CN107177499B

CN107177499B - Gene loop remote regulation and control system

Info

Publication number: CN107177499B
Application number: CN201610136489.4A
Authority: CN
Inventors: 叶海峰; 邵佳伟; 余贵玲; 薛帅; 杨雪平; 于袁欢; 朱苏承; 白郁
Original assignee: East China Normal University
Current assignee: East China Normal University
Priority date: 2016-03-10
Filing date: 2016-03-10
Publication date: 2020-08-04
Anticipated expiration: 2036-03-10
Also published as: CN107177499A

Abstract

The invention discloses a far-red light gene loop expression control system, which comprises a photoreceptor for sensing a far-red light source; a processor for processing the signals transmitted by the photoreceptors; and an effector responsive to signals communicated by the processor. The invention also discloses a gene loop remote regulation and control system, which comprises the far-red light gene loop expression control system; the control device is used for sending a remote control instruction; and a far-red light source device. The invention also discloses an expression control system of the far-red light gene loop and application of the gene loop remote control system in treating diabetes. The invention also discloses a eukaryotic expression vector, an engineered cell or an engineered cell transplantation vector containing the far-red light gene loop expression control system. The invention can quickly regulate and control gene expression, and has the characteristics of ultra-remote control, intelligent regulation and control of gene expression quantity, high regulation and control expression multiple, high space-time specificity, strong tissue penetrating power, good insulation, no toxic or side effect and the like.

Description

Gene loop remote regulation and control system

Technical Field

The invention relates to the interdisciplinary fields of synthetic biology, optogenetics, electronic engineering and the like, in particular to a far-red light gene loop expression control system, a gene loop remote regulation and control system comprising the far-red light gene loop expression control system, a regulation and control method and application thereof.

Background

Synthetic biology is a comprehensive discipline that takes engineering theory as guidance, designs and synthesizes various complex biological function modules, systems and even artificial life bodies, and is applied to specific chemical production, biological material manufacture, gene therapy, tissue engineering and the like. The rapid development of synthetic biology over the last decade has made significant progress in a number of application areas. In particular, in the field of mammalian cell synthetic biology, a variety of controllable genetic circuits have been designed and constructed for the diagnosis and treatment of metabolic diseases, cancer, immune system diseases, and the like.

In the fields of synthetic biology and disease treatment, molecular switches for artificially and precisely regulating gene expression have become an indispensable means in disease treatment. Currently, there are many systems for inducing gene expression by artificial regulation in the world, and the systems can be mainly divided into two categories: (1) inducing and regulating gene expression system by chemical substance; (2) the gene expression system is induced and regulated by a physical method.

Chemical inducers are mainly small molecule substances, such as the tetracycline-inducible regulated gene expression system [ Gossen, M. et al, Proc Natl Acad Sci USA,1992,89: 5547-; 1440 (1444), benzoic acid and vanillic acid inducible regulatory gene expression systems [ Xie, M. et al, Nucleic Acids Res.2014 Aug; 42(14), etc. These systems usually use chemicals as inducers, and some systems have been optimized for many years to obtain very excellent inducing properties. Therefore, in the past years, chemical substances are widely used for inducing and regulating gene expression systems to effectively regulate gene expression in time. However, the chemical substance to induce and regulate the small molecule inducer involved in the gene expression system has some potential problems, such as toxicity, pleiotropic property, non-specificity, and poor tissue permeability. And the precise control on time and space is difficult to achieve by inducing and controlling a gene expression system by chemical substances.

The regulation system induced by the physical method comprises: ultraviolet induced regulation of the "caging cage" technology [ Keyes, WM. et al, Trends Biotechnol.2003 Feb; 21(2):53-5 ], far infrared light control heat shock effect induction regulation gene expression system [ Kamei, y, et al, Nat methods.2009 Jan; 79-81 ], a radio-controlled temperature-induced induction regulated gene expression system [ Stanley, SA. et al, science.2012 May 4; 336(6081), 604-8, etc. These systems for inducing gene expression by physical means are generally very toxic to cells, may cause irreversible damage to cells and even death of cells, and involve expensive and complicated equipment.

Light is an ideal inducer of gene expression because it is ubiquitous in nature, readily available, highly controllable, non-toxic, and most importantly, it enables precise regulation in time and space, and thus regulation of gene expression and thus various metabolic activities of living organisms is an ongoing goal of biologists in our earlier research work, attempts to design and synthesize a gene loop for blue light regulation and transgene expression using synthetic biology Methods, and deactivation, regulation and control of the expressed gene using blue light as a switch [ Ye, h, et al, Science,2011.332(6037): p.1565-8. ]. the teaching group of populus university of china, professors, reports on L igg on blue light regulation and gene expression switch [ Wang, x, et al, Nat Methods,2012.9(3): p.266-9. ]. blue light is used to regulate gene expression in vivo, however, there is a great limitation on transdermal efficiency, difficulty in the deactivation of skin or deactivation, and greatly limits the development of the gene expression of target genes and red light control systems in vivo, and development of the gene expression of pigment synthesis in vitro gene expression control systems [ 5-8 ], and the blue light-red light synthesis system has not been shown to be very low in the experimental control of genes of phytochrome-red light (the research).

The diabetes mellitus is a chronic lifelong disease affecting human health, mainly aims at high blood sugar level after the onset of the diabetes mellitus, and can affect various important organs and tissues of the body along with the aggravation of the disease, various complications are caused and cannot be completely cured, and only can be controlled, the number of the diabetes mellitus patients in the whole world is increased at an alarming speed in recent decades.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention provides a novel far-red light gene loop expression control system and a gene loop remote regulation and control system comprising the far-red light gene loop expression control system, a control device and a far-red light source device for the first time. In the invention, the far-red light transdermal efficiency is far better than that of blue light, and the far-red light transdermal efficiency can permeate 7-8 cm of skin and muscle tissues, so that the target cell transplanted in the abdominal cavity can be remotely and tracelessly regulated and controlled to express a target gene, and even a specific tissue organ in a body can be regulated and controlled to express the target gene. The photon energy of far-red light is much lower than that of blue light, and the toxic and side effect on cells is far less than that of the blue light. Moreover, the far-red light control system can be directly activated by far-red light without adding any photosensitive pigment additionally. The system and the far-red light control switch have great potential application value and can be widely popularized in clinical application.

The invention provides a gene loop control system which is artificially designed and synthesized and based on a smart phone platform for ultra-remote regulation and control of transgene expression. The system consists of an intelligent control system for controlling a far-red light source by the smart phone through a local area network WiFi or 2G/3G/4G network and a gene loop control system for regulating and controlling transgene expression of far-red light. The invention provides an intelligent control system for controlling a far-red light source by a smart phone, a eukaryotic expression vector containing a smart phone regulation transgenic expression system, a smart phone regulation transgenic expression method in host cells and a smart phone regulation transgenic expression method transplanted into a mouse. The invention provides a kit for regulating and controlling each component of a transgenic expression system by using the smart phone. The invention also provides a novel diabetes therapy based on the ultra-remote regulation of the smart phone platform. The invention can quickly regulate and control gene expression, and has the characteristics of ultra-remote control, intelligent regulation and control of gene expression quantity, high regulation and control expression multiple, high space-time specificity, strong tissue penetrating power, good insulation, no toxic or side effect and the like.

The invention provides a far-red light gene loop expression control system, which comprises: a photoreceptor that senses a far-red light source; a processor for processing the signals transmitted by the photoreceptors; an effector responsive to a signal delivered by the processor.

Wherein the photoreceptors comprise photosensitive diguanylate cyclase Bphs that convert GTP to c-di-GMP under far-red light conditions. Wherein, the photosensitive diguanylate cyclase Bphs is one of the most key proteins and is used as a core component.

Wherein the photoreceptor photosensitive diguanylate cyclase Bphs are prepared by fusing the 1 st-511 th amino acid of BphG protein and the 175 st-343 st amino acid of Slr1143 protein and mutating the 587 th arginine of the fusion protein into alanine (R587A); wherein, the gene for encoding the BphG protein can be synthesized by artificial chemistry and can also be from Rhodobacter sphaeroides (Rhodobacter sphaeroides); the Slr1143 protein can be from Synechocystis sp or be artificially and chemically synthesized, and the gene coding Slr1143 protein adopted by the invention is artificially and chemically synthesized.

Wherein the construction form of the photoreceptor comprises:

a) artificially synthesized bacterial photosensitive diguanylate cyclase Bphs coding gene (Bphs);

b) the artificially synthesized bacterial photosensitive diguanylate cyclase Bphs coding gene is connected with the c-di-GMP degrading enzyme YhjH coding gene through a 2A sequence (Bphs-2A-YhjH);

c) the artificially synthesized bacterial photosensitive diguanylate cyclase Bphs coding gene is connected with a photosensitive pigment synthetase Bphos coding gene through a 2A sequence (Bphs-2A-Bphos);

d) the artificially synthesized bacterial photosensitive diguanylate cyclase Bphs coding gene is connected with a photosensitive pigment synthetase Bpho coding gene through a 2A sequence, and then is connected with a c-di-GMP degrading enzyme YhjH coding gene through a 2A sequence (Bphs-2A-Bpho-2A-YhjH);

wherein the 2A sequence can be replaced by an internal ribosome entry site sequence IRES; the photosensitive pigment synthetase Bpho has the function of synthesizing photosensitive pigment biliverdin; the degrading enzyme YhjH of the c-di-GMP has the function of degrading the c-di-GMP into pGpGpGpG.

Wherein the amino acid sequence of the Bphs, Bpho and YhjH is selected from the sequences 45, 46 and 48.

The promoter for expressing photoreceptors comprises a) SV40, b) CMV, c) hEF1 α, d) mPGK and e) CAG.

Wherein the processor comprises a polypeptide as a DNA binding domain and a c-di-GMP binding domain, a polypeptide as a nuclear localization signal N L S, a polypeptide as a linking domain, and a polypeptide as a transcriptional regulatory domain.

The polypeptide as the DNA binding domain and the c-di-GMP binding domain is a protein capable of binding to a specific DNA sequence after binding to c-di-GMP, for example, a BldD protein, which may be a BldD protein derived from Streptomyces venezuelae (Streptomyces venezuelae) or may be artificially synthesized, and the amino acid sequence thereof is selected from the sequence 49, and the BldD protein of the present invention is artificially synthesized.

Wherein, the polypeptide as the nuclear localization signal N L S can be in various forms of 1-3 copies, and the amino acid sequence thereof is selected from the sequence 54.

Wherein, the polypeptide as the connecting functional domain (L inker) can be in various forms with the length of 0-30 amino acids, and the amino acid sequence thereof is selected from the sequence 55.

Wherein the polypeptide as a transcription regulatory domain is a domain protein having a transcription activation function, and comprises an NF-k B p65 subunit transcription activation domain, a heat shock transcription factor HSF1 transcription activation domain, a herpes simplex virus granule protein VP16 transcription activation domain and 4 copies of the transcription activation domain VP 64; the amino acid sequence is selected from the

sequences

50, 51, 52 and 53.

Wherein the polypeptide as a transcriptional regulatory domain is placed N-terminal or C-terminal to the DNA binding domain and the polypeptide BldD of the C-di-GMP binding domain.

Wherein, the polypeptides of different functional domains in the processor can be directly connected or connected through a connecting peptide.

Wherein the effector comprises P_FRL-a reporter; the effector comprises a promoter sequence and a nucleic acid sequence of the protein to be transcribed.

Wherein the promoter sequence comprises a processor BldD protein binding DNA sequence and a weak promoter that promotes gene expression.

