WO2023050644A1

WO2023050644A1 - Chimeric allosteric transcription factor for mammalian system, regulatory element group, and inducible expression system

Info

Publication number: WO2023050644A1
Application number: PCT/CN2022/071023
Authority: WO
Inventors: 娄春波; 项延会; 王卫军; 李婷婷; 陈茜
Original assignee: 中国科学院深圳先进技术研究院
Priority date: 2021-09-29
Filing date: 2022-01-10
Publication date: 2023-04-06
Also published as: CN115873125A

Abstract

The present invention relates to a chimeric allosteric transcription factor for regulating genes of interest in mammalian cells and a regulatory element group comprising the chimeric allosteric transcription factor, and an inducible expression system. The chimeric allosteric transcription factor comprises a regulatory domain and a transcriptional activation domain, wherein the regulatory domain is selected from the group consisting of RpaR, BjaR, and BraR; the transcriptional activation domain is selected from P65, VP16, VP64, VTR1, VTR2, and VTR3; the chimeric allosteric transcription factor uses a natural amino acid-derived acyl homoserine lactone as an inducer; and the natural amino acid-derived acyl homoserine lactone is selected from the group consisting of the following compounds: p-coumaroyl-homoserine lactone (pC-HSL), isovaleryl-homoserine lactone (IV-HSL), and cinnamoyl-homoserine lactone (Cinn-HSL). The system can be used in a modular manner in a variety of mammalian cells, tissues or organisms to regulate the expression of genes of interest.

Description

Chimeric allosteric transcription factors, sets of regulatory elements and inducible expression systems for use in mammalian systems

References to related applications

This application claims the rights and interests of the Chinese patent application No. 202111150967.4 submitted to the State Intellectual Property Office of the People's Republic of China on September 29, 2021, the entire contents of which are hereby incorporated herein by reference.

technical field

The invention belongs to the field of synthetic biology and bioengineering, and relates to a chimeric allosteric transcription factor capable of regulating gene expression in a mammalian system, a regulatory element group and an induced expression system. In particular, the present invention relates to the use of natural amino acid-derived acyl homoserine lactones as inducers to regulate the transcription of a gene of interest in a host cell.

Background technique

Precise regulation of gene expression in mammalian hosts is extremely important for gene function analysis, biopharmaceuticals, vaccine preparation, animal model design, and gene therapy. Various gene expression regulation systems for mammals have been reported in the art, for example, using immunosuppressants or steroid hormones as inducers to regulate the transcription of target genes. However, these systems generally have problems such as difficulty in controlling the concentration of the inducer or causing interference to the host gene network. In order to construct an inducible expression system orthogonal to the host gene regulatory network, attempts have been made to transform the regulatory elements derived from prokaryotic cells and transfer them to eukaryotic systems to induce the expression of genes in eukaryotic hosts. For example, the Streptomyces coelicolor quorum sensing receptor ScbR or the Streptomyces pristinaespiralis transcription factor SpbR can be fused to the eukaryotic transcriptional activation domain VP16 of herpes simplex virus as a transcriptional activator. factor, and the corresponding operator sequence was assembled to the 5' end of the CMVmini promoter to construct a mammalian inducible expression system using butyrolactone compounds as inducers ^[1] . However, the above expression systems are all positive control repressor systems, in which allosteric transcription factors are detached from DNA in the presence of small molecules. WO2007058527A2 reports the transfer of a prokaryotic-derived Tet regulatory system to a mammalian system. In Escherichia coli, the repressor protein TetR specifically binds to its operator sequence tetO to inhibit downstream gene transcription, while the binding of tetracycline or doxycycline (dox) to TetR triggers a conformational change, releases the promoter region, and turns on gene transcription. For eukaryotic hosts, VP16 can be fused with TetR to construct an allosteric transcription factor tTA, which can regulate eukaryotic promoters containing tetO sequences. However, the binding of tetracycline or dox to tTA disengages the transcription factor from the promoter region, and the system remains a positively controlled repressive system. As an improvement, some amino acids of TetR can be mutated so that it binds to DNA after being combined with an inducer. This negative control system can be activated by a lower concentration of inducer (such as 44ng/ml doxycycline).

The quorum sensing system is a system that exists in bacteria and responds to bacterial population density signals. It realizes intercellular communication by synthesizing, secreting, detecting, and responding at the population level to coordinate gene expression within the population. This process is closely related to bioluminescence, antibiotic synthesis and biofilm formation. The signaling molecules secreted by different species of bacteria have different chemical structures, which provide more options for constructing artificial expression systems that are orthogonal to the gene regulatory network of mammalian hosts. For example, the quorum sensing system of Proteus uses acyl homoserine lactone (N-acyl homoserine lactone, AHL) as a signal molecule, and the LuxR family allosteric transcription factor acts as a receptor to transduce the concentration information of the signal molecule. It has been reported in the field that the LuxR family allosteric transcription factor was fused with the eukaryotic transactivation domain NF-κB p65 or VP16 to construct a chimeric transcription factor; and the operator sequence was added to the CMVmini promoter to construct an inducible promoter responsive to AHL ^[2]-[3] . The EC50 levels of such mammalian induction systems reported in the art are above 10 μM, and the inducers are limited to 3-oxo-octanoyl-L-homoserine lactone (3OC8) or 3-oxo- Hexanoyl-L-homoserine lactone (3OC6); the acyl side chains of these two straight-chain fatty acid-type AHLs are highly similar, so the multichannel communication system based on this type of AHL exists at both the signal level and the promoter level Extensive crosstalk.

In the present invention, the corresponding allosteric transcription factors and operating sequences are identified by data mining of the quorum sensing system in prokaryotes using acyl homoserine lactone derived from natural amino acids as an inducer, and are compared with the eukaryotic transcriptional regulation system. Integrating, a novel inducible expression system for mammalian cells was constructed.

Contents of the invention

In a first aspect, the present invention provides a chimeric allosteric transcription factor for regulating a gene of interest in a mammalian cell, the chimeric allosteric transcription factor comprising a regulatory domain and a transcriptional activation structure domain; wherein, the regulatory domain is selected from the group consisting of RpaR, BjaR and BraR; the transcriptional activation domain is selected from P65, VP16, VP64, VTR1, VTR2 and VTR3; the chimeric type Constitutive transcription factors use natural amino acid derived acyl homoserine lactones as inducers; said natural amino acid derived acyl homoserine lactones are selected from the group consisting of the following compounds: p-coumaroyl-homoserine lactone (pC -HSL), isovaleryl-homoserine lactone (IV-HSL) and cinnamoyl-homoserine lactone (Cinn-HSL).

In a second aspect, the present invention provides a set of regulatory elements using natural amino acid-derived acyl homoserine lactones as inducers selected from the group consisting of : p-coumaroyl-homoserine lactone (pC-HSL), isovaleryl-homoserine lactone (IV-HSL) and cinnamoyl-homoserine lactone (Cinn-HSL); the set of regulatory elements comprises:

A chimeric allosteric transcription factor, said chimeric allosteric transcription factor comprising a regulatory domain and a transcriptional activation domain; wherein said regulatory domain is selected from the group consisting of RpaR, BjaR and BraR; said The transcriptional activation domain is selected from the group consisting of P65, VP16, VP64, VTR1, VTR2 and VTR3; and

An inducible promoter active in mammalian cells, said inducible promoter comprising a core sequence and at least one operator site pair interacting with said chimeric allosteric transcription factor.

In a third aspect, the present invention provides a system for inducing expression of a gene of interest in mammals, the inducible expression system utilizes acyl homoserine lactone derived from a natural amino acid as an inducer, and the acyl homoserine lactone derived from a natural amino acid Acyl homoserine lactones are selected from the group consisting of p-coumaroyl-homoserine lactone (pC-HSL), isovaleryl-homoserine lactone (IV-HSL) and cinnamoyl-homoserine lactone Lactone (Cinn-HSL); the inducible expression system comprises:

A first gene expression cassette, the first gene expression cassette comprising a first promoter sequence and a coding sequence of a chimeric allosteric transcription factor, wherein the chimeric allosteric transcription factor comprises a regulatory domain and a transcriptional activation structure domain; the regulatory domain is selected from the group consisting of RpaR, BjaR and BraR; the transcriptional activation domain is selected from the group consisting of P65, VP16, VP64, VTR1, VTR2 and VTR3; and

A second gene expression cassette, the second gene expression cassette comprising an inducible promoter sequence and the coding sequence of the gene of interest, the inducible promoter sequence comprising an allosteric transcription factor interacting with the chimeric type At least one operator site pair for .

In the fourth aspect, the present invention provides the chimeric allosteric transcription factor described in the first aspect, the set of regulatory elements described in the second aspect, and the inducible expression system described in the third aspect in the construction of single-cell or multi-cell signaling Communication systems and use in models of human disease.

Beneficial effect

In bacteria, the interaction of natural amino acid-derived acyl homoserine lactones with the LuxR family transcriptional activators RpaR, BjaR or BraR changes the conformation of these transcriptional activators, allowing them to bind to the corresponding operator sequence on DNA and activate downstream genes transcription. In the present invention, by identifying and characterizing the operator sequences of the above-mentioned transcription factors, and adding the operator sequences to eukaryotic promoters, constructs capable of responding to pC-HSL, IV-HSL and/or Cinn-HSL in mammalian hosts The inducible promoter of HSL; on the other hand, the eukaryotic transactivator is fused as the transcriptional activation domain to the above-mentioned transcriptional activator of the LuxR family to construct a chimeric allosteric transcription factor capable of regulating gene expression in a eukaryotic host factor. Since pC-HSL, IV-HSL and Cinn-HSL can freely diffuse in and out of cells, the regulatory domain of the chimeric allosteric transcription factor constructed in the present invention is combined with the above-mentioned inducer to change its conformation, so that it can be compatible with the corresponding The operator sequence is combined, the transcriptional activation domain of the chimeric allosteric transcription factor is positioned to a promoter containing the corresponding operator sequence, and the gene controlled by the promoter is induced to express. In the absence of an inducer, the chimeric allosteric transcription factor dissociates from the DNA, turning off gene transcription. This is the first report in the field of using natural amino acid-derived acyl homoserine lactone as an inducer to induce a gene of interest in mammalian cells. The induction system can be used in various types of mammalian cells, tissues or organisms in a modular manner to regulate the expression of genes of interest.

In fact, in mammalian hosts, transcription cannot be completely shut down when repressor proteins are used as transcriptional regulators because the transcriptional regulatory sites are difficult to be 100% occupied. Therefore, the repressor-based induction system not only requires the expression of a higher concentration of transcription factors in the cell, which increases the metabolic burden of the host cell, but also makes it difficult to improve the signal-to-noise ratio and achieve rapid induction. The chimeric allosteric transcription factor of the present invention is a transcription activator without the above-mentioned systemic defects. On the other hand, there is no signaling pathway associated with prokaryotic AHL in mammalian cells, so when the chimeric allosteric transcription factor or inducible expression system of the present invention is introduced into a mammalian host, the chimeric allosteric transcription factor is highly Specifically combined with the corresponding operator sequence, there is no undesired pleiotropic effect. The currently reported induction system using straight-chain fatty acid type AHL as an inducer responds to 3OC8 signal by means of VP16-TraR fusion protein, and the half-maximum effective concentration EC50 of the system is 7-14 μM ^[3] , while the pC-TraR of the present invention The EC50 of HSL and Cinn-HSL induction system are 0.8μM and 1.25nM, respectively. Compared with the 3OC8 induction system, the induction system of the present invention can achieve a hypersensitive response. Since it can be induced at a lower working concentration, the cytotoxicity of the inducer to cells is effectively reduced. In addition, the maximum induction multiple of the 3OC8 induction system is about 20 times, while the maximum induction multiple of the pC-HSL induction system of the present invention can reach 100 times, and the signal-to-noise ratio is higher. In addition, since there is no signal crosstalk between the induction system of the present invention and the straight-chain fatty acid-type AHL induction system ^[5] , the induction system of the present invention can be used in combination with the straight-chain fatty acid-type AHL induction system to construct multiple A complex regulatory network or gene circuit of signals.

In a preferred embodiment, the chimeric allosteric transcription factor further comprises a multimerization domain CarH. Without wishing to be bound by theory, the addition of a multimerization domain to an allosteric transcription factor increases steric hindrance near the promoter, increases the steepness of the induction curve, reduces background expression, and shifts the induction threshold. High for better transcription switch performance. For example, in the process of vaccine production, excessive background expression will lead to the loss of inducer dependence, thereby resulting in excessive immunogenicity, so reduced background expression is extremely important. As demonstrated in the examples, fusion of the CarH multimerization domain can increase the sensitivity of the pC-inducible system by more than 5 times.

It is known in the art that linearization can reduce signal distortion and improve linearity of the amplifier in the circuit, which is very important for peak frequency adaptation and conversion of non-linear transient trigger signals into stable linear responses. In gene circuits, the linear transformation of the reporter level can be achieved by adding negative feedback in the regulatory network, reducing the heterogeneity of expression and changing the shape of the sigmoid response curve. In a preferred embodiment of the present invention, the dynamic range of the inducible expression system is expanded by adding a TetR-based negative feedback system, so that the concentration of the inducer has a linear relationship with the expression level of the target protein within a wide range. Linearization of such regulatory elements is critical for the rational design of regulatory networks.

