBD of LexA, a well-characterized transcriptional repressor which regulates the transcription of genes involved in E. coli SOS response. Upon induction of the SOS response, RecA promotes LexA inactivation through self-cleavage at residue 85, a flexible hinge between DNA-binding and dimerization domains. The repressive activity of the monomeric LexA DBD can be restored by forcing its dimerization through fusion with leucine zippers. LexA DBD was used in two-hybrid screens to probe protein-protein. In order to prevent interference from endogenous E.coli LexA, we used the mutant LexA-408, and its corresponding promoter which is not recognized by the wild type LexA. We fused LexA DBD to a monomeric LBD that undergoes ligand-induced homodimerization. Thus, in the presence of ligand, LexA DBD should be able to bind DNA and repress target gene expression.
[0070] Choice of a synthetic ligand binding domain. In order to develop a scalable receptor platform, we set several criteria for selecting an ideal LBD scaffold: (i) potential for further engineering to bind many different ligands, and of different types (e.g. proteins, small molecules); (ii) high solubility and stability; and (iii) low propensity to aggregate to ensure monomeric state before ligand binding. Antibodies are an ideal scaffold to detect various kinds of ligands and can be applied to different therapeutic or diagnosis applications. However, IgGs have poor expression levels in prokaryotic system. To counter this problem, researchers have developed single domain antibodies, such as Camelid VHHs, or antibody mimetics like monobodies, DARPins, or alphaReps that are well expressed in prokaryotic systems with high stability and solubility, and forwhich combinatorial libraries can be selected to target many different antigens. We thus decided to use a synthetic antibody that could undergo monomer-to-dimer transition upon ligand binding. To establish our proof-of-concept, and because of the limited membrane permeability to proteins, we searched for an antibody targeting a small diffusible molecule. We used a single-domainVHH camelid antibody that can be dimerized upon binding to its ligand caffeine (termed VHH-Caffeine from herein). As a negative control we chose a VHH targeting RNase A (termed VHH-Control from herein).
[0071] Detection of caffeine using a LexA-VHH fusion. We built two chimeric proteins, expressed in E. coli cytoplasm, composed of an N-terminal LexA DBD and a C-terminal VHH, and placed their expression under the control of the pLacO1 promoter induced by isopropyl -D-1-thiogalactopyranoside (IPTG). As a reporter, we used the Green Fluorescent Protein (GFP) driven by the LexA promoter. As positive and negative controls, we expressed full-length LexA and LexA DBD. As expected, we observed that full-length LexA mediated-repression increased with IPTG concentration and was not affected by caffeine (data not shown). We also observed that the LexA DBD was capable of gene repression a high concentration (14% and 35% repression at 50 M and 100 M IPTG, respectively, data not shown). We then characterized the behavior of cells expressing LexAVHH- Caffeine and LexA-VHH-Control in response to increasing concentrations of IPTG and caffeine (data not shown). We confirmed by Western blot (WB) that both fusion proteins were expressed at similar levels (data not shown). We first observed that similarly to LexADBD, the fusion proteins LexA-VHH-Caffeine and LexA-VHHControl displayed a concentration-dependent repressive activity (30% and 45% at 50 M and 100 M IPTG induction, respectively, data not shown). LexA-VHH-Caffeine and LexAVHH- Control had a comparable repressive activity to LexADBD at a similar IPTG concentration, suggesting that this repressive effect is primarily due to the DBD and that VHHs are in a monomeric state in the cytoplasm. We then monitored the response of the LexA-VHH fusions to increasing concentrations of caffeine at different IPTG concentrations. While no change was detectable in response to cafeine for LexA-VHH-Control (data not shown), LexA-VHHCaffeine had a dose-dependent response to caffeine starting at 25 M IPTG concentration and 100 nM caffeine (data not shown). These results show that in the presence of its ligand, VHH-Caffeine dimerizes and restores LexA DNA-binding activity, leading to reporter gene repression. Response to caffeine was homogenous among the whole cell population (data not shown). We calculated the fold repression of