Wherein, the DNA sequence which is specifically recognized and combined by the polypeptide of the DNA binding domain and the c-di-GMP binding domain can be part sequences of bldM and whiG promoter regions of Streptomyces venezuelae (S.venezuelae) or artificially synthesized chemically, and the DNA sequence which is specifically recognized and combined by the BldD protein of the processor is artificially synthesized chemically.

Wherein the nucleotide sequence of bldM is selected from the group consisting of SEQ ID NO 18; the nucleotide sequence of whiG is selected from the group consisting of SEQ ID NO. 19.

Wherein, the partial sequences of the bldM and whiG promoter regions are 1-10 copies.

Wherein, the weak promoter for promoting gene expression comprises all weak promoters including TATA box, cytomegalovirus CMV minimum promoter and mutant CMVmin 3G thereof.

Wherein the nucleic acid sequence of the promoter sequence is selected from the group consisting of

sequences

20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39.

The protein to be transcribed and encoded and expressed by the nucleic acid sequence can be any meaningful protein, and comprises a protein serving as a reporter gene and/or a nucleic acid sequence serving as a drug protein or a small peptide for treating diseases, wherein the protein serving as the reporter gene comprises a nucleic acid sequence of secreted alkaline phosphatase (SEAP), enhanced green fluorescent protein (EGFP, EYFP) and luciferase (L uciferase), and the nucleic acid sequence serving as the drug protein or the small peptide for treating diseases comprises Insulin (Insulin) and glucagon-like peptide (G L P-1).

Wherein all the interesting proteins can simultaneously express a plurality of proteins on one expression vector through 2A sequences, including SEAP-2A-Insulin, EGFP-SEAP-2A-Insulin; wherein the 2A sequence may be replaced by an internal ribosome entry site sequence IRES.

In one embodiment, the far-red light gene loop expression control system of the present invention comprises: photoreceptors Bphs which perceive far-red light; a processor P65-VP64-BldD for processing signals transmitted by the photoreceptors; effectors P responsive to signals delivered by processors_FRL-reporter。

The far-red light gene loop expression control system of the invention can be loaded by three plasmids.

First, a far-red light-sensing photoreceptor expression vector plasmid is loaded, the photoreceptors comprising Bphs, Bpho, YhjH and 2A.

Wherein Bphs is obtained by fusing 1-511 th amino acid of BphG and 175-343 rd amino acid of Slr1143, and mutating 587 th amino acid arginine of the fusion protein into alanine (R587A); the gene for coding the BphG can be from Rhodobacter sphaeroides (Rhodobacter sphaeroides) and also can be synthesized by artificial chemistry, and the gene for coding the BphG protein adopted by the invention is synthesized by artificial chemistry; the Slr1143 protein gene can be from Synechocystis sp, and can also be synthesized by artificial chemistry, and the Slr1143 protein gene fragment adopted by the invention is synthesized by artificial chemistry. Amino acids 1-511 of Bphs are PAS-GAF-PHY structural domains and can sense far-red light; the 512-680 amino acid is DGC structure domain, which can convert GTP into c-di-GMP after activation. The photoreceptor may be a protein having the same functional domain from other genera, such as BphG et al [ Ryu M H. et al, ACS synthetic biology,2014,3(11): 802-.

Bpho is a red blood oxidase present in Rhodobacter sphaeroides (Rhodobacter sphaeroides), and its encoding gene can also be synthesized by artificial chemistry, which is capable of synthesizing the photosensitive pigment biliverdin (biliverdin), providing the photosensitive pigment for Bphs to synthesize c-di-GMP [ Ryu M H. et al, ACS synthetic biology,2014,3(11):802-810 ].

YhjH is derived from E.coli (E.coli.) and its gene can be synthesized by artificial chemistry, which has the function of degrading c-di-GMP to pGpG [ Ryu M H. et al, ACS synthetic biology,2014,3(11):802-810 ], and its degrading enzyme can be any other protein having the function of degrading c-di-GMP to pGpG.

2A is an amino acid sequence of two different proteins expressed by the same promoter, and can be T2A, F2A,

P2A et al [ Doronina VA et al, Molecular and cellular biology,2008,28(13): 4227-. Wherein the 2A sequence used may be replaced by an internal ribosome entry site sequence IRES.

When Bphs, Bphos and YhjH are expressed in mammalian cells, under the irradiation of far-red light and the action of photosensitive pigment biliverdin, the Bphs can sense the far-red light to synthesize GTP into c-di-GMP. When the illumination intensity or illumination time is different, the system can synthesize different amounts of c-di-GMP, and the illumination intensity or illumination time is positively correlated with the amount of c-di-GMP generated within a certain range. When c-di-GMP is not required, it can be degraded by YhjH.

The BldD protein can be obtained from Streptomyces venezuelae [ Tschwari N. et al, Cell,2014,158 (1135): 1136 and 1147 ] and can also be synthesized by artificial chemistry, the gene encoding BldD protein of the invention is synthesized by artificial chemistry, VP16 is herpes simplex virion protein transcription activation domain, P65 is NF-B P65 subunit transcription activation domain, and HSF1 is heat transcription factor transcription activation domain [ Konerrma S. 517. 517.29.517.7538; Jane 7536-7538).

N S can be 1-3 copies and the like, and is directly connected to the N end of BldD, P, VP, HSF and the like as transcription activation domains can be respectively connected to the N end or the C end of BldD in series or in a two-by-two combination or three series connection mode, and polypeptide inker (0-30 amino acids are different) as a connection function is connected among the components.

The processor cannot recognize and combine with specific sequences in an effector in the absence of c-di-GMP; when c-di-GMP is present and forms a tetramer, the two molecules bind to the tetramer of c-di-GMP to form a hexameric complex [ Tschowri N. et al, Cell,2014,158(5): 1136-.

Third, effector-bearing plasmids. The effector consists of an insulating signal, a promoter sequence and a nucleic acid sequence to be transcribed.

The insulating signal may be simian vacuolating virus polyA (SV40 polyA), bovine auxin gene PolyA (BGH polyA), etc., and has a function of blocking the influence of an upstream promoter. The promoter sequence consists of a BldD-binding DNA sequence and a weak promoter sequence that promotes gene expression. The DNA sequence combined by the BldD is a partial sequence from the bldM and whiG promoter regions of streptomyces venezuelae (S.venezuelae), and various wild-type or mutant forms such as 1-10 repeats, and is characterized by a sequence which can be specifically recognized and combined by the BldD of an upstream processor, and the partial sequence of the bldM and the whiG promoter regions can also be artificially and chemically synthesized.

Weak promoters to initiate gene expression comprising TATA box, cytomegalovirus CMV minimal promoter and mutant (CMV)_min3G) In the absence of an upstream processor, it does not express or hardly expresses the nucleic acid sequence to be transcribed downstream.

The nucleic acid sequence to be transcribed can be any meaningful protein, such as reporter gene secreted alkaline phosphatase (SEAP), enhanced green fluorescent protein (EGFP, EYFP), luciferase (L uciferase) and the like, or functional protein for treating diseases, such as Insulin (Insulin), glucagon-like peptide (G L P-1) and the like, different required proteins can be connected by 2A, so that one promoter can express a plurality of proteins at the same time, such as SEAP-2A-Insulin, EGFP-2A-SEAP-2A-Insulin and the like.

When c-di-GMP exists, the formed tetramer forms a hexamer with two molecular processors, a specific sequence in an effector is identified and combined, and transcription factors are recruited to start transcription and expression of downstream genes. In the absence of c-di-GMP, the processor is unable to bind to the effector and is unable to turn on transcriptional expression of the gene.

Another objective of the invention is to provide a novel gene loop remote control system. With the rapid development of the mobile internet technology, the remote control of the mobile phone causes a revolution in various equipment applications, and the market prospect is huge. The mobile phone ultra-remote control system integrates hardware resources and an internet cloud platform, hardware equipment can be accessed into the internet of things cloud through a local area network WiFi, and the mobile phone APP software can conveniently and rapidly control the equipment, even control across continents. The invention combines an ultra-remote intelligent control system of a mobile phone with the far-red light gene loop expression control system, and provides a gene loop remote control system for ultra-remote regulation and control of gene expression based on an intelligent mobile phone platform.

The gene loop remote regulation system comprises: the far-red light gene loop expression control system, a control device for sending a remote control instruction and a far-red light source device are arranged; the control device remotely regulates and controls the far-red light source device by sending a control instruction, and regulates and controls the far-red light gene loop expression control system by the far-red light sent by the far-red light source device to realize the regulation and control of transgenic expression.

The control device sends a remote control instruction through a local area network WiFi or a 2G/3G/4G network to regulate and control different working states of the far-red light source device.

Wherein, the different working states comprise the on or off of the far-red light source, the illumination intensity, the illumination time or the illumination method which can be adjusted according to the requirement.

Wherein the illumination intensity is 0-5mW/cm²(ii) a The irradiation time is 0-72 h; the irradiation method comprises pulse irradiation, continuous irradiation, direct irradiation or irradiation for spatially controlling the gene expression level of cells at different positions by using a projection card with hollow depiction.

The control device comprises a smart phone App and/or a smart remote controller. The intelligent remote controller comprises all controllers with remote control functions, and the controllers comprise intelligent remote controllers with single-path output and/or multiple-path output.

In a specific embodiment, the invention relates to a smart phone platform-based ultra-remote gene loop remote control system, comprising: the intelligent mobile phone controls an intelligent control device of the far-red light source, the far-red light source device and a gene loop remote regulation and control system of the far-red light regulation and control transgene expression through a local area network WiFi or a 2G/3G/4G network. The intelligent control device for controlling the far-red light source through the local area network WiFi or the 2G/3G/4G network comprises a smart phone App and/or an intelligent remote controller.

First, the smartphone App client. The intelligent control system for controlling the far-red light source by the smart phone through the local area network WiFi or the 2G/3G/4G network inputs an instruction through an APP client on the smart phone to complete control of the far-red light source, and the instruction can be transmitted to the intelligent remote controller through the local area network WiFi or mobile phone mobile data and the cloud server. The system has the functions of inputting the far-infrared light source and the on or off working state thereof selected in an ultra-remote way, and adjusting the working intensity, the working time and other instructions of the far-infrared light source in an ultra-remote way, thereby realizing the regulation type induced expression of the target gene. This App can install with any smart mobile phone, is applicable to operating systems such as android, iOS.

And secondly, the remote intelligent controller has a multi-path output function. The intelligent control system for controlling the far-red light source by the smart phone through the local area network WiFi or the 2G/3G/4G network receives a signal sent to the cloud server by the smart phone through the intelligent remote controller and executes control on the far-red light source. The control conditions may be: a) the intelligent remote control system comprises an intelligent remote controller, a cloud server and a local area network WiFi or 2G/3G/4G network, wherein the intelligent remote controller receives signals sent by a mobile phone to the cloud server to realize the ultra-remote control; b) the device supports multi-channel control, each channel can be independently controlled, and multi-channel output of the switching power supply is achieved. The control effect is pushed to the mobile phone APP client in real time, and the far-red light source is controlled to generate different illumination intensities, so that different expression quantities of the smart phone remote control genes are realized; c) the device supports a timing switch, realizes the functions of the timing switch and a one-key switch, and realizes the control of the far-red light source to generate different illumination time, thereby realizing the remote regulation of different expression quantities of genes by the smart phone.

Wherein, the far-red light source device is a light source device capable of generating far-red light with the wavelength of 600-900 nm.