Description of drawings

Figure 1 shows the design principle and characterization of the induction system of the present invention. (A) The design principle of chimeric allosteric transcription factors and the working principle of the induction system comprising said chimeric allosteric transcription factors. (B) The pC inducible system, the Cinn inducible system and the inducer of the IV inducible system, the regulatory domain of the chimeric allosteric transcription factor, and the EC50 and dynamic range of the induction curve according to an embodiment of the present invention. (C) Induction curves of the pC inducible system, Cinn inducible system and IV inducible system measured by flow cytometry, the inducers used were pC-HSL, Cinn-HSL and IV-HSL, respectively.

Figure 2 shows the identification and optimization of the operator sequence for the Cinn inducible system. (A) is the result of comparing the sequences of the putative BraI promoter regions in four Bradyrhizobium oligotrophicum S58 (APO) and ORS278 (CUL and CUS) by BLAST . Box encloses the putative operator site braO. (B) Induction curves of Cinn inducible systems containing promoters P _CUS-TRE3G , P _CUL-TRE3 , P _ORS-TRE3G , P _APO-TRE3G and P _NC-TRE3G determined by flow cytometry. Wherein, the promoters P _CUS-TRE3G , P _CUL-TRE3 , P _ORS-TRE3G , P _APO-TRE3G and P _NC-TRE3G respectively contain a pair of CUS, CUL, ORS, APO and NC operation sites. (C) For the operator sequence CUS (wt) of Bradyrhizobium ORS278 strain, it was truncated from upstream or downstream, respectively, to obtain operator sequences F1, F2, F4, F7 (5' end truncated), and R1, R4, R6 , R7, R8 (3' end truncated). (D) The fluorescent intensity of the reporter in the induction system containing each operator sequence in (C) was measured by flow cytometry at 0 and 10 ^-6 mol/L Cinn-HSL. (E) Induction curves of Cinn inducible systems containing the promoter P _2XR8-TRE3G or P _CUS-TRE3G determined by flow cytometry. (F) shows the minimal manipulation sequences corresponding to CUL, NC, ORS, APO and CUS in (A).

Figure 3 shows the induction curves of the Cinn inducible system comprising the transcriptional activation domain VTR3 or VP64 measured by flow cytometry.

Figure 4 shows the effect of the multimerization domain on the performance of the induction system. (A) shows the design principle of the chimeric allosteric transcription factor VTR3-CarH-RpaR containing the multimerization domain CarH and the working principle of the pC induction system containing the allosteric transcription factor. (B) Induction curves of pC-inducible systems containing allosteric transcription factors VTR3-CarH-RpaR or VTR3-RpaR measured by flow cytometry.

Figure 5 shows that adding negative feedback to the induction system can linearize the response. (a) The working principle of the regulation of the dual-input promoter pC-dox by the inducer pC-HSL and the allosteric transcription factor VTR3-RpaR, wherein the pC-dox promoter contains a pair of rpaO and a pair of tetO operation sites. (b) The mechanism of regulation of the dual-input promoter pC-dox by the inducer dox and the allosteric transcription factor TetR. (c) The induction curve of the pC-dox induction system was determined by flow cytometry. The data presented are the mean fluorescence values of triplicate experiments.

Figure 6 shows the signal crosstalk between the pC-inducible system and the Cinn-inducible system. (A) The dose-response curves of the Cinn-inducing system were measured by flow cytometry at each concentration of pC-HSL (ORS-pC) or Cinn-HSL (ORS-Cinn). (B) The dose-response curves of the pC-inducing system were determined by flow cytometry at various concentrations of pC-HSL (RpaO-pC) or Cinn-HSL (RpaO-Cinn).

Figure 7 shows the construction of mammalian cell-to-cell communication using the Cinn induction system of the present invention. (A) Gene circuit diagram of mammalian cell communication system B4YP-2XR8. (B) Gene circuit diagram of negative control module 2XR8. (C) Communication response of B4YP-2XR8 or 2XR8 modules in mammalian cells. From left to right are the FITC channel, ECD channel, bright field and the overlay of the three. The magnification of the fluorescence microscope is 20×, and the scale bar is 150 μm.

In Fig. 1-Fig. 4 and Fig. 6, the data given are the average fluorescence values of three repeated experiments, and the error bars correspond to the standard deviations of each measurement.

Detailed ways

Aiming at the common problems of low induction level and missing expression in existing mammalian induction systems, the present invention proposes to use natural amino acid-derived acyl homoserine lactone as an inducer to regulate gene expression in mammalian cells in a dose-dependent and reversible manner. To regulate.

As shown in Figure 1A, the chimeric allosteric transcription factor of the present invention has a regulatory domain of prokaryotic origin and a transcriptional activation domain of eukaryotic origin, and this heterologous fusion enables the allosteric transcription factor to respond to heterologous signals. Response: The response to the inducer is achieved by the regulatory domain, and the transcription of the gene in mammals is controlled by the transcriptional activation domain. Specifically, the regulatory domain is a quorum sensing response protein RpaR, BjaR or BraR, and the regulatory domain is combined with acyl homoserine lactone pC-HSL, Cinn-HSL or IV-HSL derived from a natural amino acid to produce a conformation Change, bind to the operator sequence on the DNA, thereby positioning the transcriptional activation domain to the promoter containing the operator sequence, so the expression level of the target gene can be regulated by the concentration of the inducer.

In the bacterial quorum sensing system, the production and response of signaling molecules are usually mediated by the autoinducer synthase LuxI and the response protein LuxR. LuxI catalyzes the generation of signal molecule AHL with S-adenosylmethionine and acyl carrier protein (ACP) as substrates in vivo, and this kind of signal molecule can freely diffuse through the cell membrane; the allosteric transcription factor LuxR can interact with the AHL binds to regulate the transcription of quorum sensing-dependent genes. The positions of LuxI and LuxR genes on the bacterial genome are usually close to each other ^[6] , and the promoter of LuxI contains an operator site that can be regulated by LuxR and AHL, so that the synthesis pathway of AHL includes positive feedback, so bacteria can be in a short time Faster synthesis of signaling molecules. In the present invention, the quorum-sensing allosteric transcription factor is fused as a regulatory domain with the transcriptional activation domain derived from eukaryotic sources, and the operator sequence of the allosteric transcription factor is added to the eukaryotic promoter, and constructed in eukaryotic cells Regulatory element sets and induction systems in response to quorum sensing signals.

In the present invention, the inducible system in which the chimeric allosteric transcription factor comprises RpaR as the regulatory domain and the inducible promoter comprises rpaO as the operating site is called the pC inducible system. Generally speaking, the The inducer is p-coumaroyl-homoserine lactone (pC-HSL). In the present invention, the inducible system in which the chimeric allosteric transcription factor comprises BraR as the regulatory domain and the inducible promoter comprises braO as the operating site is called the Cinn inducible system. Generally speaking, the The inducer was cinnamoyl-homoserine lactone (Cinn-HSL). In the present invention, the induction system in which the chimeric allosteric transcription factor includes BjaR as the regulatory domain and the inducible promoter includes bjaO as the operating site is called the IV induction system. Generally speaking, the The inducer is isovaleryl-homoserine lactone (IV-HSL). In the present invention, the terms "inducible system" and "inducible expression system" and "gene expression system" are used interchangeably.

The terms "inducer", "inducer", "signaling molecule" and "signaling small molecule" are used interchangeably herein, and refer to small chemical molecules that can regulate the binding of allosteric transcription factors and operator sequences by themselves. Source input or host own synthesis. Most of the discovered bacterial quorum sensing systems use straight-chain fatty acid AHL as the signal molecule, and its acyl side chain determines the specificity of the signal. The inducers used in the present invention are acyl homoserine lactones derived from natural amino acids, which are currently only found in bacteria that are symbiotic with plants, and these small molecules are involved in bacteria-plant interactions. In the natural host, straight-chain fatty acid-type AHL is catalyzed by Lux type I N-acyl homoserine lactone synthase, and the substrates of this reaction are S-adenosylmethionine and acyl-acyl carrier protein (Acyl-ACP) . In the biosynthetic pathway of AHL derived from natural amino acids, the substrates of Lux type I synthases RpaI, BraI and BjaI are acyl-coenzyme A (Acyl-CoA) rather than acyl-acyl carrier protein. ACP and coenzyme A have similar chemical properties, both form adducts with carboxylated substrates and present these substrates to the enzyme. The function of ACP usually lies in the biosynthesis and transfer of fatty acid substrates; while CoA is related to carboxylation substrates with more diverse structures: Coenzyme A modified compounds are usually intermediates in biosynthesis and biodegradation pathways ^[5] . Acyl homoserine lactones derived from natural amino acids can freely diffuse into eukaryotic cells, exist stably in cells, and cannot be synthesized by eukaryotic own metabolic pathways. This heterologous inducer can effectively reduce the background expression.

In the chimeric allosteric transcription factor of the present invention, the LuxR homologous protein is used as a regulatory domain to detect and respond to signal molecules. Its structure includes a domain that binds to an inducer and a domain that binds to a promoter operator sequence. Binding of the regulatory domain to the small molecule causes a conformational change that alters the transcriptional level of downstream genes controlled by the promoter. In general, allosteric transcription factors that bind to DNA to trigger or enhance the transcription of target genes are called activators, and vice versa are called repressors. The function of the chimeric allosteric transcription factor of the present invention is an activator. Inducible promoters contain operator sequences capable of interacting with regulatory domains. The operator sequence is generally located near, or spatially adjacent to, the transcription initiation site of the gene of interest. Herein, the terms "operator site", "operator sequence" and "transcriptional regulatory site" are used interchangeably. Without wishing to be bound by theory, in the present invention, an inducible promoter may comprise one or more pairs of operator sequences, and the operator site pairs may both be located upstream or downstream of the transcription initiation site, or located respectively at the transcription initiation site. both sides of the starting point.

The term "promoter" refers to a region of DNA that drives the expression or transcription of a nucleic acid sequence, located synonymously upstream of the transcription initiation site of a gene. RNA polymerase and transcription factors are able to bind DNA at the promoter. An inducible promoter generally includes a transcriptional regulatory region, an RNA polymerase recognition region, and a transcriptional initiation site. It is known in the art that the transcription of the gene can be dynamically turned on or off by operably linking the coding sequence of the gene of interest to the downstream of an inducible promoter, and adding an inducer at a desired time. The term "minimal promoter" or "promoter's core sequence" refers to the smallest transcriptional control unit capable of triggering transcription in eukaryotic cells, such as a promoter core region comprising only the RNA polymerase recognition region and the transcription initiation site . At least one operator site can be added upstream and/or downstream of the minimal promoter to construct an inducible promoter with excellent switch performance. In a preferred embodiment of the present invention, the core sequence of the inducible promoter can be selected from CMV1 promoter (SEQ ID NO: 1), CMVmini promoter (SEQ ID NO: 2), TRE3G promoter (SEQ ID NO: 3), EF1a core promoter (SEQ ID NO: 4) or hEF1a promoter (SEQ ID NO: 40); preferably, the core sequence is a CMV1 promoter or a TRE3G promoter.

In some embodiments, the inducible system is a pC inducible system. Wherein, the inducer is pC-HSL, the regulatory domain of the chimeric allosteric transcription factor is RpaR, and the operator sequence contained in the inducible promoter is rpaO (Table 1). The natural host of the pC quorum sensing system is Rhodopseudomonas palustris, which is symbiotic with plants, and its quorum sensing system includes RpaI of the LuxI family and RpaR of the LuxR family. The transcription of RpaI is jointly activated by pC-HSL and RpaR. In Rhodopseudomonas palustris RCB100 strain, the coding sequence of RpaR is the 348672-349403bp antisense strand of its whole genome sequence (NCBI accession number is WP_011155889.1). In the culture of Rhodopseudomonas palustris, the concentration of pC-HSL is on the order of 1-10 μM, and the bacterium can appear quorum sensing in the presence of nM pC-HSL ^[7] . In addition, the RpaI promoter containing the rpaO operator sequence could not be activated by straight-chain fatty acid-type AHL, indicating that there was good orthogonality between the pC quorum sensing system and the straight-chain fatty acid-type AHL quorum sensing system at working concentrations. This system can be used in combination with a linear fatty acid-type AHL induction system (for example, 3OC6 or 3OC8 as an inducer) to construct a multiple induction system. As demonstrated in the examples of the present invention, the pC induction system can also be regulated using Cinn-HSL as an inducer. In terms of the use of the chimeric allosteric transcription factor, regulatory element group and induction system of the present invention in the construction of an intercellular signal communication system, the pC-HSL synthesis pathway that exists in Rhodopseudomonas palustris is not complete, and requires The biosynthesis of pC-HSL can only be achieved by adding p-coumarate to the medium. In the present invention, tyrosine ammonia lyase TAL, 4-coumaric acid coenzyme A ligase 4CL and p-coumaroyl-homoserine lactone synthetase RpaI can be used to catalyze the synthesis of p-coumaroyl-homoserine from tyrosine Serine lactone ^[8] .