LexA-VHH-Caffeine. We found that the higher repression fold (4.3-fold) was at 100 M IPTG induction (data not shown). LexA-VHHCaffeine had a repression activity comparable to full-length lexA at similar protein expression levels (respectively 100 and 12.5 M IPTG concentration). We also calculated the swing values (i.e. the absolute change in fluorescence intensity between inactive and active states), and found that the best swing (15 RPUs) was at 25 M IPTG and 100 M caffeine (data not shown). However, higher expression of LexA-VHH-Caffeine was needed to detect lower concentration of caffeine. Because monitoring a signal increase is more practical for a biosensor assay, we then connected our system to a genetic inverter based on the BetI repressor (data not shown). Starting at 50 M IPTG induction, we observed a marked increased inGFP intensity (data not shown). While the swing was lower than with the repressor only system (2.2 RPUs vs 12.2 RPUs at 100 M IPTG, respectively), the fold change was still significant, (4-fold, data not shown), due to the very low background signal in the absence of caffeine. Interestingly, we found that there was only a slight change in GFP signal across increasing concentrations of IPTG (from 0.6 RPU at 0 M IPTG to 0.7 RPU at 100 M IPTG). We hypothesize that the inverter module buffers the non-specific repressive effect of the DBD at high concentrations, probably because of the delay required to degrade the Betl repressor. These results demonstrate that split-DBDs can be used to engineer synthetic receptors activated via ligand-induced dimerization of antibody-based LBD.
EXAMPLE 2
Artificial Dimerization of the Cadc Dna Binding Domain Via Leucine Zippers Induces Gene Expression
[0072] Here we fused the CadC DNA binding domain with the wild-type CadC transmembrane region or with an artificial transmembrane region. We then fused these two scaffolds with the GCN4 homodimerizing leucine zipper. We show that while in the abscence of GCN4, nosignal is observed, the constructs fused with GCN4 are able to trigger GFP expression (
[0073] While previously it had been demonstrated that forcing dimerization of the CadC DNA binding domain via self-dimerizing transmembrane helixes could trigger downstream gene expression, this is the first demonstration, to our knowledge, that the CadC DNA binding domain and downstream gene expression can be activated via dimerization of an extracellular dimerization domain. Moreover, we demonstrate that the transmembrane region sequence can greaty affect the efficiency of activation.
EXAMPLE 3
[0074] Engineering a synthetic transmembrane receptor using CadC DBD and a VHH ligand binding domain. One current limitation of whole cell biosensors is the difficulty to detect large biological molecules such as proteins that cannot cross the cellular membrane. While synthetic transmembrane receptors have been developed in mammalian cells, no scalable receptor platform for soluble extracellular ligand detection have been reported in microorganisms, a robust chassis for whole-cell biosensing. We thus aimed at engineering chimeric transmembrane receptors exploiting the principle of split-DBDs. We looked for existing E. coli transmembrane transcription regulators and turned to CadC, a transcriptional activator from the ToxR family. CadC is composed of a N-terminal cytosolic DBD and a C-terminal periplasmic pH sensor domain. It activates the pCadBA promoter when environmental pH decreases and in the presence of lysine. Interestingly, dimerization of transmembrane helixes bound to the cytosolic CadC DNA binding domain is sufficien to restore CadC transcriptional activity. We thus aimed to restore CadC activity through ligand-induced dimerization of a periplasmic sensor domain. In order to remove endogenous regulations by the Lysine permease LysP, we used an artificial transmembrane domain composed of 16 Leucine repeat. We fused the self-dimerizing leucine-zipper GCN4 to the periplasmic C-terminus of CadC and observed that cells CadCLeu TM-GCN4 produced a strong GFP signal, confirming that dimerization alone was able to restore the function of CadC (data not shown). We then built two CadC-VHH fusion proteins composed of CadC DBD, CadC juxtamembrane domain (JM), the Leu(16)TM, CadC wild type external linker region (EL), and the VHH ligand binding domain (data not shown), and placed their expression under the control of the pLacO1 promoter (data not shown). Both fusion proteins had similar expression