The far-red light source device can generate a light source of 600-900nm wavelength far-red light, such as 600-900nm L ED, an infrared therapeutic device, a laser lamp and the like, in a specific embodiment, 720nm far-red light L ED is adopted in an in-vitro experiment, so that the ultra-remote control, the regulation and control gene expression of different illumination time, the regulation and control gene expression of different illumination intensity and the like of the smart phone are realized, in-vivo experiment is realized by controlling a red light physiotherapy instrument or 720nm L ED to be transplanted in vivo, and the ultra-remote control, the regulation and control gene expression of different illumination time, the regulation and control gene expression of different illumination intensity and the like of the smart phone through radio power supply, the smart phone controls the far-red light source through three ways of controlling the far-red light source through a local area network WiFi or a 2G/3G/4G network, wherein the remote red light source and the cell phone control the ultra-red light source through the WiFi of the local area network, or the key of the ultra-red light source under the emergency remote control condition through the 2G/3G/4G network.

In a specific embodiment, the invention is a gene loop remote regulation and control system for controlling gene expression through a local area network WiFi or 2G/3G/4G network based on a smart phone platform, a remote controller and the like, wherein a far-red light source device is composed of an instruction receiving device and a light source, the far-red light source is controlled by a mobile phone, the remote controller and the like to generate far-red lights with different intensities, illumination times and illumination modes to induce and express the far-red light gene loop expression control system, as shown in fig. 2 (a). When the far-red light photoreceptor, the processor and the effector are expressed in a mammalian cell vector, Bphs in the photoreceptor are not activated and c-di-GMP synthesis is not performed in the absence of far-red light induction, so that the processor cannot be bound to a far-red light-induced promoter (P)_FRL) Then the expression of the downstream gene of interest cannot be driven. When induced by far-red light, Bphs in photoreceptors are activated, synthesizing c-di-GMP, and a processor is combined with a promoter (P) for the induction and initiation of far-red light_FRL) The transcription factors are recruited to start transcription and translation, driving the expression of the downstream gene of interest, as shown in FIG. 2 (b).

The invention also provides a eukaryotic expression vector, an engineered cell or an engineered cell transplantation vector containing the far-red light gene loop expression control system.

The invention also provides a method for preparing the eukaryotic expression vector, the engineered cell or the engineered cell transplantation vector containing the far-red light gene loop expression control system.

The eukaryotic expression vector comprises a mammalian cell expression vector containing the far-red light gene loop expression control system. The expression vector can be a vector containing a far-red light photoreceptor coding gene alone or a vector containing a processor coding gene alone or a vector containing an effector coding gene alone, wherein the effector contains a far-red light response promoter but does not contain a nucleic acid sequence to be transcribed. Alternatively, it may be an expression vector containing two or three of them. The construction of all the aforementioned mammalian cell expression vectors is detailed in Table 1.

The invention also provides application of the eukaryotic expression vector containing the far-red light gene loop expression control system in preparation of a medicament for treating diabetes.

Another objective of the invention is to provide a method for regulating gene expression in host cells by using the far-red light gene loop expression control system or the gene loop remote regulation system. The method comprises the following steps: a) constructing the far-red light gene loop expression control system in a eukaryotic plasmid expression vector; b) transfected into said host cell; c) and inducing and regulating the host cell by regulating or remotely regulating far-red light irradiation conditions to realize the expression of the effector coding gene. Wherein the host cell comprises a mammalian cell.

In a specific embodiment, the present invention provides a method for regulating gene expression in mammalian cells using the far-red light gene loop expression control system or the gene loop remote regulation system, comprising the steps of:

a) constructing the smart phone platform in a mammalian cell expression vector through a far-red light gene loop expression control system for ultra-remote regulation and control of gene expression through a local area network WiFi or a 2G/3G/4G network;

b) introducing the plasmid into a mammalian cell;

c) the intelligent mobile phone induces and regulates the mammalian cells through the WiFi (wireless fidelity) of the local area network or the 2G/3G/4G network to super-regulate certain far-infrared irradiation conditions, so that the effector P in the mammalian cells_FRLExpression of reporter-encoding genes (e.g. SEAP, EGFP, L uciferase, Insulin, G L P-1, etc.).

d) Detecting the expression condition of the target gene at three time points of 24h, 48h and 72h respectively

The plasmid construction method of the present invention is referred to the materials method and table 1. The method of introducing a plasmid into a mammalian cell comprises: calcium phosphate transfection, PEI transfection, lipofection electroporation transfection, viral infection, and the like.

The method comprises the steps that a smart phone regulates and controls certain far-red light irradiation conditions through a local area network WiFi or a 2G/3G/4G network in an ultra-remote mode, wherein the far-red light irradiation conditions comprise selection of a light source and selection of irradiation intensity, time and an irradiation method, the light source comprises an L ED lamp, a far-red light physiotherapy instrument and the like, and the irradiation intensity is 0-5mW/cm²Etc.; irradiation time of 0 to 72 hours, continuous irradiation or discontinuous irradiation; the irradiation method comprises the steps of directly irradiating and using a projection card with hollow pictures to spatially control the gene expression level of cells at different positions.

The invention also provides a method for carrying out transgenic regulation and expression in a transplantation carrier by utilizing the far-red light gene loop expression control system or the gene loop remote regulation and control system.

The method comprises the following steps: a) preparing a eukaryotic plasmid expression vector containing the far-red light gene loop expression control system; b) preparing an engineered cell containing the far-red light gene loop expression control system; c) preparing an engineered cell transplantation carrier containing the far-red light gene loop expression control system; d) inducing the engineering cell transplantation carrier by remotely regulating and controlling a far-red light sourceExpressing effector P in said transplantation vector_FRLExpression of reporter encoding genes (such as SEAP, EGFP, L uciferase, Insulin, G L P-1 and the like). e) the expression of the target genes is detected at three time points of 24h, 48h and 72h respectively.

In a specific embodiment, the method for regulating and controlling the expression of genes in an engineered cell transplantation carrier by using the far-red light gene loop expression control system or the gene loop remote regulation and control system comprises the following steps:

a) preparing a eukaryotic plasmid expression vector containing the far-red light gene loop expression control system;

b) preparing an engineered cell containing the far-red light gene loop expression control system;

c) preparing an engineered cell transplantation carrier containing the far-red light gene loop expression control system;

d) inducing and expressing a transplantation carrier containing engineered cells by remotely regulating and controlling a far-red light source to ensure that an effector P in the transplantation carrier_FRL-reporter encoding gene (e.g. SEAP, EGFP, L uciferase, Insulin, G L P-1, etc.) expression;

e) and detecting the expression condition of the target gene at three time points of 24h, 48h and 72h respectively.

The method for constructing the eukaryotic expression vector is detailed in table 1; methods of engineering the cells include calcium phosphate transfection, PEI transfection, lipofection electroporation transfection, or viral infection; the preparation method of the engineered cell transplantation carrier comprises the following steps: preparing microcapsules, preparing sodium alginate gel block skins and preparing hollow fiber membrane transplanting tubes.

The method comprises the steps that a smartphone regulates and controls certain far-red light irradiation conditions through a local area network WiFi or a 2G/3G/4G network in an ultra-remote mode, wherein the far-red light irradiation conditions comprise selection of a light source and selection of irradiation intensity, time and an irradiation method, the light source comprises an L ED lamp, a far-red light physiotherapy instrument and the like, and the irradiation intensity is 0-5mW/cm²Etc.; the irradiation time is 0 to 72 hours or the like, and the irradiation time is more than 0 hours, and may be continuous irradiation or discontinuous irradiation or the like.

The invention also provides a method for implanting the far-red light gene loop expression control system or the gene loop remote regulation and control system transplanting carrier into a mouse body, and a method for performing transgenic regulation and expression in the mouse body by the far-red light gene loop expression control system or the gene loop remote regulation and control system, wherein the method comprises the following steps:

a) preparing a eukaryotic plasmid expression vector containing the far-red light gene loop expression control system; b) preparing an engineered cell containing the far-red light gene loop expression control system; c) preparing a transplanting carrier containing the far-red light gene loop expression control system or the gene loop remote regulation and control system; d) implanting a transplantation carrier containing the far-red light gene loop expression control system or the gene loop remote regulation and control system into a mouse body; e) inducing and expressing a transplantation carrier containing engineered cells by remotely regulating and controlling a far-red light source to ensure that an effector P in the transplantation carrier_FRLExpression of reporter coding genes (such as SEAP, EGFP, L uciferase, Insulin, G L P-1 and the like), and f) detection of the expression condition of target genes at three time points of 24h, 48h and 72h respectively.

In a specific embodiment, the engineered cell transplantation vector containing the far-red light gene loop expression control system is transplanted into a mouse body for far-red light induced expression, and the steps are as follows:

c) preparing a transplanting carrier containing the far-red light gene loop expression control system or the gene loop remote regulation and control system;

d) implanting a transplantation carrier containing the far-red light gene loop expression control system or the gene loop remote regulation and control system into a mouse body;

e) inducing and expressing a transplantation carrier containing engineered cells by remotely regulating and controlling a far-red light source to ensure that an effector P in the transplantation carrier_FRL-reporter encoding gene (e.g. SEAP, EGFP, L uciferase, Insulin, G L P-1, etc.) expression;

f) and detecting the expression condition of the target gene at three time points of 24h, 48h and 72h respectively.

Wherein the preparation method of the transplantation carrier comprises the steps of preparing microcapsules, preparing sodium alginate gel block skins and preparing hollow fiber membrane transplantation tubes; the transplantation method can be abdominal cavity transplantation or subcutaneous transplantation, etc.;

the method for regulating gene expression in mouse body includes selection of light source and control of light source, the light source includes L ED lamp, physiotherapeutic instrument and laser lamp, and the lighting method includes selection of lighting time, lighting intensity and lighting frequency.

The invention also provides an application of the far-red light gene loop expression control system or the gene loop remote regulation and control system in preparing a diabetes treatment drug, wherein the diabetes comprises type I diabetes and/or type II diabetes, the system of the invention provides a novel strategy which is safe and reliable and can accurately regulate and control the release of Insulin and glucagon-like peptide in time and space to treat the diabetes, the invention provides a novel method and a novel strategy for treating the diabetes, the system can regulate and control the expression of the Insulin and/or glucagon-like peptide G L P-1, the expression construction of the Insulin comprises SEAP-2A-Insulin, EGFP-2A-SEAP-2A-Insulin, and the expression of the glucagon-like peptide G L P-1 comprises G L P-1-Fc and the like.

The invention also provides a kit which contains the far-red light gene loop expression control system or contains the gene loop remote regulation and control system. The invention also provides a kit which is provided with the eukaryotic expression vector containing the far-red light gene loop expression control system or/and transfected with the host cell containing the eukaryotic expression vector and corresponding instructions.

The kit comprises a plasmid kit for each component of the far-red light gene loop expression control system regulated and controlled by the smart phone platform in an ultra-remote manner, and a mammalian cell kit containing the far-red light gene loop expression control system regulated and controlled by the smart phone platform in an ultra-remote manner. The kit comprises a plasmid kit for ultra-remotely regulating and controlling each component of the far-red light gene loop expression control system by the smart phone platform through a local area network WiFi or 2G/3G/4G network, a mammalian cell kit containing the smart phone platform for ultra-remotely regulating and controlling the far-red light gene loop expression control system through the local area network WiFi or 2G/3G/4G network, and corresponding instructions.

Drawings

FIG. 1 is a schematic diagram of the remote control far-red light gene loop expression control system of the present invention in mammalian cells.

In fig. 2: FIG. 2(a) is a schematic diagram of an apparatus for controlling a far-red light source by a control device of the present invention through a local area network WiFi or a 2G/3G/4G network; FIG. 2(b) is a schematic diagram of far-red light gene loop expression control system for far-red light regulation of transgene expression in mammalian cells.

FIG. 3 is an APP schematic diagram of a gene loop expression control system for controlling a far-red light source by a smart phone through a local area network WiFi or a 2G/3G/4G network according to the present invention.

Fig. 4 is a schematic diagram and a physical diagram of an intelligent controller of an intelligent control system for controlling a far-red light source by a smart phone through a local area network WiFi or a 2G/3G/4G network according to the present invention.