In some embodiments, the induction system is a Cinn induction system. Wherein, the inducer is Cinn-HSL, the regulatory domain of the chimeric allosteric transcription factor is BraR, and the operator sequence contained in the inducible promoter is braO (Table 1). In the present invention, the operator sequences capable of responding to Cinn-HSL and BraR are collectively referred to as braO, for example, braO can be NC (SEQ ID NO: 5), ORS (SEQ ID NO: 6), APO (SEQ ID NO: 7), CUS (SEQ ID NO: 8), CUL (SEQ ID NO: 9), R1 (SEQ ID NO: 10), R4 (SEQ ID NO: 11), R7 (SEQ ID NO: 12) or R8 (SEQ ID NO: 13) (Figure 2), preferably R8. As mentioned above, there is only a complete quorum sensing signal detection and response module in Rhodopseudomonas ponderosa symbiotic with plants, and the generation of its pC-HSL signal requires the addition of p-coumarate. Studies have shown that the small molecule Cinn-HSL secreted by photoheterotrophic Bradyrhizobium can stimulate the quorum sensing of Rhodopseudomonas palustris. In Bradyrhizobium, the biosynthesis of Cinn-HSL is catalyzed by LuxI homologous protein BraI (NCBI accession number: CAL74857.1), and the signal molecule is detected by LuxR homologous protein BraR. For example, in Bradyrhizobium ORS278, the coding sequence of BraR is its genome (NCBI accession number: CU234118.1) antisense strand 1004717-1005442bp), and the coding sequence of BraI is its genome antisense strand 1004631-1003951bp. In Bradyrhizobium reporter strains, the concentration of Cinn-HSL required to achieve a half-saturated quorum sensing response can be as low as 10 pM, while the concentration of linear fatty acid-type AHL required to achieve a half-saturated quorum sensing response is 10 ⁴ -10 ⁷ pM, indicating that there is a good orthogonality between the Cinn quorum sensing system and the linear fatty acid AHL quorum sensing system at the working concentration. A multiple induction system can be constructed by reducing the concentration of Cinn-HSL and using this system in combination with a linear fatty acid-type AHL induction system (for example, using 3OC6 or 3OC8 as an inducer). In Rhodopseudomonas palustris, a higher concentration of Cinn-HSL can also activate the RpaI promoter containing the rpaO operator sequence. By adjusting the concentration of Cinn-HSL, the host cells containing both the pC inducible system and the Cinn inducible system can Spatiotemporal specific regulation. As far as the chimeric allosteric transcription factor of the present invention, regulatory element group and induction system are used in the construction of the intercellular signal communication system, although Bradyrhizobium biosynthesis of Cinn-HSL does not require the addition of cinnamate, Cinn The yield of -HSL was only 20nM. Considering that BraI catalyzes the production of Cinn-HSL from S-adenosylmethionine and the active form of cinnamate, the production of the intermediate product cinnamate can be increased by introducing the cinnamate biosynthesis pathway, thereby increasing the production of Cinn-HSL . It is known in the art that phenylalanine ammonia lyase catalyzes the deamination of phenylalanine to cinnamic acid. The phenylalanine ammonia lyase can be derived from, for example, Streptomyces maritimus or Rhodosporidium toruloides. In the present invention, phenylalanine ammonia-lyase PAL, 4-coumaric acid coenzyme A ligase 4CL and BraI are used to catalyze the synthesis of cinnamoyl-homoserine lactone from phenylalanine ^[9] .

In some embodiments, the induction system is an IV induction system. Wherein, the inducer is IV-HSL, the regulatory domain of the chimeric allosteric transcription factor is BjaR, and the operator sequence contained in the inducible promoter is bjaO (Table 1). IV response elements exist in a variety of Bradyrhizobium japonicum, Bradyrhizobium diazoefficiens, Bradyrhizobium niftali, Bradyrhizobium liaoningense, etc. In Bradyrhizobium japonicum, the first discovered natural host, its quorum sensing system includes BjaI of the LuxI family and BjaR of the LuxR family. The transcription of BjaI is jointly activated by IV-HSL and BjaR. In Bradyrhizobium japonicus, the BjaI-lacZ reporter can respond to IV-HSL as low as 10pM, while the concentration of linear fatty acid-type AHL required to produce quorum sensing signals is nM, indicating that the working concentration is low. There is good orthogonality between the IV induction system and the linear fatty acid type AHL induction system ^[5] . In Diazorhizobium amplified, the coding sequence of BjaR is the sense strand 1105945-1106673 bp of its genome (the whole genome NCBI accession number of 110spc4 strain: NZ_CP032617.1). In terms of the use of the chimeric allosteric transcription factor, regulatory element group and induction system of the present invention in the construction of an intercellular signal communication system, the branched chain α-ketoacid dehydrogenase complex BCDH and isovaleryl-homoserine Lactone synthase BjaI catalyzes the synthesis of IV-HSL from isoleucine ^[8] .

Table 1 Elements of pC, Cinn and IV induction systems

It is known in the art that transcriptional regulators of prokaryotic origin can be fused to eukaryotic transcriptional activation domains to regulate gene expression in eukaryotic cells. US8383364B2 discloses that the VP16 activation domain of the herpes simplex virus is fused with the prokaryotic repressor protein TetR to prepare the transcriptional activator tTA. In eukaryotic cells, tTA regulates the transcriptional activity of the promoter P _tet containing tetO. Neddermann et al. fused the LuxR family allosteric transcription factor TraR with the eukaryotic transcriptional activation domain p65, and the transcription factor TraR-p65 can induce the expression of mammalian genes ^[3] . In addition, eukaryotic transcriptional activation domains such as VP16 (ie VP64), p65 ^[10] or SAM ^[11] with four repeats in tandem can be fused to dCas9 to guide target gene activation by sgRNA. Ma et al. fused the known eukaryotic transcription activation domains p65, RTA, and VP64 to construct a series of VTR domains, and their fusion proteins with dCas9 can be used to regulate eukaryotic gene transcription ^[12] . Transcription activation domains that can be used as chimeric allosteric transcription factors of the present invention include but are not limited to the above-mentioned p65, VP16, VP64, VTR1, VTR2 and VTR3, preferably VP64 or VTR3.

It is well known in the art that if a nucleic acid molecule (such as DNA) comprises a nucleotide sequence containing transcriptional and translational regulatory information, and such a sequence is "operably linked" to a nucleotide sequence encoding a polypeptide, then the nucleic acid molecule ( such as DNA) is said to be "capable of expressing" the polypeptide. An operable link is a link between the regulatory DNA sequence and the coding sequence of each structural domain in a manner that "allows the gene to be expressed as a peptide or protein in a recoverable amount". As far as the regulatory domain is operably linked to the transcriptional activation domain, as long as the functions of the regulatory domain and the transcriptional activation domain are not destroyed, the regulatory domain can be directly linked to the N-terminal or C-terminal of the transcriptional activation domain, Or a linker peptide is used to link the regulatory domain to the N- or C-terminus of the transcriptional activation domain. The connecting peptide is flexible, does not contain any active domains, and preferably consists of glycine and/or serine. In fact the length of the connecting peptide has very limited effect on the chimeric protein and can be of any length. In one embodiment, the connecting peptide is 20-60 bp. In one embodiment, the sequence of the connecting peptide is GGAGGAGGCGGCTCCGGAGGCGGAGGAAGC (SEQ ID NO: 16).

In a preferred embodiment, the chimeric allosteric transcription factor of the present invention may further comprise a nuclear localization signal peptide (NLS), which localizes the allosteric transcription factor to the nucleus of eukaryotic cells. In fact, in the original report, adding NLS had little effect on the performance of the Tet induction system; however, when Tet was used in the adenovirus expression system, the addition of NLS greatly improved the performance of the induction system (US 7745592B2). Furthermore, deletion of the NLS included in the TraR-p65 junction region had little effect on the performance of the chimeric protein ^[3] . The chimeric allosteric transcription factor of the present invention may comprise a nuclear localization signal peptide, the nuclear localization signal peptide comprises 1-4 nuclear localization sequences, and each nuclear localization sequence may have a flanking sequence of 5-40bp at both ends, said The flanking sequences are flexible. Each flanking sequence may be the same or different in length. In fact, the flanking sequence has very limited influence on the function of the chimeric protein and can be any sequence. The nuclear localization signal peptide can be located at the N-terminal or C-terminal of the chimeric allosteric transcription factor, or between the regulatory domain and the transcriptional activation domain. In a preferred embodiment, the nuclear localization signal peptide is located between the regulatory domain and the transcriptional activation domain, and the nuclear localization signal peptide has two nuclear localization sequences, with 5 -20bp flanking sequence. Nuclear localization sequences that can be used in the present invention include: the NLS of the SV40 virus large T antigen with the amino acid sequence of PKKKRKV; the NLS derived from the nucleoplasmic protein (such as the bipartite nucleoplasmic protein NLS with the KRPAATKKAGQAKKKK sequence); with PAAKRVKLD or RQRRNELKRSP NLS of c-myc with amino acid sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY NLS of hRNPA1M9; sequence of importin-α derived IBB domain RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV; sequence of myoma T protein VSRKRPRP and PPKKARED; sequence SALIKKKKKMAP of mouse c-abl IV; sequence DRLRR and PKQKKRK of influenza virus NS1; sequence RKLKKKIKKL of hepatitis D antigen; sequence REKKKFLKRR of mouse Mx1 protein; sequence KRKGDEVDGVDEVAKKKKSKK of human poly(ADP-ribose) polymerase; or Steroid hormone receptor glucocorticoid sequence RKCLQAGMNLEA RKTKK, but not limited thereto. In a preferred embodiment, the nuclear localization sequence is gctgaccccaagaagaagaggaaggtg (SEQ ID NO: 17).

In a preferred embodiment, the chimeric allosteric transcription factor of the present invention may further comprise a multimerization domain, enabling the multimerization of the chimeric allosteric transcription factor. This ability to multimerize does not depend on the structure of the regulatory domain or the transcriptional activation domain, and does not affect the interaction of the regulatory domain with the corresponding operator site. As shown in the examples of the present invention, even if there is only a pair of operator sites on the promoter region, the inducible system switch performance of the allosteric transcription factor containing the multimerization domain is better. In some embodiments, the multimerization domain is CarH (SEQ ID NO: 18). CarH is a coenzyme B12-dependent bacterial photoreceptor protein that controls carotenoid synthesis in response to light. Monomer CarH can form a tetramer after combining with coenzyme B12 in the dark, and the tetramer disintegrates under light. It has been reported in the field that the C-terminal adenosylcobalamin binding domain of CarH was used to prepare gels by utilizing the tetramerization property of CarH ^[13] . For the purposes of the present invention, the addition of a multimerization domain between the regulatory domain and the transcriptional activation domain of a chimeric allosteric transcription factor increases the steepness of the induction curve and reduces background expression. In some embodiments, the multimerization domain is located between the regulatory domain and the transcriptional activation domain. Preferably, the regulatory domain, the multimerization domain and the transcriptional activation domain are linked using a linker peptide. In a preferred embodiment, the chimeric allosteric transcription factor further comprises a nuclear localization signal peptide, and the nuclear localization signal peptide may be located at the N-terminal or C-terminal of the chimeric allosteric transcription factor; or in the regulatory domain Between the multimerization domain, or between the transcription activation domain and the multimerization domain. The features of the linker peptide and the nuclear localization signal peptide are as described above.

Those skilled in the art can understand that the first promoter of the induction system of the present invention (ie, the promoter driving the expression of chimeric allosteric transcription factors) can be selected according to the desired expression level of transcription factors. The first promoter may be a promoter commonly used in mammals, a constitutive promoter, an inducible promoter, or a tissue-specific promoter. In the present invention, the first promoter may be the same as or different from the inducible promoter driving the expression of the gene of interest. In some embodiments, the first promoter is selected from CMV1 promoter, CMVmini promoter, TRE3G promoter, EF1a core promoter or hEF1a promoter.