levels across the IPTG concentration range (data not shown). We observed, as the LexA system, that the CadC-VHH-Caffeine construct exhibited nonspecific activation at high IPTG concentrations (from 0.6 to 5.6 RPUs, about a 9-fold increase), while the CadC-VHH-Control fusion did not (data not shown). Because both receptors are expressed at the same levels, these results suggest that the VHHCaffeine expressed at the membrane has a higher propensity to oligomerize than the VHH-Control. Despite receptor self-activation, we observed a strong response of CadC-VHH-Caffeine to increasing concentrations of caffeine, starting at 25 M IPTG concentration and above (data not shown). VHH Control did not show any change in GFP signal. We calculated the signal swing and fold change in response to caffeine (data not shown). We found that at 100 M caffeine, the swing increased with increasing receptor expression (swing=6, 19, 21 RPU at 25, 50 and 100 M IPTG, respectively, data not shown). However, the fold change was maximal at 25 M IPTG (10-fold), and decreased with IPTG concentrations above (Fold change=6-and 4.6-fold at 50 and 100 M IPTG respectively, data not shown). This decrease in fold change is explained by a higher background noise due to non-specific receptor activation at higher expression levels. Thus, even though a higher level of expression of CadC-VHH-Caffeine increases the cell sensitivity to caffeine, this overexpression also increases non-specific self activation, and consequently decreases the signal-to-noise ratio. However, cell induced at 25 M IPTG induction had a bimodal distribution while at 50 M the response to cafeine was homogeneous over the whole cell population (data not shown). Therefore, receptor expression needs to be balanced to satisfy two criteria: support homogenous response while minimizing self activation and background noise. These results demonstrate that a synthetic transmembrane receptor can be engineered by fusing a split- DBDs with a periplasmic VHH scaffold. Receptor expression levels strongly influences sensitivity and signal-to-noise ratios.
[0075] Optimizing transmembrane receptor signal-to-noise ratio through linkers engineering. We then wanted to improve the signal-to-noise ratio of our system by educing its background noise. We assumed that improper linker sequence composition may favor receptor non-specific oligomerization and activation. We thus modified the amino acid composition of the external linker region, which was shown to influence receptor basal activation rate as well as signal-to-noise ratio. We switched the wild type CadC external linker (DTRLPMS (SEQ ID NO:16), wt) to a flexible (GGGSG (SEQ ID NO:17)) or rigid (EAAAK (SEQ ID NO:18)) linker sequences. We also completely removed the linker region (no linker, NL) (data not shown). We tested the self-activation level of the external linker variants along with their response to 100 M caffeine at 50 M IPTG (data not shown). We observed similar responses to caffeine from CadC-VHHCaffeine with wt or flexible external linkers while the construct with a rigid external linker had no response (data not shown). Finally, we observed a significant reduction of self-activation along with a slight decrease of signal activation from the construct in which the external linker region had been removed (data not shown). While the signal swing of the NL construct was slightly lower, the fold change was more than tripled compared to the wt construct (5 vs 16-fold change, respectively, data not shown). We found via WB analysis that the wt, flexible, and NL constructs had similar expression levels while the construct with the rigid linker was strongly proteolysed, explaining the total loss in activity of this variant (data not shown). We mapped in more details the response range of CadCVHH- Caffeine NL version (data not shown). We observed a marked improvement of the receptor fold change in response to lower caffeine concentration. For example, at 50 M IPTG induction, the NL version displayed a 3.6-fold change in response to 100 nM caffeine, while the wt construct showed only a 2.8-fold change. This effect was even stronger at 1 M caffeine (NL=8-foldchange vs wt=5), 10 M caffeine (12.6 vs 5.7). We were therefore able to obtain receptors showing strong signal swing in response to caffeine while having a better S/N ratio. These results demonstrate that chimeric transmembrane receptor response can be tuned by optimizing interdomain linker sequences.