Fig. 5 is a schematic diagram and a real object diagram of a multi-output switching power supply of an intelligent control system for controlling a far-red light source by a smart phone through a local area network WiFi or a 2G/3G/4G network.

FIG. 6 is a system wiring diagram of a super-remote intelligent controller with a multi-output function of an intelligent control system for controlling a far-red light source by a smart phone through a local area network WiFi or a 2G/3G/4G network.

FIG. 7 is a diagram of a super-remote intelligent controller with a multi-output function of an intelligent control system for controlling a far-red light source by a smart phone through a local area network WiFi or a 2G/3G/4G network according to the present invention.

Fig. 8 is a design diagram of 24 far infrared L ED circuits of an intelligent control system for controlling a far infrared light source by a smartphone through a local area network WiFi or a 2G/3G/4G network.

Fig. 9 is a layout diagram of 24 far infrared L ED circuits of an intelligent control system for controlling a far infrared light source by a smart phone through a local area network WiFi or a 2G/3G/4G network.

Fig. 10 is a real diagram of 24 far infrared L ED circuits of an intelligent control system for controlling a far infrared light source by a smartphone through a local area network WiFi or a 2G/3G/4G network.

Fig. 11 is a schematic diagram of an overall system of an intelligent control system for controlling a far-red light source by a smart phone through a local area network WiFi or a 2G/3G/4G network according to the present invention.

FIG. 12 is a graph showing the results of experiments in which red light of the present invention induces photoreceptor pWS189 to produce c-di-GMP in mammalian cells.

FIG. 13 is a diagram showing the results of experiments to demonstrate the components of the far-red light gene loop expression control system of the present invention.

FIG. 14 is a diagram showing experimental results of photoreceptors with different configurations of the far-red light gene loop expression control system of the present invention.

FIG. 15 is a diagram showing the experimental results of photoreceptors expressed by different promoters of the far-red light gene loop expression control system of the present invention.

FIG. 16 is a diagram showing experimental results of processors constructed by different configurations of the far-red light gene loop expression control system of the present invention.

Fig. 17 and fig. 18 are graphs showing experimental results of different processor recognition sites and different numbers of repetitions of the recognition sites in effectors with different configurations of the far-red light gene loop expression control system according to the present invention.

FIG. 19 is a diagram showing the experimental results of adding an insulation signal before a recognition site of a processor having a different number of repeats in effectors having different configurations of the far-red gene loop expression control system according to the present invention.

FIG. 20 is a diagram showing experimental results of different kinds of weak promoters in effectors with different configurations of the far-red light gene loop expression control system according to the present invention.

FIG. 21 is a diagram showing the results of experiments on the expression of far-red light gene loop control system in different mammalian cells.

FIG. 22 is an experimental result diagram of the smartphone of the present invention setting different illumination times to regulate different expression levels of the far-red gene loop expression control system via a local area network WiFi or a 2G/3G/4G network over-long distance.

FIG. 23 is an experimental result diagram of the smartphone of the present invention setting different illumination intensities to regulate different expression levels of the far-red gene loop expression control system via a local area network WiFi or a 2G/3G/4G network over-long distance.

FIGS. 24, 25, 26 and 27 are experimental results of the invention demonstrating that the smart phone can express all meaningful proteins by controlling the far-red light gene loop expression control system through WiFi or 2G/3G/4G network ultra-remote control, with the expression of SEAP, L uciferase, EGFP and G L P1 as examples.

Fig. 28 and fig. 29 are graphs showing experimental results of the smart phone of the present invention that can simultaneously express two or more all-significant proteins by ultra-remote regulation of the far-red gene loop expression control system via the local area network WiFi or 2G/3G/4G network.

FIG. 30 is a diagram showing the experimental results of the preparation of the microcapsule transplantation carrier containing the engineered cells of the smart phone super-regulated far-red light gene loop expression control system via a local area network WiFi or a 2G/3G/4G network according to the present invention.

FIG. 31 is a diagram showing the experimental results of far-red light regulated expression of cells engineered by the far-red light gene loop expression control system in a microcapsule by a smartphone through WiFi (wireless fidelity) or 2G/3G/4G network over a long distance.

FIG. 32 is a graph showing the experimental results of the preparation of a hollow fiber membrane graft vessel graft carrier containing far-red light gene loop expression control system engineered cells for ultra-remote regulation by a smartphone via a local area network WiFi or 2G/3G/4G network in accordance with the present invention.

FIG. 33 is a graph showing the results of experiments in which the far-red light-regulated expression of the far-red light-regulated gene loop expression control system-engineered cells in a hollow fiber membrane graft vessel is regulated and controlled by the far-red light in a local area network WiFi or 2G/3G/4G network-containing smartphone according to the present invention.

Fig. 34 and 35 are experimental result diagrams of the situation that the smart phone regulates the far-red light gene loop expression control system in the mouse body under the regulation and control of the far-red light through the local area network WiFi or the 2G/3G/4G network.

FIG. 36 shows the fasting blood glucose level of a smart phone of the present invention precisely regulating insulin expression in a type I diabetic mouse by a WiFi or 2G/3G/4G network ultra-remote regulation far-infrared light gene loop expression control system through a local area network or a 2G/3G/4G network.

FIG. 37 shows the glucose tolerance test results of the smart phone of the present invention for precisely regulating insulin expression in type I diabetes model mice by using the WiFi or 2G/3G/4G network ultra-remote regulation far-red light gene loop expression control system to treat type I diabetes.

FIG. 38 shows the fasting blood glucose level of the smart phone of the present invention in type I diabetic mice by using the WiFi or 2G/3G/4G network to regulate the far-red light gene loop expression control system in an ultra-remote manner to regulate G L P-1 expression for the treatment of type II diabetes.

FIG. 39 shows sugar tolerance experimental results of the smart phone of the present invention accurately regulating G L P-1 expression in type I diabetic mice by the ultra-remote regulation far-red light gene loop expression control system through WiFi or 2G/3G/4G network for the treatment of type II diabetes.

FIG. 40 shows the results of insulin resistance experiments in which the smart phone of the present invention regulates G L P-1 expression precisely in type I diabetic mice by the ultra-remote regulation far-red light gene loop expression control system through WiFi or 2G/3G/4G network to treat type II diabetes.

FIG. 41 shows that the smart phone of the present invention precisely regulates the amount of glucagon expressed by G L P-1 expression therapy for type II diabetes in a type I diabetic mouse through a WiFi or 2G/3G/4G network ultra-remote regulation far-red light gene loop expression control system.

Detailed Description

The invention is further illustrated by the following examples. These examples are intended to illustrate the invention and do not set any limit to the scope of the invention. The implementation process, conditions, reagents, experimental methods and the like of the present invention are general knowledge and common general knowledge in the field except for the contents specifically mentioned below, and the present invention is not particularly limited. The reagents, instruments, etc. used in the following examples, as well as the experimental methods not specified for specific conditions, were conducted according to the conditions conventionally or proposed by commercial suppliers.

Materials and methods

Manufacturing of intelligent control system for controlling far-red light source by smart phone through WiFi (wireless fidelity) or 2G/3G/4G network in ultra-remote mode through local area network

Intelligent controller

The intelligent controller used for manufacturing is purchased from an intelligent household studio, and the parameters and functions are as follows:

1. the remote control of the mobile phone is supported, and WiFi, 2G/3G/4G control modes are supported;

2. the maximum allowable 10A large current supports multi-channel control, each channel can be independently controlled, and the control effect is pushed to the mobile phone App client in real time;

3. the timing switch and the scene mode are supported, and the functions of the timing switch and the one-key switch are realized;

4. supporting android, apple mobile phones and tablets; the software supports custom attributes;

5. the same software supports a plurality of devices and a plurality of switches;

6. supporting data backup and data recovery; the two-dimensional code scanning import equipment is supported, the equipment can be shared with other personnel, and the operation is convenient and fast.

Multi-output switch power supply

The used multi-output switching power supply is purchased from a cloud particle electronic working room, and the parameters and functions of the multi-output switching power supply are as follows: 1. input voltage of 5-23V, maximum 23V, 20V internal use is recommended, input is prevented from being reversely connected (the input voltage is higher than the output voltage by more than 1V)

2. The adjustable output voltage range is 0V-16.5V and can be continuously adjusted, and the last set voltage is automatically stored.

3. Peak current 3A, suggested for use within 2A. The precision is 1%, the minimum display is 0.01, the unit is A, the heat generation is larger than 2A, and a user wants to solve the heat dissipation problem by a self-thought method.

4. High conversion efficiency (95% efficiency related to input and output voltage, current and voltage difference)

5. The load regulation rate S (I) is less than or equal to 0.8 percent, and the voltage regulation rate S (u) is less than or equal to 0.8 percent

6. Size 62mm × 44mm × 18mm

7. Weight 45g

Far infrared L ED lamp

The used far infrared L ED lamp bead is purchased from a hundred-light photoelectricity, the design of the far infrared L ED lamp circuit board is completed by a microelectronic engineer in white depression, the processing is completed by Shenzhen Jie Multi-help science and technology Limited, and other materials are all common domestic materials.

1. Input voltage: 1.5V-4.8V, and the maximum is 6V;

2.6 different voltage can be switched at will;

3. with independent switches, with potentiometers (normally not recommended) to regulate the brightness as a whole;

4. the diode can be independently disassembled;

molecular cloning

The construction reagents, specific construction systems and procedures of all expression plasmids of the present invention are shown below.

All primers used for PCR are synthesized by Jinwei Biotechnology Inc. the expression plasmids constructed in the embodiment of the invention are subjected to sequence determination, and the sequence determination is completed by Jinwei Biotechnology Inc. the Phanta Max Super-Fidelity DNA polymerase used in the embodiment of the invention is purchased from Nanjing Nodezam Biotechnology Inc. endonuclease and T4DNA ligase are purchased from Takara, homologous recombinase is purchased from Shanghai Biotechnology (Shanghai) GmbH, Phanta Max Super-Fidelity DNA polymerase is purchased and is accompanied by corresponding polymerase buffer solution and dNTP endonuclease, T4DNA ligase and recombinase homologous purchase and is accompanied by corresponding buffer solution, Yeast extract (Yeast extract), Trypton (Trypose), agar powder and ampicillin (Amp) are purchased from Shanghai Biotechnology technology Inc. DNA Mark L5000, DNA MarD L2000 (treasure Bio-engineering kit), the nucleic acid extraction kit (Trypose Bio-Rad Biotechnology Inc. (Australin Biotechnology Inc.), the other pure products of DNA extraction and the Kangkui Biotechnology kit (Otsu Biotechnology, Kangji Biotechnology, Kanji Biotechnology, Inc. and other purified products are purchased from Kanji Biotechnology Inc. (Kanji Bio.

Polymerase Chain Reaction (PCR)

Amplification step (bp represents the number of nucleotides of the amplified fragment).

(II) endonuclease cleavage reaction

1. System for double digestion of plasmid vector (x is the volume at which the plasmid mass is 1. mu.g; n is the amount of sterilized ultra pure water. mu. L required to bring the system to the total volume)

2. System for double digestion of PCR product fragments (x is the volume at which the plasmid mass is 1. mu.g; n is the amount of sterilized ultra pure water. mu. L required to bring the system to the total volume)

3. Connecting the PCR product fragment subjected to double enzyme digestion into a plasmid vector subjected to double enzyme digestion to form a plasmid packaging system:

note that the mass ratio of the PCR product fragment to the vector double-enzyme digestion product is approximately between 2:1 and 6: 1.