In a preferred embodiment, negative feedback is added to the set of regulatory elements and the induction system of the present invention, so that the linear transformation of the reporter level can be realized. In a design without negative feedback, the dose-response curve exhibits an S-shape, that is, reporter gene expression saturates rapidly as the inducer concentration increases. However, after adding negative feedback, the dose-response curves were linear in a wide range of inducer concentrations, and the dynamic range increased. Without wishing to be bound by theory, the structure of the linearized gene circuit resembles a bacterial operon regulated by a repressor protein. However, since in bacteria the function of effector proteins is often to catalyze biochemical reactions, or the effector proteins encode carrier proteins that affect the level of inducers, other feedbacks will occur, so that a completely linear response rarely exists in natural gene circuits ^[14] . Considering that the prokaryotic gene circuit is orthogonal to the signal pathway of the eukaryotic host, negative feedback can be added in the induction system of the present invention to linearize the sigmoidal dose-response curve and reduce the bimodal state, thereby obtaining Induced systems with a large linear range. These linear induction systems are beneficial for constructing complex transcriptional regulatory networks. In such embodiments, the set of regulatory elements of the present invention further comprises a repressor protein (such as TetR (US8383364B2) or CymR (WO2019175600A1)) and a third promoter regulating the repressor protein, said third promoter comprising the same A second operator site for repressor interaction (eg tetO or cymO). In addition, besides the operating site interacting with the chimeric allosteric transcription factor, the inducible promoter further includes a second operating site interacting with the repressor protein. There can be one or more second manipulation sites, and they can be located upstream or downstream of the transcription initiation site, or multiple second manipulation sites are distributed upstream or downstream of the transcription initiation site. In such embodiments, the inducible expression system of the present invention further comprises a third gene expression cassette comprising a third promoter sequence and a coding sequence of a repressor protein for expressing said gene expression cassette in a host cell. repressor protein, the third promoter sequence includes a core sequence and a second operator site interacting with the repressor protein; the inducible promoter sequence includes an operator site interacting with a chimeric allosteric transcription factor In addition to the site, it further contains a second operator site that interacts with the repressor protein. In a preferred embodiment, the repressor protein is TetR, and the second operator site is tetO. For the TetR and its operating sequence tetO, see, for example, US8383364B2. In an embodiment of the invention, tetO is tccctatcagtgatagaga (nucleotides 173-191 of SEQ ID NO: 37). The gene sequence of TetR is as described in literature ^[17] . In one embodiment, the repressor protein is CymR and the second operator site is cymO. For the CymR and its operating sequence cymO, see CN107344962A, for example.

In view of the fact that the regulatory domain of the chimeric allosteric transcription factor of the present invention is derived from the signal detection and response protein of the quorum sensing system, the induction system of the present invention can be used as a signal receiving module of the intercellular communication system. Specifically, the biosynthetic gene expression cassette of the signal molecule is placed in a cell as a signal sending element as a whole, so that the signal sending cell expresses all the enzymes required for the synthesis of the signal molecule in vivo. On the other hand, the induction system of the present invention is placed in another cell as a signal receiving element, so that the signal molecule is used as a "wire" to couple the signal sending module and the signal receiving module to construct a multicellular gene circuit. The prokaryotic quorum sensing system can also be imitated, and the signal sending module and the induction system of the present invention can be introduced into the same mammalian cells to regulate the functions of the mammalian cells at the population level. The signaling modules (complete biosynthetic pathways of signaling molecules) corresponding to the pC, Cinn and IV induction systems are described above and summarized in Table 2.

Table 2 pC, Cinn and IV signal transmission modules

Due to its orthogonality, modularity, adjustability and combinability, the chimeric allosteric transcription factor, regulatory element group and induction system of the present invention can be transiently or stably transfected into mammalian cells, and the host gene Expression is regulated. Methods of transient transfection and stable transfection are well known in the art. Based on this, the chimeric allosteric transcription factor, regulatory element group and induction system of the present invention are also useful in the construction of artificial tissues, artificial organs, animal models of human diseases, gene function analysis, biopharmaceuticals, vaccine preparation, gene therapy, etc. advantageous.

Those skilled in the art can understand that, as far as the protein of the present invention is concerned, proteins that are substantially homologous to the above-mentioned enzymes that catalyze the biosynthesis of signal molecules and allosteric transcription factors can also be used. The term "substantially homologous" means that the protein sequences are at least 95%, at least 98% or at least 99% identical at the sequence level. A homologous sequence may be a functionally identical sequence in a different species. Homologues of a gene or protein of the present invention can be readily determined by those skilled in the art. In addition, codons can be humanized and optimized based on the protein sequence to improve the expression strength of the protein in a heterologous host, which is well known in the art.

The term "expression cassette" as used herein refers to a DNA segment consisting of one or more genes and sequences that control the expression of the genes so that the protein encoded by the genes can be expressed in a desired host cell (i.e., a eukaryotic cell). ) expression. It is known in the art that the expression control sequences included in the expression cassette optionally include promoter sequences, 3' non-coding regions, transcription termination sites, polyadenylation and the like. In the present invention, an expression cassette comprising a promoter and a coding sequence can be integrated into the genome or present in a plasmid vector.

Implementations of the aspects described herein can be illustrated by the following numbered paragraphs:

1. A chimeric allosteric transcription factor for regulating a gene of interest in a mammalian cell, said chimeric allosteric transcription factor comprising a regulatory domain and a transcriptional activation domain; wherein said regulation The structural domain is selected from the group consisting of RpaR, BjaR and BraR; the transcription activation domain is selected from P65, VP16, VP64, VTR1, VTR2 and VTR3; the chimeric allosteric transcription factor is derived from natural amino acids The acyl homoserine lactone derived from a natural amino acid is selected from the group consisting of the following compounds: p-coumaroyl-homoserine lactone (pC-HSL), isovaleryl - homoserine lactone (IV-HSL) and cinnamoyl-homoserine lactone (Cinn-HSL).

2. The chimeric allosteric transcription factor as described in paragraph 1, wherein the transcriptional activation domain is VTR3.

3. The chimeric allosteric transcription factor as described in

paragraph

1 or 2, wherein the regulatory domain is RpaR, and the inducer is pC-HSL or Cinn-HSL; preferably, the inducer is pC -HSL.

4. The chimeric allosteric transcription factor as described in

paragraph

1 or 2, wherein the regulatory domain is BjaR, and the inducer is IV-HSL.

5. The chimeric allosteric transcription factor according to

paragraph

1 or 2, wherein the regulatory domain is BraR, and the inducer is Cinn-HSL.

6. The chimeric allosteric transcription factor of any one of paragraphs 1-5, wherein a connecting peptide is used to connect the regulatory domain to the transcriptional activation domain; preferably, the connecting peptide The length is 20-60bp; preferably, the connecting peptide has the sequence of SEQ ID NO: 16.

7. The chimeric allosteric transcription factor of any one of paragraphs 1-5, wherein the chimeric allosteric transcription factor further comprises a nuclear localization signal peptide, wherein the nuclear localization signal peptide is located at the The N-terminal or C-terminal of the chimeric allosteric transcription factor or between the regulatory domain and the transcriptional activation domain, the nuclear localization signal peptide comprises 1-4 nuclear localization sequences; preferably, each The 5' end and the 3' end of the nuclear localization sequence independently have a flanking sequence of 5-40bp; preferably, the nuclear localization signal peptide is located between the regulatory domain and the transcriptional activation domain; preferably, the The nuclear localization signal peptide comprises two nuclear localization sequences, and the 5' end and the 3' end of each nuclear localization sequence independently have a flanking sequence of 5-20bp; preferably, the nuclear localization sequence has the sequence of SEQ ID NO: 17 .

8. The chimeric allosteric transcription factor according to any one of paragraphs 1-5, wherein the chimeric allosteric transcription factor further comprises a multimerization domain, preferably, the multimerization structure The domain is CarH.

9. The chimeric allosteric transcription factor of paragraph 8, wherein the regulatory domain, the multimerization domain and the transcriptional activation domain are linked using a linking peptide; preferably, the linking The length of the peptide is 20-60bp; preferably, the connecting peptide has the sequence of SEQ ID NO: 16.

10. The chimeric allosteric transcription factor as described in paragraph 8, wherein the chimeric allosteric transcription factor further comprises a nuclear localization signal peptide, wherein the nuclear localization signal peptide is located in the chimeric allosteric The N-terminal or C-terminal of the transcription factor is either located between the regulatory domain and the multimerization domain, or between the transcription activation domain and the multimerization domain, and the nuclear localization The signal peptide contains 1-4 nuclear localization sequences; preferably, the 5' and 3' ends of each nuclear localization sequence independently have flanking sequences of 5-40bp; preferably, the nuclear localization signal peptide contains 2 nuclear localization sequences Sequences, the 5' and 3' ends of each nuclear localization sequence independently have flanking sequences of 5-20bp; preferably, the nuclear localization sequence has the sequence of SEQ ID NO: 17; preferably, the nuclear localization signal peptide Located between the regulatory domain and the multimerization domain, or between the transcriptional activation domain and the multimerization domain; preferably, the 5' end of the multimerization domain And 3' end further comprises connecting peptide, preferably, described connecting peptide each has the length of 20-60bp; Preferably, described connecting peptide has the sequence of SEQ ID NO:16.

11. The chimeric allosteric transcription factor of any of paragraphs 1-10, wherein the mammalian cell is a human cell.

12. The chimeric allosteric transcription factor of any of paragraphs 1-10, wherein the mammalian cell is an immortalized cell, a primary cell, or a stem cell.

13. A set of regulatory elements using natural amino acid-derived acyl homoserine lactones as inducers selected from the group consisting of p-coumaroyl-homoserine lactones Serine lactone (pC-HSL), isovaleryl-homoserine lactone (IV-HSL) and cinnamoyl-homoserine lactone (Cinn-HSL); the set of regulatory elements comprises:

14. The set of regulatory elements of paragraph 13, wherein the transcriptional activation domain of the chimeric allosteric transcription factor is VTR3.

15. The set of regulatory elements as described in paragraph 13 or 14, wherein the regulatory domain of the chimeric allosteric transcription factor is RpaR, the operating site of the inducible promoter is rpaO, and the inducer is pC-HSL or Cinn-HSL; preferably, the inducer is pC-HSL.

16. The set of regulatory elements as described in paragraph 13 or 14, wherein the regulatory domain of the chimeric allosteric transcription factor is BjaR, the operator site of the inducible promoter is bjaO, and the inducer is IV-HSL.

17. The set of regulatory elements as described in paragraph 13 or 14, wherein the regulatory domain of the chimeric allosteric transcription factor is BraR, and the operating site of the inducible promoter is braO, preferably R8; The inducer is Cinn-HSL.

18. The set of regulatory elements of any one of paragraphs 13-17, wherein the regulatory domain in the chimeric allosteric transcription factor is linked to the transcriptional activation domain using a linker peptide; preferably Preferably, the length of the connecting peptide is 20-60bp; preferably, the connecting peptide has the sequence of SEQ ID NO: 16.

19. The set of regulatory elements of any one of paragraphs 13-17, wherein the chimeric allosteric transcription factor further comprises a nuclear localization signal peptide, wherein the nuclear localization signal peptide is located in the chimeric allosteric transcription factor The N-terminal or C-terminal of the allosteric transcription factor or located between the regulatory domain and the transcriptional activation domain, the nuclear localization signal peptide comprises 1-4 nuclear localization sequences; preferably, each nuclear localization sequence The 5' end and the 3' end independently have a flanking sequence of 5-40bp; preferably, the nuclear localization signal peptide is located between the regulatory domain and the transcriptional activation domain; preferably, the nuclear localization signal The peptide comprises two nuclear localization sequences, and the 5' and 3' ends of each nuclear localization sequence independently have flanking sequences of 5-20 bp; preferably, the nuclear localization sequence has the sequence of SEQ ID NO: 17.

20. The set of regulatory elements of any of paragraphs 13-19, wherein the chimeric allosteric transcription factor further comprises a multimerization domain, preferably, the multimerization domain is CarH.

21. The set of regulatory elements of paragraph 20, wherein the regulatory domain, the multimerization domain, and the transcriptional activation domain in the chimeric allosteric transcription factor are linked using a linker peptide ; Preferably, each of the connecting peptides has a length of 20-60bp; Preferably, the connecting peptide has the sequence of SEQ ID NO: 16.

22. The set of regulatory elements as described in paragraph 20, wherein the chimeric allosteric transcription factor further comprises a nuclear localization signal peptide, wherein the nuclear localization signal peptide is located at the N of the chimeric allosteric transcription factor terminal or C-terminal, or between the regulatory domain and the multimerization domain, or between the transcriptional activation domain and the multimerization domain, and the nuclear localization signal peptide contains 1 - 4 nuclear localization sequences; preferably, the 5' end and 3' end of each nuclear localization sequence independently have a flanking sequence of 5-40bp; preferably, the nuclear localization signal peptide comprises 2 nuclear localization sequences, each nuclear localization sequence The 5' end and the 3' end of the localization sequence independently have flanking sequences of 5-20bp; preferably, the nuclear localization sequence has the sequence of SEQ ID NO: 17; preferably, the nuclear localization signal peptide is located in the regulatory domain and the multimerization domain, or between the transcriptional activation domain and the multimerization domain; preferably, the 5' end and the 3' end of the multimerization domain Further comprising a connecting peptide, preferably, each of the connecting peptides has a length of 20-60bp; preferably, the connecting peptide has the sequence of SEQ ID NO: 16.

23. The set of regulatory elements according to any one of paragraphs 13-22, wherein the core sequence of the inducible promoter is selected from CMV1 promoter, CMVmini promoter, TRE3G promoter, EF1a core promoter or hEF1a Promoter; preferably, the core sequence is a CMV1 promoter or a TRE3G promoter.

24. The set of regulatory elements of any one of paragraphs 13-23, wherein the pair of manipulation sites is one or more pairs; preferably, the pair of manipulation sites is one pair; preferably, each The operator sites are located upstream and downstream of the transcription start site, respectively.