EXAMPLE 4
[0076] Transmembrane receptor mediated-detection of extracellular proteins using L-form bacteria. Many biosensing applications like medical diagnostics require the detection of large molecules like protein biomarkers of diseases. However, the CadC transmembrane receptor system is expressed in the inner membrane of E. coli, limiting its accessibility to these large molecules. Nonetheless, the transmembrane receptor should be able to detect such ligands if its sensing domain was directly exposed to the extracellular environment. To demonstrate this possibility, we decided to use L-form E. coli, an outer membrane deficient form of E. coli. L-form bacteria were first isolated in 1935 as osmosensitive, spherical bacterial due to their lack of intact cell wall. L-forms can be generated transiently with antibiotics that inhibit peptidoglycan synthesis or disrupt cell wall formation or permanently by mutation of genes related to peptidoglycan synthesis. Because of their lack of outer membrane, Lform bacteria could be suitable candidates for developing whole cell biosensors to detect large molecules such as proteins. We prepared L-form E. coli by growing cells in osmoprotective media with the antibiotic cefsulodin, which disrupt cell wall synthesis by inhibiting penicillin binding protein (PBP) crosslinking of peptidoglycan (data not shown). We confirmed via microscopic observation that cells had the characteristic spherical shape of L-form (data not shown). We confirmed that our receptor was still functional by adding IPTG and caffeine to the cells and monitoring expression of the fluorescent reporter (data not shown). Thanks to the outer membrane deficiency, we also confirmed that the receptor was effectively targeted to the inner membrane via immunofluorescent labeling (data not shown). We then tested if our receptor could bind and be activated by large protein ligands by monitoring its response to an antibody. Antibodies are a relevant class of biomarkers that can be used to diagnose infections or autoimmune diseases. Because antibodies are multivalent, binding of an antibody to the extracellular domain of the receptor should trigger its multimerization and lead to receptor activation. As a proof-of-concept, because our VHHs have a c-Myc tag in C-terminus, we aimed at detecting the anti-c-Myc antibody. We incubated L-form E.coli expressing NL version of CadC-VHHCaffeine with 10 g/ml (66 nM) anti-c-Myc antibody (data not shown). We observed an increase in GFP signal of 3-fold from Lform bacteria incubated with the c-Myc antibody (data not shown). This response was dependent on the presence of the receptor as signal increase was observed only in the presence of IPTG. Finally, we did not observe any signal increase when we incubated untreated E. coli with the anti-c-Myc antibody (data not shown). These results demonstrate that a bacterial chimeric transmembrane receptor can be used to detect protein ligands in the extracellular environment, and that L-form bacteria might be a suitable format for such applications.
EXAMPLE 5
Introduction
[0077] First of all, in order to validate the usefulness of our chimeric receptor polypeptide in a cell free system, we tested a system used in vivo based on dimerization activation. Mono-domain antibodies, VHH-caffeine, are activated by binding a ligand, caffeine, which induces their homodimerization. These antibodies are fused with monomeric DNA binding domains (LexA-DBD) which, with their dimerization, bind to the operator of the pLexA promoter repress the expression of a GFP reporter gene (
Methods
[0078] Plasmid Preparation: Genes encoding the GFP reporter, LexA-DBD-VHHcafe as well as LexA-DBD-Lam4 were cloned as described in Birnboim HC, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979;7: 1513-1523. All the amplification was done in E. coli. Plasmid purification was performed with Qiagen kits.
[0079] Preparation of cell extracts: The process took 4 to 5 days, according to the protocol made available on jove.com (Sun ZZ, Hayes CA, Shin J, Caschera F, Murray RM Noireaux V. Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology. J Vis Exp. 2013; e50762).First of all, the strain of E. coli was cultured on agar plate in presence of an antibiotic for selection, and the culture medium is prepared. Then the amplification was done in the culture medium. The cultures were centrifuged and washed out. Finally, the cells were lysed. The calibration of the extracts, the optimization of glutamate, DTT as well as magnesium concentrations are finally carried out.
[0080] Kinetic data collection by plate reading: The samples are put in a 384 well plate. Cytation3 is used to record the fluorescence emitted from the GFP wavelength (528nm) and the RFP for DsRed fluorescence (620nm). The machine is programmed at 30 C. and the fluorescence is read for 16 hours at 10 minute intervals.
Results
[0081] Validation of the in vitro system: We analysed the intensity of the fluorescence emitted by the expression of the deGFP by the reporter gene. We tested the influence of a growing range of LexA-VHHcafe plasmid on the expression of deGFP, with and without caffeine in the medium (
[0082] Detection of a larger ligand, DsRed: As for the previous system, we detected the fluorescence emitted by the expression of deGFP by the pLexA-deGFP reporter gene (
REFERENCES
[0083] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.