(III) homologous recombination ligation reaction

According to the instructions of the kit for seamless cloning (assembly) of the Hetaobiotech (Shanghai) GmbH: adding 15bp nucleotide sequences homologous with the 15bp nucleotide sequences on two sides of the linearized vector on two sides during PCR product fragment amplification, wherein the 15bp nucleotide sequences on two sides of the amplified PCR product are homologous with the nucleotide sequences on two sides of the linearized vector, and the PCR product fragment and the linearized vector are recombined and connected into a ring shape under the action of homologous recombinase.

Note that the value of n depends on the size and concentration of the PCR product fragment.

(IV) preparation of competent cells of Escherichia coli

All solutions and consumables used for competent cell preparation were autoclaved.

1. Streaking the strain of the Escherichia coli DH5 α strain on a flat plate without antibiotics, and performing inverted culture at 37 ℃ for 12-16 h;

picking a single colony in a L B shake tube without any antibiotic at 2m L, and shaking and culturing at 37 ℃ and 180rpm overnight;

2. sucking 1m L bacterial liquid, transferring the bacterial liquid into a 250m L triangular flask L B culture medium for amplification culture (amplification culture according to a ratio of 1: 100), and carrying out shaking culture on a shaking table at 180rpm at 37 ℃ for 2-3 h until OD600 is 0.4-0.5;

3. transferring the culture solution into a centrifuge tube, standing on ice for 10min, centrifuging at 4 deg.C and 3000rpm for 10min (the centrifuge needs precooling in advance), and carefully discarding the supernatant;

4. adding pre-cooled 0.1M CaCl₂4mL(0.1M CaCl₂Precooling in advance), quickly suspending thalli by using a vortex mixer after full precooling, then carrying out ice bath for 10min, and combining two tubes for 1 tube;

centrifuging at 4000rpm at 5.4 deg.C for 6 min;

6. the supernatant was discarded, and 4M of L ice-cold 0.1M CaCl was added₂And 0.1m L precooled sterilized pure glycerol, and suspending and precipitating;

7. the suspension is subpackaged into PCR tubes (the PCR tubes are preferably pre-cooled on ice) in 100 mu L/tube, and stored in liquid nitrogen for later use;

(V) transformation of the ligation product into E.coli

1. The prepared competent cells were thawed (thawed on ice), added with the ligation product in an appropriate volume and mixed well, and then ice-cooled for 30 min. 1/10, which is generally added to a volume of ligation product less than the volume of competent cells;

heat shock is carried out for 90s in water bath at the temperature of 2.42 ℃, and then the mixture is rapidly placed on ice for 5 min;

3. adding the bacterial liquid into 800 μ L L B liquid culture medium (without antibiotic), mixing, and shake culturing at 37 deg.C for 40-60 min;

4. transferring the bacterial liquid into a centrifugal tube with the diameter of 1.5m L, centrifuging for 5min at 4000rpm, discarding part of supernatant, reserving about 100 mu L of supernatant, and blowing the cells into cell suspension;

5. spreading the suspension on L B solid culture medium containing Amp, and inverting in 37 deg.C incubator for overnight culture;

other experimental procedures, such as gel recovery and purification recovery of DNA fragments, were performed according to the protocol of DNA gel recovery kit and PCR product purification kit (Kangji Biotech Co., Ltd.); the plasmid extraction step was performed according to the protocol of the extraction kit for plasmid Mini-drawer (Tiangen Biochemical technology, Beijing, Ltd.).

Selection and fabrication of light sources

In the embodiment of the invention, L ED (L720- __ AU, epitex, Japan) with the wavelength of 720nm and an infrared therapeutic device (CQ-61, Chongqing space rocket electronics Co., Ltd. in China) are taken as examples to illustrate the regulation and control method of the gene loop remote regulation and control system, but the invention is not limited in scope.

L ED used in the experiment is purchased from EPITex company of Japan, the infrared ray therapy device is purchased from electronic technology Limited company of Chongqing space rocket, China, and other accessories are all domestic conventional consumables.

L EDs with the wavelength of 720nm are used for providing a far-red light emitter with low power.4 4 × 6L ED plates are connected in a mode that each L ED corresponds to the central position of each hole according to the arrangement of cell culture plates (24 holes), and each L ED is connected in parallel.

An infrared therapeutic device is a far-infrared light emitter with high power. And adjusting the illumination time according to the experiment requirement, and carrying out the experiment according to the illumination intensity.

Cell culture and transfection

In the embodiment of the invention, the following cell line and PEI transfection are used as examples to illustrate the working condition of the gene loop remote control system for regulating the transgene expression by far-red light in cells and animals, but the invention is not limited in scope.

10cm cell culture dishes, cell culture plates (24 wells), 15m L and 50m L centrifuge tubes for cell culture were purchased from Thermo Fisher Scientific, USA (L abserv), modified Eagle medium, fetal bovine serum, penicillin and streptomycin solutions were purchased from Gibico, USA, PEI used for transfection was purchased from Polysciences, USA, cell culture boxes were purchased from Thermo Fisher Scientific, USA, and the rest of the consumables were common domestic consumables.

Cell culture human embryonic kidney cells (HEK-293T, ATCC: CR L-11268), continuous expression RTP1, RTP2, REEP1 and G_αoλφHEK-293(Hana3A), telomerase immortalized human mesenchymal stem cells (hMSC-TERT, ATCC: CR L-3220), human embryonic kidney epithelial cells (HEK-293A), human cervical cancer cells (He L a, ATCC: CC L-2), murine neuroma blast cells (Neuro-2A, ATCC: CC L-131) were cultured in modified Eagle medium supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin and streptomycin solution, all types of cells were cultured in an incubator at 37 ℃ containing 5% carbon dioxide.

Transfection of all cell lines the procedure for optimized PEI was used (Wieland M, Methods 56(3): 351). briefly, 6X10 was plated per well in a 500. mu. L24 well plate culture system⁴After culturing for 16h, the cells were incubated for 6h with the optimal ratio of DNA mixed with PEI in a mass ratio of 3:1(PEI: DNA) dissolved in 50. mu. L of culture medium.

Detection of reporter Gene secreted alkaline phosphatase (SEAP)

Homoarginine, magnesium chloride, diethanolamine and HCl for preparing a detection reporter gene reaction buffer solution are purchased from Biotechnology engineering (Shanghai) GmbH; chromogenic substrates (pNPP: p-Nitrophenylphosphate) were purchased from Shanghai Crystal pure science and technology, Inc. (Aladdin).

(1) Reagent preparation:

2x buffer:

20mM homoarginine notes: the function of the inhibitor is to inhibit the activity of endogenous alkaline phosphatase

1mM magnesium chloride

2% diethanolamine

Adjusting the pH to 9.8 with HCl

Substrate solution:

120mM chromogenic substrate (pNPP: p-Nitrophenylphosphate)

·In 2x assay buffer

(2) The experimental steps are as follows:

1. pipette 200u L of cell culture supernatant into a centrifuge tube (Note: generally over 150u L because subsequent heating will lose a portion of its volume)

Water bath at 2.65 deg.c for 30min (note: heating mainly inactivates endogenous alkaline phosphatase, while SEAP is high temperature resistant and does not inactivate at this temperature).

3. Pipette 80u L (diluted by itself depending on the experimental conditions) into a 96-well plate, and add rapidly 100. mu.l of previously preheated 2xbuffer and 20. mu.l of substrate solution.

4. The enzyme-linked immunosorbent assay is carried out 10 times at 405nm, and each time interval is 1min (conditions can be set according to the experiment situation).

(3) Calculation of enzyme Activity

The enzyme activity of alkaline phosphatase is defined as that of disodium p-nitrophenylphosphate (PNPP-Na) which reacts with the substrate at 37 ℃ and pH 9.8 within 1min₂) The alkaline phosphatase for producing 1 mol/L p-nitrophenol is defined as 1 activity unit (1U). the p-nitrophenol has bright yellow color, and when the wavelength is 405nm, the p-nitrophenol with different concentrations corresponds to different light absorption values.

Detection of reporter Gene luciferase (L luciferase)

The specific operation steps are detailed in the specification of the dual-luciferase reporter gene detection kit (Cat: B17001, L ot71005, Bitool, Houston, TX, USA).

Preparation method of sodium alginate-P LL-sodium alginate microcapsule

Sodium alginate and microcapsule granulating apparatus for preparing microcapsule are from Buchi of Switzerland, polylysine is from Sigma of America, and trisodium citrate (Na)₃Citrate), sodium chloride, calcium chloride, mops (morpholino phenol sulfonic acid) were purchased from shanghai bio-engineering technology ltd. The details of the reagent preparation and the operation steps used in the experiment are described in the operating manual of a microcapsule granulator (Inotech Encapsulater Research Unit IE-50R).

The main parameters in the preparation process are that the diameter of an assembled spray head is 200 mu m, the frequency of ultrasonic vibration flow breaking is 1300Hz, the static electricity for dispersion use is 1100V, the injection speed is 20m L/min, the number of microcapsules formed per second is 500, the size of each microcapsule is 300-350 mu m, and the number of cells wrapped by each microcapsule is 200-250.

Preparation method of hollow fiber membrane transplanting tube

Hollow fiber membrane graft tube for experiment (

Implant Membrane) purchased Spectrum L laboratories Inc. hollow fiber Membrane graft preparation is described in detail in

Implant Membrane product Specification.

Method for detecting Insulin (Insulin)

The Insulin detection kit (Mouse Insulin E L ISA kit) used for the experiment is purchased from Mercodia, Sweden, and the detailed determination method is described in the product instruction.

Method for detecting glucagon (G L P-1)

Glucagon detection kits for the experiments (Millipore Corporation, Billerica, MA01821 USA, Cat. No. EG L P-35K, L ot. No.2639195) were purchased from Millipore Corporation, USA, and the specific assay procedures are described in the product specification.

Embodiment 1, manufacture of intelligent control system for controlling far-infrared light source by smart phone through WiFi (wireless fidelity) or 2G/3G/4G network in ultra-remote mode

In the invention, an intelligent controller manufactured by an intelligent household studio, an APP matched with the intelligent controller, 6 multi-output switching power supplies and 24 far infrared L ED lamps are taken as an example of a far-red light source, so that the manufacturing method of the intelligent control system for controlling the far-red light source by the smart phone through a local area network WiFi or a 2G/3G/4G network in an ultra-remote mode is described, but the protection scope of the invention is not limited.

In the first step, the APP client is realized. The specific setting and the using method of the matched APP provided by the purchased intelligent controller manufacturer (see the experimental materials and methods in detail) are shown in the manufacturer's operating instruction. The APP schematic diagram is shown in attached figure 3in the specification in detail.

And secondly, realizing the remote intelligent controller. The intelligent controller (detailed functions and parameters are shown in experimental methods and materials) is purchased from an intelligent household working room, and the intelligent mobile phone can be used to realize the ultra-remote control of the far-red light source by utilizing local area network WiFi or 2G/3G/4G network resources. The schematic diagram and the physical diagram of the intelligent controller are shown in the attached figure 4 in the specification in detail.

And thirdly, realizing the multi-output switching power supply. The multi-output switch power supply (detailed functions and parameters are shown in an experimental method and materials) is purchased from a cloud particle side, 6 paths of independent switch power supplies of an electronic working chamber of the cloud particle side are respectively connected to an intelligent controller, and the 6 paths of power supplies are controlled by a mobile phone APP to set different brightnesses of the same far-red light source, so that the design of the multi-output switch power supply is realized. The schematic diagram and the physical diagram of the multi-output switch power supply are shown in the attached figure 5in the specification in detail.