25. The set of regulatory elements of any one of paragraphs 13-24, further comprising a repressor protein and a third promoter, wherein the third promoter drives expression of the repressor protein, wherein The third promoter comprises a second operator site interacting with the repressor protein; wherein the inducible promoter further comprises the second operator site.

26. The set of regulatory elements of paragraph 25, wherein the repressor protein is TetR and the second operator site is tetO; or, the repressor protein is CymR and the second operator site is cymO.

27. The set of regulatory elements of any of paragraphs 13-26, wherein the mammalian cells are human cells.

28. The set of regulatory elements of any of paragraphs 13-26, wherein the mammalian cell is an immortalized cell, a primary cell, or a stem cell.

29. A system for inducing expression of genes of interest in mammalian cells, the inducible expression system utilizing natural amino acid-derived acyl homoserine lactone as an inducer, the natural amino acid-derived acyl homoserine lactone selected from From the group consisting of the following compounds: p-coumaroyl-homoserine lactone (pC-HSL), isovaleryl-homoserine lactone (IV-HSL) and cinnamoyl-homoserine lactone (Cinn-HSL ); The inducible expression system comprises:

30. The inducible expression system according to paragraph 29, wherein the transcriptional activation domain of the chimeric allosteric transcription factor in the first gene is VTR3.

31. The inducible expression system as described in paragraph 29 or 30, wherein the regulatory domain of the chimeric allosteric transcription factor in the first gene is RpaR, and the inducible promoter in the second gene is The operator site is rpaO, and the inducer is pC-HSL or Cinn-HSL; preferably, the inducer is pC-HSL.

32. The inducible expression system as described in paragraph 29 or 30, wherein the regulatory domain of the chimeric allosteric transcription factor in the first gene is BjaR, and the inducible promoter in the second gene The operator site is bjaO, and the inducer is IV-HSL.

33. The inducible expression system as described in paragraph 29 or 30, wherein the regulatory domain of the chimeric allosteric transcription factor in the first gene is BraR, and the inducible promoter in the second gene The operator site is braO, preferably R8, and the inducer is Cinn-HSL.

34. The inducible expression system of any one of paragraphs 29-33, wherein, in the first gene, a linker peptide is used to link the regulatory domain to the transcriptional activation domain; preferably, the The length of the connecting peptide is 20-60bp; preferably, the connecting peptide has the sequence of SEQ ID NO: 16.

35. The inducible expression system according to any one of paragraphs 29-33, wherein the chimeric allosteric transcription factor of the first gene further comprises a nuclear localization signal peptide, wherein the nuclear localization signal peptide Located at the N-terminal or C-terminal of the chimeric allosteric transcription factor or between the regulatory domain and the transcriptional activation domain, the nuclear localization signal peptide comprises 1-4 nuclear localization sequences; preferably , the 5' and 3' ends of each nuclear localization sequence independently have flanking sequences of 5-40 bp; preferably, the nuclear localization signal peptide is located between the regulatory domain and the transcriptional activation domain; preferably , the nuclear localization signal peptide comprises two nuclear localization sequences, the 5' end and the 3' end of each nuclear localization sequence independently have a flanking sequence of 5-20bp; preferably, the nuclear localization sequence has SEQ ID NO: 17 the sequence of.

36. The inducible expression system according to any one of paragraphs 29-35, wherein said chimeric allosteric transcription factor of said first gene further comprises a multimerization domain, preferably said multimerization The K domain is CarH.

37. The inducible expression system of paragraph 36, wherein the regulatory domain, the multimerization domain and the transcriptional activation domain in the chimeric allosteric transcription factor are linked using a linking peptide ; Preferably, each of the connecting peptides has a length of 20-60bp; Preferably, the connecting peptide has the sequence of SEQ ID NO: 16.

38. The inducible expression system as described in paragraph 36, wherein the chimeric allosteric transcription factor further comprises a nuclear localization signal peptide, wherein the nuclear localization signal peptide is located at the N of the chimeric allosteric transcription factor terminal or C-terminal, or between the regulatory domain and the multimerization domain, or between the transcriptional activation domain and the multimerization domain, and the nuclear localization signal peptide contains 1 - 4 nuclear localization sequences; preferably, the 5' end and 3' end of each nuclear localization sequence independently have a flanking sequence of 5-40bp; preferably, the nuclear localization signal peptide comprises 2 nuclear localization sequences, each nuclear localization sequence The 5' end and the 3' end of the localization sequence independently have flanking sequences of 5-20bp; preferably, the nuclear localization sequence has the sequence of SEQ ID NO: 17; preferably, the nuclear localization signal peptide is located in the regulatory domain and the multimerization domain, or between the transcriptional activation domain and the multimerization domain; preferably, the 5' end and the 3' end of the multimerization domain Further comprising a connecting peptide, preferably, each of the connecting peptides has a length of 20-60bp; preferably, the connecting peptide has the sequence of SEQ ID NO: 16.

39. The inducible expression system of any of paragraphs 29-38, wherein the first promoter in the first gene is a constitutive promoter or an inducible promoter.

40. The inducible expression system according to any one of paragraphs 29-39, wherein the core sequence of the inducible promoter in the second gene is selected from CMV1 promoter, CMVmini promoter, TRE3G promoter, EF1a core promoter or hEF1a promoter; preferably, the core sequence is CMV1 promoter or TRE3G promoter.

41. The inducible expression system according to any one of paragraphs 29-40, wherein the pair of operating sites in the second gene is 1 or more pairs; preferably, the pair of operating sites is 1 Right; preferably, each operator site is located upstream and downstream of the transcription initiation site, respectively.

42. The inducible expression system of any one of paragraphs 29-41, further comprising a third gene expression cassette, wherein the third gene expression cassette comprises a third promoter sequence and a repressor protein A coding sequence for expressing the repressor protein in a host cell, the third promoter sequence comprising a second operator site interacting with the repressor protein; the second gene's The inducible promoter sequence further comprises a second operator site.

43. The inducible expression system of paragraph 42, wherein the repressor protein is TetR, and the second operator site is tetO; or, the repressor protein is CymR, and the second operator site is cymO.

44. The inducible expression system as described in paragraph 42 or 43, wherein the core sequence of the third promoter in the third gene is selected from CMV1 promoter, CMVmini promoter, TRE3G promoter, EF1a core promoter or hEF1a promoter; preferably, the core sequence is CMV1 promoter or TRE3G promoter.

45. The inducible expression system of any of paragraphs 29-44, wherein the mammalian cells are human cells.

46. The inducible expression system of any of paragraphs 29-44, wherein the mammalian cells are immortalized cells, primary cells or stem cells.

47. The chimeric allosteric transcription factor of any one of paragraphs 1-12, the set of regulatory elements of any one of paragraphs 13-28, the inducible expression system of any one of paragraphs 29-46 Use in the construction of a single-cell or multi-cell signal communication system, wherein the signal communication system includes a signal sending element and a signal receiving element, and the signal receiving element includes a chimeric allosteric transcription factor and a mammalian cell having active inducible promoter.

48. The use as described in paragraph 47, wherein the signal molecule of the signal communication system is pC-HSL; the regulatory domain in the chimeric allosteric transcription factor is RpaR, and the inducible promoter comprises A core sequence and at least one pair of rpaO; the signaling elements include TAL, 4CL and RpaI.

49. The use as described in paragraph 47, wherein the signal molecule of the signal communication system is Cinn-HSL; the regulatory domain in the chimeric allosteric transcription factor is BraR, and the inducible promoter comprises A core sequence and at least one pair of braO; the signaling elements include PAL, 4CL and BraI.

50. purposes as described in paragraph 47, wherein, the signal molecule of described signal communication system is IV-HSL; Said regulatory domain in said chimeric allosteric transcription factor is BjaR, and said inducible promoter comprises A core sequence and at least one pair of bjaO; the signaling element includes a branched-chain α-ketoacid dehydrogenase complex BCDH and BjaI.

51. The chimeric allosteric transcription factor of any one of paragraphs 1-12, the set of regulatory elements of any one of paragraphs 13-28, the inducible expression system of any one of paragraphs 29-46 Uses in gene function analysis, construction of human disease models, artificial tissues or organs.

52. The use according to paragraph 51, wherein the chimeric allosteric transcription factor, the set of regulatory elements or the inducible expression system are stably transfected into mammalian cells.

53. The use according to paragraph 51, wherein the chimeric allosteric transcription factor, the set of regulatory elements or the inducible expression system is transiently transfected into mammalian cells.

Example

As shown in Figure 1A, the chimeric allosteric transcription factor of the present invention has a regulatory domain of prokaryotic origin and a transcriptional activation domain of eukaryotic origin, the response to the inducer is realized by the regulatory domain, and the transcriptional activation domain is triggered. Transcription of genes in mammalian cells.

Identification and optimization of the operator sequence for the Cinn inducible system

In Bradyrhizobium, the biosynthesis of Cinn-HSL is catalyzed by the LuxI homolog protein BraI, and the signal is detected by the LuxR homolog protein BraR. In the quorum sensing system, in order to respond to the population density, the inducer small molecule can usually positively regulate the transcription of the autoinducer synthase LuxI, in other words, the promoter region of LuxI should contain the LuxR operating site, and the LuxI Transcription is co-activated by AHL and LuxR. The promoter region of acyl homoserine lactone-dependent genes usually has an inverted repeat sequence of 18-20 bp, however, the operator site interacting with BraR could not be identified in the BraI promoter region according to the above characteristics ^[5] .

In response to this problem, we used the BLAST method to compare the BraR-BraI operon regions of four Bradyrhizobium bacterium BTAi1, ORS285, Bradyrhizobium oligotrophicum S58 and ORS278 (NCBI sequence numbers are CP000494.1, LT859959.1, AP012603.1 and sequence similarity of CU234118). Figure 2A shows the regions between the BraR terminator and the BraI promoter-10 region in BTAi1, ORS285, Bradyrhizobium oligotrophicum S58 and ORS278 (indicated by NC, ORS, APO and CUS, respectively), we speculate that the The sequence contains the operator site braO that interacts with BraR. In addition, to ensure that the operator site was fully included within the putative region, the CUL sequence was further tested, which is the CUS comprising the upstream 45 bp. NC, ORS, APO, CUS and CUL were respectively inserted into the upstream of the TRE3G core promoter in pairs, and the Citrine yellow fluorescent protein coding sequence was connected downstream as a reporter gene, and Cinn-HSL was used to induce and discover the above-mentioned induction systems. There was little difference in sensitivity and dynamic range (Fig. 2B, EC50 was 1.1-1.4nM, dynamic range was 18.5-37.7), indicating that the performance of the Bra operator sequence of different Bradyrhizobium bacteria was similar. In a further embodiment, further optimization is performed based on the CUS sequence.

As shown in Figure 2C, the CUS was truncated from different positions. Among them, F1, F2, F4, F7 are truncated from the 5'end; R1, R4, R6, R7 and R8 are truncated from the 3' end. Insert the above operating sequence into the upstream of the TRE3G core promoter to construct an inducible promoter, and perform inducible expression. Under the induction conditions of 0 and 10 ^-6 mol/L Cinn-HSL, the fluorescence intensity changes of the reporter genes downstream of the above nine promoters are shown in Figure 2D. Inducible systems containing F2, F4, and F7 promoters did not respond to induction by Cinn-HSL, indicating that its Bra operator site was disrupted, while fluorescence intensities induced by R1, R4, R7, and R8 did not change much, indicating that they were almost completely retained manipulated sequence. As shown in Figure 2E, the promoter P _2XR8-TRE3G containing a pair of R8 operating sequences was similar to the promoter P _CUS-TRE3G containing a pair of full-length CUS operating sequences (EC50 were 4.9nM and 9.3nM, dynamic range 35.5 and 74.1, respectively). Considering the length of the operator sequence and the induction strength, R8 was used as the minimum operator sequence (18bp). In a further embodiment, for the Cinn induction system, the P _2XR8-TRE3G promoter containing two R8 operator sequences is used as an inducible promoter; the eukaryotic transcription activation domain VTR3 is fused with the regulatory domain BraR to construct VTR3- BraR chimeric allosteric transcription factor, the dose-response curve of each Cinn-HSL concentration was detected by flow cytometry.