The method comprises the steps of respectively connecting output ports of 6 independent switching power supplies with 6 input ports of an intelligent controller, connecting corresponding 6 output ports of the intelligent controller with an input port of a far infrared L ED to serve as a direct current power supply port of a far infrared L ED, wherein power supply of the whole system is completed by an AC/DC power adapter, a fuse with a parameter of 3A is connected in series with a negative connecting wire of the AC/DC power adapter, the ultra-remote intelligent controller with the multi-output function is protected to normally work within a safe current range, a system wiring diagram shows in detail in figure 6, when APP software matched with the intelligent controller is installed on the intelligent mobile phone, the intelligent mobile phone and the intelligent controller are configured to be jointly connected to a local area network or a 2G/3G/4G network resource, the opening and closing of the 6 independent switching power supplies can be controlled by operating the APP software, the opening and closing of the 6 independent switching power supplies can be set, the ultra-remote intelligent controller with different remote illumination control functions in different remote illumination time states, such as a WiFi 7 remote control with different remote illumination control functions.

The printed circuit board of the far-red light source is provided with 6 power supply ends, wherein a voltage clamping diode is added at each of the 6 power supply ends to effectively protect a L ED circuit, and a master control self-locking switch is added in the whole power supply, so that the circuit of the lamp panel is manually controlled to be switched on and off.

In summary, after the APP client, the remote intelligent controller, the multi-output switching power supply and the far infrared L ED lamp are integrated and connected, the smart phone is formed to manufacture an intelligent control system for controlling the far-red light source through the local area network WiFi or the 2G/3G/4G network in an ultra-remote manner, and the overall system schematic diagram is shown in fig. 11 in detail.

Example 2 construction of Gene Loop remote regulatory System elements for far-Red light Regulation of transgene expression

The embodiment of the invention includes a method for constructing a representative element in a gene loop remote control system for regulating transgene expression by far-red light, but the invention is not limited to the protection scope of the invention. The detailed design scheme and procedure are shown in table 1.

Example 3 verification that far-red light induces photoreceptor pWS189 to produce c-di-GMP in mammalian cells

In the first step, plasmid construction. The plasmid construction in this example is detailed in Table 1.

Second, seeding the cells good HEK-293T cells in growth state after digestion with 0.25% pancreatin were seeded in 7 24-well plates, 6 × 10 per well⁴Cells were plated and 500 μ L DMEM medium containing 10% FBS was added.

And thirdly, transfecting, namely uniformly mixing 0.1 mu g of photoreceptor pWS189 and PEI transfection reagent with serum-free DMEM within 16 to 24 hours after inoculating the cells, standing at room temperature for 15min, and uniformly dripping the mixture into a 24-well culture plate, wherein the preparation total volume of each well is 50 mu L, the mass ratio of the plasmid to the PEI is 1:3, and culturing by changing into 500 mu L DMEM culture medium containing 10% FBS after 6 hours of transfection.

And fourthly, illuminating. After the liquid is changed for 14-18 hours, the liquid is divided into 7 groups (respectively numbered 1, 2, 3,4, 5, 6 and 7), and all the groups except the 1 are placed at the wavelength of 720nm and the illumination intensity of 1mW/cm²L ED (see test materials and methods for specific attachment) for 1 h.

Fifthly, collecting cells, harvesting the cells at 0, 5, 10, 15, 30 and 60min (corresponding to

numbers

2, 3,4, 5, 6 and 7 respectively; the 24-hole plate of number 1 is always in dark condition) after illumination for one hour, removing the culture medium in the 24-hole plate, adding 200 mu L PBS, repeatedly blowing and beating the cells one by using a pipette in sequence to enable the cells to fall off, transferring the cells into a new 2m L centrifugal tube, and centrifuging the cells for 5min at 1000 × g.

Sixthly, cell lysis is carried out, 200 mu L lysis buffer solution is added into the collected cells, the cells are placed for 15min at minus 80 ℃, the cells are quickly taken out and placed for 5min at 37 ℃, the operations are repeated for 3 times, the cells are uniformly shaken and mixed in the middle, the cells are centrifuged for 5min at 5000 × g, and supernatant is taken out to be detected.

And seventhly, measuring the amount of the c-di-GMP, and measuring the expression amount of the c-di-GMP of each group by using a c-di-GMPE L ISA kit, wherein the experimental data are shown in the attached figure 12 of the specification in detail.

Example 4 composition of Gene Loop remote control System for far-Red light control of transgene expression

In this example, SEAP is used as a reporter gene to illustrate the components of the gene loop remote control system for regulating the expression of the transgene with far-red light, but the scope of the invention is not limited. The method comprises the following specific steps:

Second, seeding the cells good HEK-293T cells in growth state after digestion with 0.25% pancreatin were seeded in 2 24-well plates, 6 × 10 per well⁴Cells were plated and 500 μ L DMEM medium containing 10% FBS was added.

In the step 16 to 24h of inoculated cells, 0.3. mu.g of pcDNA3.1(+) in the 1 st group of the dark group and the light group, 0.1. mu.g of pWS189 and 0.2. mu.g of pcDNA3.1(+) in the 2 nd group, 0.1. mu.g of pGY32 and 0.2. mu.g of pcDNA3.1(+) in the 3 rd group, 0.1. mu.g of pXY34 and 0.2. mu.g of pcDNA3.1(+) in the 4 th group, 0.1. mu.g of pXY34 and 0.2. mu.g of pcDNA3.1(+) in the 5 th group, 0.1. mu.g of pWS189, 0.1. mu.g of pGY32 and 0.1. mu.g of pcDNA3.1(+) in the 5 th group, 0.1. mu.g of pWS 34 and 0.1. mu.1. mu.g of pcDNA3.1. mu.1. mu.g of (+), 0.1. mu.g of PEI 3.1. mu.1. mu.g of pcDNA0.1. mu.1. mu.10. mu.g of PEI, 0.1. mu.1. mu.g of pWS 34 and 0.1. mu.1. mu.10. mu.g of PEI, 0.1. mu.10. mu.g of PEI, 0.1. mu.10. mu..

And fourthly, illuminating. After the liquid is changed for 14-18h, the illumination group is placed at the wavelength of 720nm and the illumination intensity of 1mW/cm²L ED (reference material and method for connection) for 4h, and the dark group was cultured in the dark.

And fifthly, detecting the reporter gene. After culturing for 48 hours, cell culture supernatants of the dark group and the light group were each removed to determine the expression level of SEAP (see materials and methods for details).

The results show that the far-red light gene loop expression control system works normally only when photoreceptors, processors and effectors are simultaneously present in the host cell and induced by far-red light. The system fails to work if either component is missing or under dark conditions. The experimental data are shown in figure 13 of the specification.

Example 5 photoreceptors with different constructions of Gene Loop control System for far-Red light regulating transgene expression

In this embodiment, the conditions that photoreceptors differently constructed by a far-red light gene loop expression control system for far-red light regulation and control of transgene expression are regulated and expressed by far-red light in mammalian cells are demonstrated by way of example, but the scope of the invention is not limited. The method comprises the following specific steps:

In the second step, cells were seeded (the same procedure as in example 3).

And thirdly, within 16 to 24 hours of inoculating cells, dividing 2 pieces of 24-well plates into a dark group and a light group, wherein each group is divided into 1-4 groups, and within 16 to 24 hours of inoculating cells, 0.1 mu g of pWS46 in the 1 st group of the dark group and the light group, 0.1 mu g of pWS48 in the 2 nd group, 0.1 mu g of pWS 1in the 3 rd group, and 0.1 mu g of pWS50 in the 4 th group, respectively, and 0.1 mu g of Y32, 0.1 mu g of pXY34 and PEI transfection reagent are mixed with serum-free DMEM uniformly, and the mixture is dripped into the 24-well culture plate after standing for 15min at room temperature, wherein the total volume of each well is 50 mu L, the mass ratio of PEI to pG3 is 1, and the culture medium of 500 mu L containing 10% of FBS is changed after transfection for 6 hours.

And step four, illuminating (the concrete steps are the same as those of the example 3).

And the fifth step, detecting the reporter gene (the specific steps are the same as those in example 3).

The result shows that photoreceptors which are constructed by different far-red light gene loop expression control systems for regulating transgene expression by far-red light can work normally in mammalian cells under the induction of the far-red light. But photoreceptors constructed differently under the same far-red light induction give different intensities of response in the effector. The experimental data are shown in figure 14 in the specification.

Example 6 photoreceptors expressed by different promoters of the far-red light Gene Loop expression control System for far-red light-regulated transgene expression

In this embodiment, for example, it is demonstrated that the photoreceptors of the far-red light gene loop expression control system for far-red light regulation of transgene expression are expressed by different promoters, and the expression of the photoreceptors is regulated by far-red light in mammalian cells, but the scope of the present invention is not limited. The method comprises the following specific steps:

In the second step, cells were seeded (the same procedure as in example 3).

And thirdly, within 16 to 24 hours of inoculating cells, dividing 2 pieces of 24-well plates into a dark group and a light group, wherein each group is divided into 1-5 groups, and within 16 to 24 hours of inoculating cells, wherein 0.1 mu g of pWS189 is used in the 1 st group of the dark group and the light group, 0.1 mu g of pWS50 is used in the 2 nd group, 0.1 mu g of pWS51 is used in the 3 rd group, 0.1 mu g of pWS55 is used in the 4 th group, 0.1 mu g of pWS59 is used in the 5 th group, and 0.1 mu g of processor pGY32, 0.1 mu g of effector pXY34 and PEI transfection reagent are mixed with serum-free DMEM, and the mixture is uniformly dripped into the 24-well culture plate after standing for 15 minutes at room temperature, wherein the total preparation volume of each well is 50 mu L, the mass ratio of plasmid to PEI is 1:3, and the culture is changed into 10% FBS-containing culture medium after 6 hours of transfection with 500 mu of DMEM L.

The results show that in the gene loop control system for regulating transgene expression by far-red light, photoreceptors expressed by different promoters can normally work in mammalian cells under the induction of the far-red light. Photoreceptors expressing different promoters under the same far-red light induction, however, cause the response intensity of the effectors to differ. The experimental data are shown in figure 15 of the specification.

Example 7 processors with different constructs of Gene Loop control System for far-Red light Regulation of transgene expression

In this embodiment, the examples show the condition that the processors with different configurations of the gene loop control system for far-red light-regulated transgene expression are regulated by far-red light in mammalian cells, but the scope of the invention is not limited. The method comprises the following specific steps:

In the second step, cells were seeded (the same procedure as in example 3).

In the inoculated cells 16 to 24 hours, 2 pieces of 24-well plates were divided into a dark group and a light group, each group was divided into 1-8 groups, and in the inoculated cells 16 to 24 hours, 0.1. mu.g of pWS200 in the 1 st group, 0.1. mu.g of pXY34 in the 2 nd group, 0.1. mu.g of pXY35 in the 3 rd group, 0.1. mu.g of pXY36 in the 4 th group, 0.1. mu.g of Y28 in the 5 th group, 0.1. mu.g of pGY32 in the 6 th group, 0.1. mu.g of pGY33 in the 7 th group, 0.1. mu.g of pGY 7in the 8 th group, and 0.01. mu.g of far-red light receptor pWS189, 0.1. mu.g of effector pXY24, PEI transfection reagent was mixed with serum-free pGY, homogeneously mixed with DMEM, and PEI was added to the 24-well plates after 15min at room temperature, PEI was added to the 24-well plates, and PEI was added to the total volume of the transfection medium was changed to 500. mu.10 mass of DMEM containing 500. mu.3.

The results show that in the gene loop control system for regulating transgene expression by far-red light, different processors can work normally in mammalian cells under the induction of the far-red light. However, different processors constructed under the same far-red light induction cause different effector responses, and different processors can be selected according to experimental needs. The experimental data are shown in figure 16 of the specification.

Example 8 Effector with different constructs of Gene Loop control System for far-Red light Regulation of transgene expression

In this example, it is shown that effectors with different configurations of a gene loop control system for far-red light-regulated transgene expression are expressed in mammalian cells under the control of far-red light. Taking different recognition sites of the processor, different numbers of repetitions of the recognition sites, whether or not to contain an insulating signal, and different types of weak promoters as examples, the situation that different effectors are regulated by far-red light in mammalian cells is illustrated, but the scope of the present invention is not limited thereto.

Different processor recognition sites and different numbers of repetitions of recognition sites. The method comprises the following specific steps:

In the second step, cells were seeded (the same procedure as in example 3).

In the inoculated cells 16 to 24 hours, the 2 pieces of 24-well plates were divided into a dark group and a light group, each group was divided into 1-10 groups, in the inoculated cells 16 to 24 hours, 0.1. mu.g of pXY19 in the 1 st group of the dark group and the light group, 0.1. mu.g of pXY20 in the 2 nd group, 0.1. mu.g of pXY21 in the 3 rd group, 0.1. mu.g of pXY22 in the 4 th group, 0.1. mu.g of pXY23 in the 5 th group, 0.1. mu.g of pXY16 in the 6 th group, 0.1. mu.g of pXY17 in the 7 th group, 0.1. mu.g of pXY18 in the 8 th group, 0.1. mu.g of pXY31 in the 9 th group, 0.1. mu.g of pXY32 in the 10 th group, 0.1. mu.g of pXY32 and 0.1. mu.g of pWS, 0.1. mu.g of PEI 189, 24. mu.1. mu.g of PEI, 24. mu.g of PEI-well, and 500. mu.1. mu.g of DMEM, after the transfection medium, the transfection, the well was mixed with the red light-well.

The results show that in the gene loop control system for regulating transgene expression by far-red light, effectors with different processor recognition sites and different repetition number recognition sites can work normally in mammalian cells under the induction of the far-red light. However, the response intensity of the effectors with different processor recognition sites and different numbers of recognition sites under the same far-red light induction is different, and different processors can be selected according to the experiment requirements. The experimental data are shown in the attached figures 17 and 18 in the specification.

An insulating signal is added before the recognition sites of the processor containing different numbers of repeats. The method comprises the following specific steps:

In the second step, cells were seeded (the same procedure as in example 3).

In the inoculated cells 16 to 24 hours, the 2 pieces of 24-well plates are divided into a dark group and a light group, each group is divided into 1-5 groups, in the inoculated cells 16 to 24 hours, 0.1 mu g of pXY33 in the 1 st group of the dark group and the light group, 0.1 mu g of pXY28 in the 2 nd group, 0.1 mu g of pXY34 in the 3 rd group, 0.1 mu g of pXY39 in the 4 th group, and 0.1 mu g of pXY40 in the 5 th group, respectively, and 0.1 mu g of far-red light pWS189, 0.1 mu g of processor pGY32, PEI transfection reagent and serum-free DMEM are mixed, and uniformly dropped into the 24-well culture plates after standing at room temperature for 15 minutes, wherein the total volume of each well is 50 mu L, the mass ratio of plasmid to PEI is 1:3, and 10% of DMEM containing medium is replaced into the 500 mu FBS L after 6 hours for culture.

The results show that in the gene loop control system for regulating transgene expression by far-red light, effectors with insulating signals added before processor recognition sites with different repetition numbers can work normally in mammalian cells under the induction of the far-red light. However, the response intensity of the effector with the addition of the insulation signal before the recognition sites of the processor with different repetition numbers under the induction of the same far-red light is different, and different processors can be selected according to the experimental needs. The experimental data are shown in figure 19 of the specification in detail.

Different kinds of weak promoters. With the promoter h _ CMV_min3GBy way of example, different weak promoters may be used. The method comprises the following specific steps:

In the second step, cells were seeded (the same procedure as in example 3).

In the inoculated cells 16 to 24 hours, the 2 pieces of 24-well plates were divided into a dark group and a light group, each group was divided into 1-5 groups, and in the inoculated cells 16 to 24 hours, 0.1. mu.g of pGY36 in the 1 st group, 0.1g of pGY37 in the 2 nd group, 0.1. mu.g of pGY38 in the 3 rd group, 0.1. mu.g of pGY39 in the 4 th group, and 0.1. mu.g of pGY40 in the 5 th group, respectively, and 0.1. mu.g of far-red light-emitting receptor pWS189, 0.1. mu.g of processor pGY32, and a PEI transfection reagent were mixed with serum-free DMEM, and uniformly dropped into the 24-well culture plates after standing at room temperature for 15 minutes, wherein the total volume of the preparation per well was 50. mu. L, the mass ratio of plasmid to PEI was 1:3, and 10% of PEI-containing DMEM was changed into the 500. mu.FBS L after 6 hours.

The results show that in the gene loop control system for controlling transgene expression by far-red light, different weak promoters contained in the effector can normally work in mammalian cells under the induction of the far-red light. But the weak promoter responds differently in intensity from other weak promoters. The experimental data are shown in the attached figures 17 and 20 in the specification.

Example 9 Gene Loop control System for far-Red light-regulated transgene expression in different mammalian cells

In this embodiment, the case of far-red light-regulated expression of the gene loop control system for far-red light-regulated transgene expression in different mammalian cells is demonstrated by way of example, but the scope of the present invention is not limited thereto. The method comprises the following specific steps:

Second, Neuro-2A cells, He L a cells, hMSC-TERT cells, Hana3A cells, HEK-293A cells and HEK-293T cells in good growth state were digested with 0.25% trypsin and seeded into 2 different 24-well plates (4 wells for each plate) of each cellIn the plate, each well was 6 × 10⁴Cells were plated and 500 μ L DMEM medium containing 10% FBS was added.

And thirdly, transfecting, namely uniformly mixing 0.1 mu g of pWS189, 0.1 mu g of pGY32, 0.1 mu g of pXY34 and PEI transfection reagent with serum-free DMEM within 16 to 24 hours after inoculating the cells, uniformly dripping the mixture into a 24-well culture plate after standing for 15min at room temperature, wherein the total preparation volume of each well is 50 mu L, the mass ratio of the plasmid to the PEI is 1:3, and culturing by replacing with 500 mu L of DMEM medium containing 10% of FBS after transfecting for 6 hours.

The results show that the gene loop control system for regulating transgene expression by far-red light can be induced to express by far-red light in different mammalian cells. Therefore, the far-red light transgenic gene loop control system is suitable for various mammalian cells. The experimental data are shown in figure 21 of the specification. Example 10, the smartphone regulates different expression levels of the gene loop control system that regulates the far-red light to regulate transgene expression through the WiFi of the local area network or the 2G/3G/4G network by controlling different illumination times at a very high distance

In this embodiment, the smartphone controls different illumination times through the local area network WiFi or the 2G/3G/4G network in a very remote manner, and the illumination time may be pulse illumination, continuous illumination, or discontinuous illumination, and may be long or short. This example demonstrates the relationship between the smart phone controlling different illumination time (continuously) and the effector regulation expression amount through the WiFi or 2G/3G/4G network, but does not define the protection scope of the present invention. The method comprises the following specific steps:

Second, seeding the cells good HEK-293T cells in growth state after digestion with 0.25% pancreatin were seeded in 12 24-well plates, 6 × 10 per well⁴Cells were plated and 500 μ L DMEM medium containing 10% FBS was added.

And thirdly, transfecting, namely uniformly mixing 0.1 mu g of photoreceptor pWS189, 0.1 mu g of pGY32, 0.1 mu g of pXY34 and PEI transfection reagent with serum-free DMEM within 16 to 24 hours after inoculating the cells, standing at room temperature for 15min, and uniformly dripping the mixture into a 24-well culture plate, wherein the total preparation volume of each well is 50 mu L, the mass ratio of the plasmid to the PEI is 1:3, and culturing by replacing with 500 mu L of DMEM medium containing 10% of FBS after 6 hours of transfection.

Fourthly, the smart phone controls different illumination time to illuminate through WiFi or 2G/3G/4G network of the local area network in an ultra-remote mode. After the liquid is changed for 14-18h, the liquid is divided into 12 groups, and the groups are placed at a wavelength of 720nm and an illumination intensity of 1mW/cm²L ED (specific connection mode refers to materials and methods). The smartphone controls different illumination time to be 0, 0.1, 0.25, 0.5, 1, 2, 4, 6, 12, 24, 48 and 72h respectively (wherein the illumination group 0h is cultured in the dark all the time).

And fifthly, detecting the reporter gene. After 72 hours of culture, the supernatant of the cell culture medium of each group was collected to determine the expression level of SEAP.

Experimental results show that the smartphone can induce different expression quantities of the far-red light regulation transgenic gene loop control system to regulate and control target genes through different illumination time controlled by the local area network WiFi or the 2G/3G/4G network in an ultra-remote mode, and the longer the illumination induction time is, the higher the expression quantity is, and illumination time dependent expression is presented. The experimental data are shown in figure 22 in the specification.

Example 11, the smartphone controls different amounts of expression of the gene loop control system that regulates the far-red light-regulated transgene expression by controlling different illumination intensities through the WiFi or 2G/3G/4G network in the local area network in an ultra-remote manner

In this embodiment, for example, it is proved that the relationship between the different illumination intensities and the effector regulation expression amounts controlled by the smartphone through the WiFi or 2G/3G/4G network in the ultra-remote manner is not limited to the protection scope of the present invention. The method comprises the following specific steps:

Second, seeding the cells good HEK-293T cells in growth state after digestion with 0.25% pancreatin were seeded in 7 24-well plates, 6 × 10 per well⁴A cell and adding500 μ L DMEM medium containing 10% FBS.

Third, transfection (the same procedure as in example 8).

Fourthly, the smart phone controls different illumination intensities to illuminate through WiFi or 2G/3G/4G network of the local area network in an ultra-remote mode. After the liquid is changed for 14-18h, the liquid is divided into 7 groups, and the groups are respectively placed at the wavelength of 720nm and the illumination intensity of 0, 50, 75, 100, 250, 500, 750, 1000, 1500 and 2000 mu W/cm²L ED (see materials and methods for connection), the light exposure time was 4h (the cells were incubated in the dark with a light intensity of 0).

Experimental results show that the smartphone can induce different expression quantities of the gene loop control system for regulating and controlling the transgene expression of the far-red light through different illumination intensities controlled by the local area network WiFi or the 2G/3G/4G network in an ultra-remote mode, and the stronger the illumination intensity is, the higher the expression quantity is, the illumination intensity dependent expression is presented. The experimental data are shown in figure 23 of the specification.

Example 12 the smartphone can express all meaningful proteins through the local area network WiFi or 2G/3G/4G network ultra-remote transgenic gene loop control system

The gene loop control system for the smartphone to regulate and control the transgene expression through the local area network WiFi or the 2G/3G/4G network in an ultra-remote way can express all meaningful proteins, in the embodiment, the gene loop control system for regulating and controlling the transgene expression in far-red light is exemplified by expression reporter gene secreted alkaline phosphatase (SEAP), Enhanced Green Fluorescent Protein (EGFP) and glucagon-like peptide (G L P-1) to regulate and control all meaningful proteins.

Second, seeding the cells good HEK-293T cells in growth state after digestion with 0.25% pancreatin were seeded in separate 24-well plates, 6 × 10 per well⁴Cells were plated and 500 μ L DMEM medium containing 10% FBS was added.

And thirdly, transfecting, namely, within 16 to 24 hours of inoculating the cells, uniformly mixing 0.1 mu g of pWS189, 0.1 mu g of pGY32, 0.1 mu g of pXY34, 0.1 mu g of pWS189, 0.1 mu g of pGY32, 0.1 mu g of pGY44, 0.1 mu g of pWS189, 0.1 mu g of pGY32 and 0.1 mu g of pWS212 with serum-free DMEM respectively, standing at room temperature for 15min, and uniformly dripping the mixture into a 24-well culture plate, wherein the total preparation volume of each well is 50 mu L, the mass ratio of the plasmid to the PEI is 1:3, and after 6 hours of transfection, 10% FBS-containing DMEM is replaced with 500 mu L for culture.