Design of pC and IV induction system

For the pC induction system, the eukaryotic transcription activation domain VTR3 was fused with the regulatory domain RpaR to construct a VTR3-RpaR chimeric allosteric transcription factor; two rpaO operating sequences were inserted into the upstream of the TRE3G core promoter to construct P _{rpaO -TRE3G} promoter; and the Citrine yellow fluorescent protein coding sequence is connected downstream as a reporter gene. In the absence of the inducer pC-HSL, VTR3-RpaR could not bind to rpaO, and the expression of the reporter gene was turned off. The combination of pC-HSL and VTR3-RpaR guides the chimeric allosteric transcription factor to the rpaO site of the P _rpaO-TRE3G promoter, and the transcription activation domain VTR3 triggers the transcription of the reporter gene Citrine, which is detected by flow cytometry Dose-response curves of the system at various inducer concentrations. For the IV induction system, the eukaryotic transcription activation domain VTR3 was fused with the regulatory domain BjaR to construct a VTR3-BjaR chimeric allosteric transcription factor; two bjaO operator sequences were inserted into the upstream of the TRE3G core promoter to construct P _{bjaO -TRE3G} promoter; and the Citrine yellow fluorescent protein coding sequence is connected downstream as a reporter gene. In the absence of inducer IV-HSL, VTR3-BjaR could not bind to bjaO, and the expression of the reporter gene was turned off. The combination of IV-HSL and VTR3-BjaR guides the chimeric allosteric transcription factor to the bjaO site of the P _bjaO-TRE3G promoter, and the transcription activation domain VTR3 triggers the transcription of the reporter gene Citrine, which is detected by flow cytometry Dose-response curves of the system at various inducer concentrations.

Characterization of each induction system

For each inducible system, the fluorescence intensity of the reporter was measured at a range of inducing concentrations (Fig. 1C). The sensitivity of the induction system was quantified by fitting the induction curve with the Hill equation to calculate the EC50 value, that is, the half-maximal effective concentration. As shown in Figure 1B and 1C, the EC50 of pC, Cinn and IV induction systems were 8×10 ^-7 mol/L, 1×10 ^-9 mol/L, 2.5×10 ^-5 mol/L, respectively. Without wishing to be bound by theory, lower EC50 values correspond to higher sensitivity. Such signal hypersensitivity and extremely low working concentrations of inducer are not found in other known AHL-based mammalian induction systems. The reported straight-chain fatty acid-type AHL induction system uses VP16-TraR fusion protein to respond to 3OC8 signal, and the EC50 of this system is 7-14μM ^[3] , while the EC50 of the Cinn-HSL induction system of the present invention is 1.25nM, 3 orders of magnitude lower than the prior art. Since it can be induced at a lower working concentration, the cytotoxicity of the inducer to cells is effectively reduced. In addition, the maximum induction multiple of the 3OC8 induction system is about 20 times, while the maximum induction multiple of the pC-HSL induction system of the present invention can reach 100 times, and the signal-to-noise ratio is higher. Although the background expression of the IV induction system of the present invention is higher, so the sensitivity is not as good as that of the pC induction system and the Cinn induction system, but the crosstalk between the IV induction system and the pC induction system and the Cinn induction system is all small, and the IV induction system can be combined with the pC induction system. Or use in combination with the Cinn induction system. In addition, since there is no signal crosstalk between the induction system of the present invention and the straight-chain fatty acid-type AHL induction system ^[5] , the induction system of the present invention can be used in combination with the straight-chain fatty acid-type AHL induction system to construct multiple signals in mammalian hosts. Complex regulatory networks or gene circuits.

Effect of different transcriptional activation domains on regulatory properties

It has been reported in the art that transcriptional regulatory factors VP16, VP64, p65, SAM, VTR, etc. can be fused as transcriptional activation domains and regulatory domains to regulate gene expression in eukaryotic cells. To compare the efficiency of different transcriptional activation domains, we constructed allosteric transcription factors of two Cinn-inducing systems, VP64-BraR and VTR3-BraR. Wherein, VP64 is four repeated tandem VP16, and said VP16 is the eukaryotic transcriptional activation domain of herpes simplex virus (WO2007058527A2). VTR3 is an optimized VTR series domain. Specifically, VP64, p65 and RTA were first fused into the eukaryotic transcriptional activation domain VTR; as an improvement, the first step was to delete the DNA binding domain (VTR1) at the N-terminal of p65 to reduce the non-targeting effect on the genome; and then for the C-terminal of p65 Two transactivation domains, TA1 and TA2, retain only the complete TA2 and part of TA1 (VTR2) in order to reduce the size of the domain; on the basis of VTR2, part of the RTA domain is further deleted to obtain only 0.8kb of VTR3 activation area. In mammalian cells, the activation efficiency of dCas9-VTR3 is 10 times that of dCas9-VP64 ^[12] . As far as the Cinn induction system of the present invention is concerned, as shown in Figure 3, when the transcription factors are VTR3-BraR and VP64-BraR respectively, the EC50 of the Cinn-HSL signal on the activation of the P _2XR8-TRE3G promoter is 3.8× 10 ^-9 mol/L and 1×10 ^-8 mol/L, the dynamic ranges are 32.2 and 9.5 respectively. In the Cinn induction system, VTR3 as a transcriptional activation domain can achieve a better transcriptional switching effect.

Effect of multimerization on regulatory properties

In prokaryotic hosts, the repression performance of regulatory elements can be enhanced by fusing the multimerization domain with a repressor protein and setting one or more pairs of operating sites at the promoter (CN107344962A). In order to investigate whether a similar strategy can be adopted in the positive control induction system of the present invention, we used a connecting peptide to connect the multimerization domain CarH to between the transcriptional activation domain and the regulatory domain of the transcription factor VTR3-RpaR, constructing VTR3-CarH-RpaR chimeric transcription factor. As shown in Figure 4B, without the multimerization domain, the EC50 of the pC-inducible system was 8.7×10 ^-7 mol/L, and the dynamic range was 52.6; while the transcription factor contained the CarH domain, the pC-inducible system The EC50 is 1×10 ^-7 mol/L, and the dynamic range is 30.8, indicating that the synergy brought about by multimerization improves the sensitivity of the induction system and reduces the working concentration of the inducer. It is known in the art that in a negative control repression system, due to the increased steric hindrance near the promoter sequence interacting with the repressor protein after multimerization of the repressor protein, the competitive inhibition of RNA polymerase is more severe. Larger, the probability of RNA polymerase binding is reduced, so by increasing the number of manipulation site pairs, the expression leakage can be effectively reduced (CN107344962A). We found that inserting 1 to 7 pairs of rpaO operator sequences upstream and/or downstream of the TRE3G promoter had little difference in the induction performance, indicating that increasing the number of operator sites in the positive control induction system did not significantly improve the induction performance of the regulatory element set . In addition, the inventors also tried to use the multimerization domain CI434 to construct a multimerizable transcription factor, but this design could not improve the sensitivity of the pC induction system.

Linearization of Regulatory Systems

It is known in the art that adding negative feedback to the induction system enables a linear transformation of reporter levels. In the present invention, the TetR expression cassette containing negative feedback (P _{tet-CMV D2i} promoter containing tetO) in the promoter region is introduced into the host cell, and the transcription start site (Inr) of the inducible promoter P _rpaO-CMV1 A tetO operator site was inserted in the upstream and downstream of , respectively, to construct a double-input promoter pC-dox, which can respond to the dual signals of pC-HSL and doxycycline. As shown in Figures 5A and 5B, the combination of the repressor protein TetR and tetO blocked the rpaO operating site of pC-dox and repressed the transcription of the reporter gene; in the presence of dox, TetR detached from tetO, exposing the operating site rpaO, thereby RpaR bound to pC-HSL can bind to rpaO, thereby recruiting RNA polymerase and reporting gene expression; since the P _{tet-CMV D2i} promoter also contains tetO, the negative regulation of TetR expression by dox makes it appear at every dox concentration TetR adaptation (adaptation), so the expression of the reporter gene is linearized with the increase of dox and pC-HSL. As shown in Figure 5C, the induction system maintains a linear response in a wide range of inducer concentration (0-100ng/mL dox, 10nM-10μM pC-HSL), and the dynamic range is as high as 500, which can realize the control of gene expression. Fine regulation.

Orthogonality of the induced system

Since the chemical structure difference between pC-HSL and Cinn-HSL is only one hydroxyl group on the aromatic ring, there is crosstalk in the quorum sensing mediated by pC-HSL and Cinn-HSL in the natural host. However, as long as the same inducer has a large difference in the working concentration of the two systems, the concentration of the inducer can be reduced so that only the more sensitive system is induced, thereby orthogonalizing the two signaling pathways. In order to test the degree of crosstalk between the two induction systems in mammalian system, we used pC-HSL and Cinn-HSL as inducers to study the signal level crosstalk between pC and Cinn induction systems. As shown in Fig. 6A, the EC50 of Cinn signaling system was 0.1 μM and 1.3 nM under the induction of pC-HSL and Cinn-HSL, respectively. As shown in Figure 6B, the EC50 of the pC signaling system was 0.7 μM and 6 μM induced by pC-HSL and Cinn-HSL, respectively. It can be seen that the lower concentration of Cinn-HSL can only successfully induce the Cinn system, so by adjusting the concentration of Cinn-HSL, the cells or tissues with both the pC-inducible system and the Cinn-inducible system can be regulated spatiotemporally. When both pC and Cinn induction systems exist, the use of pC-HSL as an inducer will lead to signal crosstalk between the two systems. However, for the pC-inducible system, using Cinn-HSL as an inducer can obtain a stronger response at a lower inducer concentration, so Cinn-HSL can also be used alone to regulate the pC-inducible system.

Mammalian cell-to-cell communication system

As an applied example, we reconstituted the quorum-sensing signaling system in mammalian cells. As mentioned above, the bacterial quorum sensing system uses freely diffusible AHL as a signal molecule, which is catalyzed by LuxI family proteins (signal sending), and detected and transduced by transcription factor LuxR family proteins (signal reception). Similarly, the mammalian cell communication system constructed in the present invention includes a signal sending module for generating signal molecules and a signal receiving module for responding to signals ( FIG. 7A ). Among them, the signaling module B4YP is composed of phenylalanine ammonia lyase PAL ^[9] , 4-coumaric acid-CoA ligase 4CL ^[9] and LuxI family protein BraI. The signal receiving module 2XR8 is the Cinn induction system of the present invention. Wherein, the first gene includes the coding sequence of the promoter hEF1a and the chimeric allosteric transcription factor VTR3-BraR, and the second gene includes the inducible promoter P _2XR8-TRE3G and the reporter gene Citrine. The negative control contained only the signal receiving module 2XR8 (Fig. 7B). As shown in the FITC channel of Figure 7C, the reporter gene Citrine of the communication system was well expressed in the cells transfected with the B4YP-2XR8 module. Control cells containing only the receptor module 2XR8 had no expression of Citrine due to the inability to synthesize signaling molecules.

method

The induction system involved in the embodiment of table 3

The reporter gene in the above induction system is Citrine ^[15] .

Plasmid construction

For the pC induction system, VTR3 (SEQ ID NO: 41) was first connected to the 5' end of the nuclear localization signal peptide (SEQ ID NO: 38) and RpaR (NCBI accession number is WP_011155889.1 antisense chain 348672-349403bp ) is connected to the 3' end of the nuclear localization signal peptide to construct the coding sequence of the allosteric transcription factor VTR3-RpaR. The nuclear localization signal peptide comprises two nuclear localization sequences (SEQ ID NO: 17), with 20 bp random sequences between the two nuclear localization sequences, 5' end and 3' end. The two rpaO operating sequences were inserted into the upstream of the TRE3G core promoter to obtain the P _rpaO-TRE3G promoter; and the Citrine yellow fluorescent protein coding sequence was connected downstream as a reporter gene. Plasmids containing VTR3-RpaR and an inducible promoter-reporter gene also constitutively express mCherry red fluorescent protein for internal monitoring of plasmid transfection. When pC-HSL exists, VTR3-RpaR can bind to rpaO to activate the expression of Citrine reporter gene. Specifically, each element was assembled onto the pcDNA3.1(+) plasmid (Invitrogen, catalog #V790-20) using Gibson assembly in the following order: P _rpaO-TRE3G promoter; Citrine reporter gene ^[15] ; hEF1a promoter (114743-113562bp segment from human clone RP11-505P4 chromosome 6 DNA (NCBI accession number AL603910.6)); VTR3-RpaR; EF1 promoter ^[8] ; mCherry reporter gene ^[16] . Wherein, each protein coding sequence is followed by a poly(A) sequence.

As far as the allosteric transcription factor VTR3-CarH-RpaR with multimerization domain is concerned, its 5' end to 3' end are respectively VTR3 (SEQ ID NO: 41), nuclear localization signal peptide (SEQ ID NO: 38) , connecting peptide (SEQ ID NO: 16), CarH (SEQ ID NO: 18), connecting peptide (SEQ ID NO: 16) and RpaR (NCBI accession number is WP_011155889.1 antisense strand No. 348672-349403bp).

As far as the IV induction system is concerned, the 5' end to the 3' end of the chimeric allosteric transcription factor VTR3-BjaR are VTR3 (SEQ ID NO: 41), nuclear localization signal peptide (SEQ ID NO: 38) and BjaR ( SEQ ID NO: 43). Among them, BjaR is obtained through human codon optimization according to the BjaR protein sequence (WP_011083882.1). Similar to the construction of the pC induction system, each element was assembled on the pcDNA3.1(+) plasmid (Invitrogen, catalog #V790-20) using Gibson assembly in the following order: P _bjaO-TRE3G promoter; Citrine reporter gene; hEF1a promoter sub; VTR3-BjaR; EF1 promoter; mCherry reporter gene, get IV induction system plasmid.

For the Cinn induction system, the 5' end to the 3' end of the chimeric allosteric transcription factor VTR3-BraR are VTR3 (SEQ ID NO: 41), nuclear localization signal peptide (SEQ ID NO: 38) and BraR (SEQ ID NO: 42). Among them, BraR is obtained through human codon optimization according to the BraR protein sequence (CAL74858.1). The components were assembled into the pcDNA3.1(+) plasmid (Invitrogen, catalog #V790-20) using Gibson assembly in the following order: Cinn-HSL inducible promoter; Citrine reporter gene; hEF1a promoter; VTR3-BraR or VP64 - BraR (NCBI accession number of VP64: ASW25882.1); EF1 promoter; mCherry reporter gene, resulting in Cinn induction system plasmid. Among them, the Cinn-HSL inducible promoter has its operating sequence from the promoters P _CUS-TRE3G , P _APO-TRE3G , P _NC-TRE3G , P _ORS-TRE3G , P _CUL-TRE3G of different strains; the upstream truncated promoter Promoters P _F1-TRE3G , P _F2-TRE3G , P _F4-TRE3G , P _F7-TRE3G ; downstream truncated promoters P _R1-TRE3G , P _R4-TRE3G , P _R6-TRE3G , P _R7-TRE3G , P _{R8- TRE3G} and the optimized promoter P _2XR8-TRE3G .

For the pC-dox double-inducible system, the _PRpaO-TRE3G promoter of the pC induction system was replaced with the _PRpaO-CMV1 promoter (SEQ ID NO: 39) by inverse PCR first, and then the P _RpaO-CMV1 A tetO was added to the upstream and downstream of the transcription initiation site (Inr) of the promoter, and a pC-dox inducible promoter capable of simultaneously responding to pC-HSL and dox signals was obtained. The GFP coding region on the plasmid pDN-D2irTNG4kwh (addgen#44722) was removed by inverse PCR, and then the entire TetR expression element of the plasmid (from the beginning of CMV enhancer to the end of bGH poly A, which contained the promoter P _{tet- CMV D2i} ). Next, the PCR-amplified TetR expression element with NotI sites at both ends was inserted into the pC inducible system containing the pC-dox promoter through NotI single enzyme digestion to construct the final receiver of the pC-dox inducible system.

B4YP-2XR8 signal communication system: as shown in Figure 7, the signal receiving module 2XR8 of the signal communication system is a Cinn inducible system (the inducible promoter is P _2XR8-TRE3G ). The signal sending module B4YP of the signal communication system includes three genes of BraI, 4CL and PAL. Use Gibson assembly to replace the entire gene expression frame sequence from the CMV promoter on the PB vector (PB513B-1) to SV40 with the EF1a core promoter (SEQ ID NO: 40), BraI (SEQ ID NO: 44) and hGH-PA (polyA region) to obtain the EF1a-Bral expression plasmid. Similarly, use Gibson assembly to replace the entire gene expression frame sequence from the CMV promoter on the PB vector to SV40 with the EF1a core promoter (SEQ ID NO: 40), 4CL (NCBI accession number: CP066699.1 No. 1990899-1992794bp) and hGH-PA to obtain the EF1a-4CL expression plasmid. Use Gibson assembly to replace the entire gene expression frame sequence from the CMV promoter on the PB vector to SV40 with the EF1a core promoter (SEQ ID NO: 40), PAL (SEQ ID NO: 45) and hGH-PA to obtain EF1a-PAL expression plasmid. Among them, BraI was obtained by human codon optimization based on the BraR protein sequence (CAL74857.1); PAL was obtained by human codon optimization based on the PAL protein sequence (XP_016272209.1). As far as the assembly of the three expression plasmids was concerned, ES plasmids were constructed first: RFP expression cassettes were inserted between the 5' insulator of the PB plasmid and the CMV promoter as bacterial selection markers, and the EF1 promoter, mCherry and SV40PA are used as mammalian screening markers, and the restriction sites at both ends of the expression element are I-CeuI and I-SceI; at the same time, the entire sequence from the CMV promoter to SV40PA on the PB vector is deleted. For the expression plasmids EF1a-BraI, EF1a-4CL and EF1a-PAL, EF1a-BraI-hGH-PA, EF1a-4CL-hGH-PA and EF1a-PAL-hGH-PA and three fragments were obtained by PCR amplification. The three fragments were assembled and integrated on the ES vector through the golden gate according to the sequence of BraI-4CL-PAL, and finally PB-EF1a-BraI-EFa-4cl-EF1a-PAL (B4YP) was obtained. For the assembly of the signaling and receiving modules, the Citrine/VTR3-BraR/mCherry expression cassette of the 2XR8 module was amplified and I-CeuI and I-SceI restriction sites were added at both ends. The amplified product and plasmid pEF1a-B4YP were digested with I-CeuI and I-SceI and ligated with T4DNA ligase to obtain the single-plasmid cell communication system p2XR8-B4YP.

Mammalian cell culture and transfection

The small molecule inducer pC-HSL was purchased from Sigma (Product No. 7077), and Cinn-HSL and IV-HSL were synthesized by Shenzhen Qianyan Drug Research and Development Technology Co., Ltd.

The human embryonic kidney cell line 293T (HEK-293T, ATCC: CRL-11268) was inoculated in the culture medium supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin and streptomycin under the culture condition of 37°C and 5% CO _{2 .} (Hyclone) DMEM high glucose medium (Hyclone, containing sodium pyruvate) for culture. Inoculate 2×10 ⁵ cells/ml in each well of a 24-well cell culture plate, and use Lipofectamine (Thermo) standard protocol for transfection after 1 day of culture: Mix 1 μg plasmid DNA with 25 μl DMEM, then add 2 μl P3000 and mix well , Add the mixed DNA/P3000/DMEM into 25 μl DMEM mixed with 2 μl Lipofectamine 3000, and incubate at room temperature for 15 minutes. The incubated DNA/Lipofectamine complex was added dropwise into each well of the 24-well plate in which 293T cells were cultured, and the cells were returned to the incubator to continue culturing. Six hours after transfection, different concentrations of small molecule inducers were added to the cells, and the samples were collected for flow cytometric analysis after induction in a 5% CO ₂ , 37° C. incubator for about 40 hours. For the pC-inducible system containing the multimerization domain, different concentrations of pC-HSL were added to the cells 6 h after transfection together with vitamin B12 at a final concentration of 15 nM.

Flow sample collection and data processing

For the induced cells, after washing twice with PBS buffer to remove the residual medium, add 25 μl 0.25% trypsin (Gibco) to each well and incubate at room temperature for 1 min, then add 150 μl DMEM complete medium, blow and beat the cells repeatedly It becomes a unicellular state. Add the dispersed cell suspension to the 96-well plate, centrifuge at 1000rpm for 5min, suck off 130μl supernatant, resuspend the cells with 110μl 4% paraformaldehyde fixative (Boster), and then use Beckman CytoFLEX S flow cytometer The FITC channel and ECD channel detect the fluorescence signals of all samples.

The flow cytometry data were analyzed using CytExpert software that comes with the Beckman CytoFLEX S flow cytometer, and the gate was set based on the fluorescence intensity of untransfected 293T cells. The average FITC of the ECD+ population was used as the output for each sample. For pC-dox inducible systems, background autofluorescence was subtracted. All flow cytometric data presented herein are the mean of at least three replicates, S.D. is the standard deviation of individual measurements.

microscope observation

B4YP-2XR8 and 2XR8 were respectively transfected into 293T cells according to the Lipofectamine transfection method described herein, and the expression of the receiver reporter gene Citrine was detected with a fluorescence microscope EVOS7000 (Thermo) 48 hours after transfection.

Quantitative fitting of induced system sensitivity

In order to determine the sensitivity and dynamic range of each induction system, the following Hill equation was used to fit the experimentally measured dose-response curves.

Wherein, y is the fluorescence intensity (output) of the reporter Citrine, y _min is the minimum value of the output, and y _max is the maximum value of the output. The K obtained by fitting is the input signal concentration when the output value is at half maximum, that is, the theoretical value of EC50. The dynamic range of this induction curve is y _max /y _min .

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Claims

A chimeric allosteric transcription factor for regulating a gene of interest in mammalian cells, said chimeric allosteric transcription factor comprising a regulatory domain and a transcriptional activation domain; wherein said regulatory domain selected from the group consisting of RpaR, BjaR, and BraR; the transcriptional activation domain is selected from P65, VP16, VP64, VTR1, VTR2, and VTR3; the chimeric allosteric transcription factor is derived from an acyl group derived from a natural amino acid Homoserine lactone is used as an inducer; the acyl homoserine lactone derived from a natural amino acid is selected from the group consisting of p-coumaroyl-homoserine lactone (pC-HSL), isovaleryl-homoserine lactone Serine lactone (IV-HSL) and cinnamoyl-homoserine lactone (Cinn-HSL).
The chimeric allosteric transcription factor according to claim 1, wherein the transcriptional activation domain is VTR3.
The chimeric allosteric transcription factor according to claim 1 or 2, wherein the regulatory domain is RpaR, and the inducer is pC-HSL or Cinn-HSL; preferably, the inducer is pC- HSL.
The chimeric allosteric transcription factor according to claim 1 or 2, wherein the regulatory domain is BjaR, and the inducer is IV-HSL.
The chimeric allosteric transcription factor according to claim 1 or 2, wherein the regulatory domain is BraR, and the inducer is Cinn-HSL.
The chimeric allosteric transcription factor according to any one of claims 1-5, wherein the regulatory domain is connected to the transcriptional activation domain using a connecting peptide; preferably, the length of the connecting peptide is It is 20-60bp; Preferably, the connecting peptide has the sequence of SEQ ID NO: 16.
The chimeric allosteric transcription factor according to any one of claims 1-5, wherein the chimeric allosteric transcription factor further comprises a nuclear localization signal peptide, wherein the nuclear localization signal peptide is located in the The N-terminal or C-terminal of the chimeric allosteric transcription factor, or between the regulatory domain and the transcriptional activation domain, the nuclear localization signal peptide contains 1-4 nuclear localization sequences; preferably, the The 5' end and the 3' end of the nuclear localization sequence each independently have a flanking sequence of 5-40bp; preferably, the nuclear localization signal peptide is located between the regulatory domain and the transcriptional activation domain; preferably , the nuclear localization signal peptide comprises two nuclear localization sequences, the 5' end and the 3' end of the nuclear localization sequence each independently have a flanking sequence of 5-20bp; preferably, the nuclear localization sequence has SEQ ID NO :17 sequence.
The chimeric allosteric transcription factor according to any one of claims 1-5, wherein the chimeric allosteric transcription factor further comprises a multimerization domain, preferably, the multimerization domain For CarH.
The chimeric allosteric transcription factor according to claim 8, wherein the regulatory domain, the multimerization domain and the transcriptional activation domain are connected using a connecting peptide; preferably, the connecting peptide Each has a length of 20-60bp; preferably, the connecting peptide has the sequence of SEQ ID NO: 16.
The chimeric allosteric transcription factor according to claim 8, wherein said chimeric allosteric transcription factor further comprises a nuclear localization signal peptide, wherein said nuclear localization signal peptide is located at said chimeric allosteric transcription The N-terminal or C-terminal of the factor, or between the regulatory domain and the multimerization domain, or between the transcriptional activation domain and the multimerization domain, the nuclear localization signal The peptide contains 1-4 nuclear localization sequences; preferably, the 5' end and the 3' end of the nuclear localization sequence each independently have a flanking sequence of 5-40bp; preferably, the nuclear localization signal peptide contains 2 nuclear Positioning sequence, the 5' end and the 3' end of the nuclear localization sequence each independently have a flanking sequence of 5-20bp; preferably, the nuclear localization sequence has the sequence of SEQ ID NO: 17; preferably, the nuclear localization sequence The positioning signal peptide is located between the regulatory domain and the multimerization domain, or between the transcriptional activation domain and the multimerization domain; preferably, the multimerization domain The 5' end and the 3' end further comprise a connecting peptide, preferably, each of the connecting peptides independently has a length of 20-60bp; preferably, the connecting peptide has a sequence of SEQ ID NO: 16.
The chimeric allosteric transcription factor according to any one of claims 1-10, wherein the mammalian cells are human cells.
The chimeric allosteric transcription factor according to any one of claims 1-10, wherein the mammalian cells are immortalized cells, primary cells or stem cells.
A set of regulatory elements using natural amino acid-derived acyl homoserine lactones as inducers selected from the group consisting of p-coumaroyl-homoserine ester (pC-HSL), isovaleryl-homoserine lactone (IV-HSL) and cinnamoyl-homoserine lactone (Cinn-HSL); the set of regulatory elements comprises:

A chimeric allosteric transcription factor, said chimeric allosteric transcription factor comprising a regulatory domain and a transcriptional activation domain; wherein said regulatory domain is selected from the group consisting of RpaR, BjaR and BraR; said The transcriptional activation domain is selected from the group consisting of P65, VP16, VP64, VTR1, VTR2 and VTR3; and

An inducible promoter active in mammalian cells, said inducible promoter comprising a core sequence and at least one operator site pair interacting with said chimeric allosteric transcription factor.
The set of regulatory elements according to claim 13, wherein the transcriptional activation domain of the chimeric allosteric transcription factor is VTR3.
The set of regulatory elements according to claim 13 or 14, wherein the regulatory domain of the chimeric allosteric transcription factor is RpaR, the operating site of the inducible promoter is rpaO, and the inducer is pC -HSL or Cinn-HSL; preferably, the inducer is pC-HSL.
The set of regulatory elements according to claim 13 or 14, wherein the regulatory domain of the chimeric allosteric transcription factor is BjaR, the operating site of the inducible promoter is bjaO, and the inducer is IV -HSL.
The set of regulatory elements according to claim 13 or 14, wherein the regulatory domain of the chimeric allosteric transcription factor is BraR, and the operating site of the inducible promoter is braO, preferably R8; The inducer was Cinn-HSL.
The set of regulatory elements according to any one of claims 13-17, wherein the regulatory domain in the chimeric allosteric transcription factor is connected to the transcriptional activation domain using a connecting peptide; preferably , the length of the connecting peptide is 20-60bp; preferably, the connecting peptide has the sequence of SEQ ID NO: 16.
The set of regulatory elements according to any one of claims 13-17, wherein the chimeric allosteric transcription factor further comprises a nuclear localization signal peptide, wherein the nuclear localization signal peptide is located in the chimeric type The N-terminal or C-terminal of the constitutive transcription factor, or between the regulatory domain and the transcriptional activation domain, the nuclear localization signal peptide comprises 1-4 nuclear localization sequences; preferably, the nuclear localization sequence The 5' end and the 3' end each independently have a flanking sequence of 5-40bp; preferably, the nuclear localization signal peptide is located between the regulatory domain and the transcriptional activation domain; preferably, the nuclear The localization signal peptide comprises two nuclear localization sequences, the 5' end and the 3' end of the nuclear localization sequence each independently have a flanking sequence of 5-20bp; preferably, the nuclear localization sequence has the sequence of SEQ ID NO: 17 .
The set of regulatory elements according to any one of claims 13-19, wherein the chimeric allosteric transcription factor further comprises a multimerization domain, preferably, the multimerization domain is CarH.
The set of regulatory elements according to claim 20, wherein the regulatory domain, the multimerization domain and the transcriptional activation domain in the chimeric allosteric transcription factor are connected using a connecting peptide; Preferably, the connecting peptides each independently have a length of 20-60bp; preferably, the connecting peptides have the sequence of SEQ ID NO: 16.
The set of regulatory elements according to claim 20, wherein the chimeric allosteric transcription factor further comprises a nuclear localization signal peptide, wherein the nuclear localization signal peptide is located at the N-terminal of the chimeric allosteric transcription factor Or the C-terminus, or between the regulatory domain and the multimerization domain, or between the transcriptional activation domain and the multimerization domain, the nuclear localization signal peptide comprises 1- 4 nuclear localization sequences; preferably, the 5' end and the 3' end of the nuclear localization sequence each independently have a 5-40bp flanking sequence; preferably, the nuclear localization signal peptide contains 2 nuclear localization sequences, so The 5' end and the 3' end of the nuclear localization sequence each independently have a flanking sequence of 5-20bp; preferably, the nuclear localization sequence has the sequence of SEQ ID NO: 17; preferably, the nuclear localization signal peptide is located at between the regulatory domain and the multimerization domain, or between the transcriptional activation domain and the multimerization domain; preferably, the 5' end of the multimerization domain and The 3' end further comprises a connecting peptide, preferably, each of the connecting peptides independently has a length of 20-60bp; preferably, the connecting peptide has a sequence of SEQ ID NO: 16.
The set of regulatory elements according to any one of claims 13-22, wherein the core sequence of the inducible promoter is selected from CMV1 promoter, CMVmini promoter, TRE3G promoter, EF1a core promoter or hEF1a promoter promoter; preferably, the core sequence is a CMV1 promoter or a TRE3G promoter.
The set of regulatory elements according to any one of claims 13-23, wherein the pair of manipulation sites is 1 or more pairs; preferably, the pair of manipulation sites is 1 pair; preferably, each manipulation site sites independently located upstream and downstream of the transcription initiation site.
The set of regulatory elements according to any one of claims 13-24, said set of regulatory elements further comprising a repressor protein and a third promoter, wherein said third promoter drives the expression of said repressor protein, said A third promoter comprises a second operator site that interacts with said repressor protein; wherein said inducible promoter further comprises said second operator site.
The set of regulatory elements according to claim 25, wherein the repressor protein is TetR, and the second operating site is tetO; or, the repressor protein is CymR, and the second operating site is cymO.
The set of regulatory elements of any one of claims 13-26, wherein the mammalian cells are human cells.
The set of regulatory elements according to any one of claims 13-26, wherein said mammalian cells are immortalized cells, primary cells or stem cells.
A system for inducing expression of a gene of interest in mammalian cells, the inducible expression system utilizing acyl homoserine lactone derived from a natural amino acid as an inducer, the acyl homoserine lactone derived from a natural amino acid being selected from The group consisting of the following compounds: p-coumaroyl-homoserine lactone (pC-HSL), isovaleryl-homoserine lactone (IV-HSL) and cinnamoyl-homoserine lactone (Cinn-HSL); The inducible expression system comprises:

A first gene expression cassette, the first gene expression cassette comprising a first promoter sequence and a coding sequence of a chimeric allosteric transcription factor, wherein the chimeric allosteric transcription factor comprises a regulatory domain and a transcriptional activation structure domain; the regulatory domain is selected from the group consisting of RpaR, BjaR and BraR; the transcriptional activation domain is selected from the group consisting of P65, VP16, VP64, VTR1, VTR2 and VTR3; and

A second gene expression cassette, the second gene expression cassette comprising an inducible promoter sequence and the coding sequence of the gene of interest, the inducible promoter sequence comprising an allosteric transcription factor interacting with the chimeric type at least one pair of operator sites.
The inducible expression system according to claim 29, wherein the transcriptional activation domain of the chimeric allosteric transcription factor in the first gene is VTR3.
The inducible expression system according to claim 29 or 30, wherein the regulatory domain of the chimeric allosteric transcription factor in the first gene is RpaR, and the inducible promoter in the second gene is The manipulation site is rpaO, and the inducer is pC-HSL or Cinn-HSL; preferably, the inducer is pC-HSL.
The inducible expression system according to claim 29 or 30, wherein the regulatory domain of the chimeric allosteric transcription factor in the first gene is BjaR, and the inducible promoter in the second gene is The operator site is bjaO, and the inducer is IV-HSL.
The inducible expression system according to claim 29 or 30, wherein the regulatory domain of the chimeric allosteric transcription factor in the first gene is BraR, and the inducible promoter in the second gene is The operator site is braO, preferably R8, and the inducer is Cinn-HSL.
The inducible expression system according to any one of claims 29-33, wherein, in the first gene, the regulatory domain is connected to the transcriptional activation domain using a connecting peptide; preferably, the The length of the connecting peptide is 20-60bp; preferably, the connecting peptide has the sequence of SEQ ID NO: 16.
The inducible expression system according to any one of claims 29-33, wherein the chimeric allosteric transcription factor of the first gene further comprises a nuclear localization signal peptide, wherein the nuclear localization signal peptide is located at The N-terminal or C-terminal of the chimeric allosteric transcription factor, or between the regulatory domain and the transcriptional activation domain, the nuclear localization signal peptide contains 1-4 nuclear localization sequences; preferably , the 5' end and the 3' end of the nuclear localization sequence each independently have a flanking sequence of 5-40bp; preferably, the nuclear localization signal peptide is located between the regulatory domain and the transcriptional activation domain; Preferably, the nuclear localization signal peptide comprises two nuclear localization sequences, and the 5' and 3' ends of the nuclear localization sequences each independently have a 5-20bp flanking sequence; preferably, the nuclear localization sequence has SEQ ID NO: Sequence of 17.
The inducible expression system according to any one of claims 29-35, wherein the chimeric allosteric transcription factor of the first gene further comprises a multimerization domain, preferably, the multimerization The structural domain is CarH.
The inducible expression system according to claim 36, wherein the regulatory domain, the multimerization domain and the transcriptional activation domain in the chimeric allosteric transcription factor are connected using a connecting peptide; Preferably, the connecting peptides each independently have a length of 20-60bp; preferably, the connecting peptides have the sequence of SEQ ID NO: 16.
The inducible expression system according to claim 36, wherein the chimeric allosteric transcription factor further comprises a nuclear localization signal peptide, wherein the nuclear localization signal peptide is located at the N-terminal of the chimeric allosteric transcription factor Or the C-terminus, or between the regulatory domain and the multimerization domain, or between the transcriptional activation domain and the multimerization domain, the nuclear localization signal peptide comprises 1- 4 nuclear localization sequences; preferably, the 5' end and the 3' end of the nuclear localization sequence each independently have a 5-40bp flanking sequence; preferably, the nuclear localization signal peptide contains 2 nuclear localization sequences, so The 5' end and the 3' end of the nuclear localization sequence each independently have a flanking sequence of 5-20bp; preferably, the nuclear localization sequence has the sequence of SEQ ID NO: 17; preferably, the nuclear localization signal peptide is located at between the regulatory domain and the multimerization domain, or between the transcriptional activation domain and the multimerization domain; preferably, the 5' end of the multimerization domain and The 3' end further comprises a connecting peptide, preferably, each of the connecting peptides independently has a length of 20-60bp; preferably, the connecting peptide has a sequence of SEQ ID NO: 16.
The inducible expression system according to any one of claims 29-38, wherein the first promoter in the first gene is a constitutive promoter or an inducible promoter.
The inducible expression system according to any one of claims 29-39, wherein the core sequence of the inducible promoter in the second gene is selected from CMV1 promoter, CMVmini promoter, TRE3G promoter, EF1a Core promoter or hEF1a promoter; preferably, the core sequence is CMV1 promoter or TRE3G promoter.
The inducible expression system according to any one of claims 29-40, wherein the pair of operating sites in the second gene is one or more pairs; preferably, the pair of operating sites is one pair ; Preferably, each operator site is independently located upstream and downstream of the transcription start site.
The inducible expression system according to any one of claims 29-41, said inducible expression system further comprising a third gene expression cassette, wherein said third gene expression cassette comprises a third promoter sequence and a coding repressor protein sequence for expressing said repressor protein in a host cell, said third promoter sequence comprising a second operator site, said second operator site interacting with said repressor protein; induction of said second gene The type promoter sequence further comprises a second operator site.
The inducible expression system according to claim 42, wherein the repressor protein is TetR, and the second operating site is tetO; or, the repressor protein is CymR, and the second operating site is cymO.
The inducible expression system according to claim 42 or 43, wherein the core sequence of the third promoter in the third gene is selected from CMV1 promoter, CMVmini promoter, TRE3G promoter, EF1a core promoter or hEF1a promoter; preferably, the core sequence is CMV1 promoter or TRE3G promoter.
The inducible expression system according to any one of claims 29-44, wherein the mammalian cells are human cells.
The inducible expression system according to any one of claims 29-44, wherein the mammalian cells are immortalized cells, primary cells or stem cells.
The chimeric allosteric transcription factor according to any one of claims 1-12, the set of regulatory elements according to any one of claims 13-28, the induced expression according to any one of claims 29-46 The application of the system in the construction of a single cell or multi-cell signal communication system, wherein the signal communication system includes a signal sending element and a signal receiving element, and the signal receiving element includes a chimeric allosteric transcription factor and in a mammalian cell Active inducible promoter.
The use according to claim 47, wherein the signal molecule of the signal communication system is pC-HSL; the regulatory domain in the chimeric allosteric transcription factor is RpaR, and the inducible promoter comprises a core sequence and at least one pair of rpaO; the signaling elements include TAL, 4CL and RpaI.
The use according to claim 47, wherein the signaling molecule of the signal communication system is Cinn-HSL; the regulatory domain in the chimeric allosteric transcription factor is BraR, and the inducible promoter comprises a core sequence and at least one pair of braO; the signaling elements include PAL, 4CL and BraI.
The use according to claim 47, wherein the signal molecule of the signal communication system is IV-HSL; the regulatory domain in the chimeric allosteric transcription factor is BjaR, and the inducible promoter comprises a core sequence and at least one pair of bjaO; the signaling element comprises a branched-chain α-ketoacid dehydrogenase complex BCDH and BjaI.
The chimeric allosteric transcription factor according to any one of claims 1-12, the set of regulatory elements according to any one of claims 13-28, the induced expression according to any one of claims 29-46 The use of the system in the analysis of gene function, or in the construction of human disease models, artificial tissues or artificial organs.
The use according to claim 51, wherein the chimeric allosteric transcription factor, the set of regulatory elements or the inducible expression system are stably transfected into mammalian cells.
The use according to claim 51, wherein the chimeric allosteric transcription factor, the set of regulatory elements or the inducible expression system is transiently transfected into mammalian cells.