Fourthly, the smart phone sets the illumination intensity to illuminate through WiFi of the local area network or 2G/3G/4G network in an ultra-remote mode. After the liquid is changed for 14-18h, the illumination group is placed at the wavelength of 720nm and the set illumination intensity is 1mW/cm²L ED (reference material and method for connection) for 4h, and the dark group was cultured in the dark.

And fifthly, detecting the reporter gene at 24h, 48h and 72h respectively and determining the amount of SEAP, taking fluorescent pictures at 0h, 6h, 12h, 24h and 48h respectively, detecting the amount of luciferase by using a dual-luciferase reporter gene detection kit at 24h, 48h and 72h, and determining the expression amount of G L P-1 at different illumination time by using an E L ISA kit at 48h (the specific method refers to experimental materials and methods).

The results show that the gene loop control system for ultra-remote regulation and control of transgene expression by the smart phone of the invention through a local area network WiFi or a 2G/3G/4G network can well induce and express different proteins. Therefore, the gene loop control system for ultra-remote regulation and control of the transgenes by the smart phone through the local area network WiFi or the 2G/3G/4G network is suitable for expressing all meaningful proteins. The experimental data are shown in the figures 24, 25, 26 and 27 in the specification in detail.

Example 13 smartphone expression of two or more proteins of all interest simultaneously via local area network WiFi or 2G/3G/4G network ultra-remote transgenic Gene Loop control System

The gene loop control system for the smartphone to regulate the transgenic expression through the WiFi or 2G/3G/4G network over the long distance can simultaneously express two different proteins (connected by 2A), and in this embodiment, expression of enhanced green fluorescent protein-2A-Insulin (EGFP-2A-Insulin) is taken as an example to demonstrate that the gene loop control system for the smartphone to regulate the transgenic expression through the WiFi or 2G/3G/4G network over the long distance can simultaneously express two different proteins (connected by 2A). But are not intended to limit the scope of the present invention. The method comprises the following specific steps:

Second, seeding the cells, good HEK-293T cells in growth state were digested with 0.25% pancreatin and plated in separate 24-well plates, 6 × 10 per well⁴Cells were plated and 500 μ L DMEM medium containing 10% FBS was added.

And thirdly, transfecting, namely uniformly mixing 0.1 mu g of pWS189, 0.1 mu g of pGY32, 0.1 mu g of pWS213 and a PEI transfection reagent with serum-free DMEM within 16 to 24 hours after inoculating the cells, uniformly dripping the mixture into a 24-well culture plate after standing for 15min at room temperature, wherein the total preparation volume of each well is 50 mu L, the mass ratio of the plasmid to the PEI is 1:3, and culturing by replacing with 500 mu L of DMEM medium containing 10% of FBS after transfecting for 6 hours.

The fourth step, illumination (the same procedure as in example 11).

And fifthly, detecting the reporter gene, shooting fluorescent protein graphs at 48h under different illumination time, and measuring the expression quantity of the Insulin under different illumination time by using an E L ISA kit (the specific method refers to experimental materials and methods).

The result shows that the gene loop control system for ultra-remote regulation and control of transgene expression by the smart phone through the local area network WiFi or the 2G/3G/4G network can well express two different proteins (connected by 2A) at the same time. Therefore, the gene loop control system for ultra-remote regulation and control of the transgenes by the smart phone through the local area network WiFi or the 2G/3G/4G network can be used for simultaneously expressing a plurality of proteins. The experimental data are shown in the attached figures 28 and 29 of the specification in detail.

Example 14: microcapsule transplantation carrier for preparing gene loop control system engineered cells containing smartphone ultra-remote regulation and control transgenic expression through local area network WiFi or 2G/3G/4G network

In this embodiment, the preparation method of the transplantation carrier is described by taking the preparation of the microcapsule transplantation carrier containing the gene loop control system engineered cells for the smartphone to regulate the transgene expression through the WiFi of the local area network or the 2G/3G/4G network in an ultra-remote manner as an example, but the protection scope of the present invention is not limited. The method comprises the following specific steps:

Second, seeding the cells good HEK-293T cells in growth state after digestion with 0.25% trypsin in 10cm cell culture dishes of 4 × 10 cells each⁶Cells were plated and 10m L DMEM medium containing 10% FBS was added.

And thirdly, uniformly mixing 4 mu g of pWS189, 4 mu g of pGY32, 4 mu g of pXY34 and PEI transfection reagent with serum-free DMEM within 16-24 h of inoculated cells, standing at room temperature for 15min, and uniformly dripping into a 10-cm cell culture dish, wherein the total preparation volume of each dish is 2m L, the mass ratio of the plasmid to the PEI is 1:3, and after transfection for 6h, replacing with 10m L of DMEM medium containing 10% of FBS for culture.

Fourthly, preparing the microcapsule. After the liquid is changed for 14-18h, pancreatin digestion and cell collection by centrifugation are carried out. Microcapsules are prepared by a microcapsule preparation instrument (see the experimental method and materials for details of specific equipment and parameters).

The prepared microcapsule has a three-layer membrane system of sodium Alginate-Polylysine-sodium Alginate (Alginate-Polylysine-Alginate-membrane), and nutrient substances required by cell growth and small molecular target proteins secreted by engineered cells can freely pass through the membrane system. But cells and other macromolecular proteins cannot pass through the membrane system. Therefore, the cells encapsulated by the microcapsule can be transplanted into a mouse to grow normally. The experimental data are shown in figure 30 of the specification.

Example 15 Gene Loop control System engineered cells containing Smart Phone transgene expression regulated by far-Red light in microcapsules by WiFi or 2G/3G/4G network over-remote

This example was carried out according to the procedure of example 14, and the microcapsules prepared were cultured in 6-well plates while usingAn infrared therapeutic device with illumination intensity of 5mW/cm²The illumination time is 2h, the microcapsule containing the gene loop control system engineered cells of the smartphone for ultra-remote regulation and control of transgenic expression through a local area network WiFi or a 2G/3G/4G network is induced and expressed, and the reporter genes are detected at 24h, 48h and 72h respectively. The gene loop control system engineering cell containing the smartphone super-remote regulation and control transgene expression through a local area network WiFi or a 2G/3G/4G network can be well regulated and controlled by far-red light to express in the microcapsule. The experimental data are shown in figure 31 of the specification.

Example 16: preparation of hollow fiber membrane transplantation vessel transplantation carrier containing gene loop control system engineered cells for ultra-remote regulation and control of transgene expression of smart phone through local area network WiFi or 2G/3G/4G network

In this embodiment, the preparation method of the transplantation carrier is described by taking the preparation of the hollow fiber membrane transplantation vessel transplantation carrier containing the gene loop control system engineered cells for the smartphone to regulate the transgene expression through the WiFi of the local area network or the 2G/3G/4G network in an ultra-remote manner as an example, but the protection scope of the invention is not limited. The method comprises the following specific steps:

Second, seeding the cells good HEK-293T cells in growth state were seeded in 24-well cell culture plates with 0.25% trypsin, 6 × 10 per well⁴Cells were plated and 500. mu. L DMEM medium containing 10% FBS was added.

And thirdly, uniformly mixing 0.1 mu g of pWS189, 0.1 mu g of pGY32, 0.1 mu g of pXY34 and PEI transfection reagent with serum-free DMEM within 16-24 h of inoculated cells, standing at room temperature for 15min, and uniformly dripping into a 24-well cell culture plate, wherein the total preparation volume of each well is 50 mu L, the mass ratio of the plasmid to the PEI is 1:3, and after 6h of transfection, changing into 10m L DMEM medium containing 10% of FBS for culture.

And fourthly, preparing the hollow fiber membrane transplanting tube. After the liquid is changed for 14-18h, pancreatin digestion and cell collection by centrifugation are carried out. The hollow fiber membrane transplanting tube is manufactured according to the manufacturing method (the detailed operation process is shown in the experimental method and materials).

The prepared hollow fiber membrane transplanting tube can freely pass through the membrane system by nutrient substances required by cell growth and small-molecule target proteins secreted by the engineered cells. But cells and other macromolecular proteins cannot pass through the membrane system. Therefore, the cells wrapped by the hollow fiber membrane transplantation tube can be transplanted into a mouse body to grow normally. The experimental data are shown in figure 32 of the specification.

Example 17 Gene Loop control System engineered cells containing Smart Phone transgene expression regulated by far-Red light in hollow fiber Membrane transplantation tubes by WiFi or 2G/3G/4G network over-remote control

In this example, the prepared hollow fiber membrane graft was cultured in a 6-well plate according to the procedure of example 16, and simultaneously irradiated with light from an infrared ray therapeutic apparatus remotely controlled by a smart phone via a local area network WiFi or 2G/3G/4G network at an intensity of 5mW/cm²The illumination time is 2h, the hollow fiber membrane transplantation tube for inducing and expressing the gene loop control system engineering cells containing far-red light regulated transgenic expression is used for detecting the reporter genes at 24h, 48h and 72h respectively. The gene loop control system engineering cell containing far-red light regulated transgenic expression can be well regulated and expressed by far-red light in a hollow fiber membrane transplanting tube. The experimental data are shown in figure 33 of the specification.

Example 18, the smartphone regulates the expression of the transgene under far-red light regulation in the mouse body by the gene loop control system of the local area network WiFi or 2G/3G/4G network ultra-remote regulation transgene expression

The method for the smartphone to regulate the expression of the transgenic expression by far-red light in the mouse body through the gene loop control system for the smartphone to regulate the expression by the local area network WiFi or the 2G/3G/4G network is various, and in the embodiment, the case of transplanting the hollow fiber membrane transplanting tube containing the engineering cells of the gene loop control system for the smartphone to regulate the expression by the far-red light through the local area network WiFi or the 2G/3G/4G network is taken as an example, so that the condition that the gene loop control system for the smartphone to regulate the expression by the far-red light in the mouse body is proved. But are not intended to limit the scope of the present invention. The method for transplanting the transplantation vector into the mouse body is provided in various ways, and in the embodiment, the back subcutaneous transplantation is taken as an example, so that the method for transplanting the smartphone into the mouse body through the gene loop control system for ultra-remote regulation and control of the transgene expression through the local area network WiFi or the 2G/3G/4G network is described. The method comprises the following specific steps:

in the first step, a hollow fiber membrane graft tube was prepared (see example 16 for details).

And secondly, implanting the hollow fiber membrane transplanting tube into the back of the mouse (the specific method refers to experimental materials and methods).

And thirdly, illuminating. Ultra-remote control infrared therapeutic device (10 mW/cm) through local area network WiFi or 2G/3G/4G network by using smart phone²) The cells were irradiated for 2h, 8h, 26h and 32h after transplantation.

And fourthly, detecting the reporter gene. The amount of the reporter gene was measured 24h and 48h after transplantation by orbital hemospasia (see experimental materials and methods for details). The detailed experimental data are shown in figures 34 and 35.

Example 19 Smart Phone Gene Loop control System for ultra remote control of transgene expression Via local area network WiFi or 2G/3G/4G network

Second, seeding the good HEK-293T cells in growth state after digestion with 0.25% pancreatin in 6 well plates of 6 × 104 cells per well and adding 500. mu. L DMEM medium containing 10% FBS.

And thirdly, transfecting, namely uniformly mixing 0.1 mu g of photoreceptor pWS189, 0.1 mu g of processor pGY32, 0.1 mu g of effector pWS213 and PEI transfection reagent with serum-free DMEM within 16 to 24 hours of inoculating the cells, standing at room temperature for 15min, and uniformly dripping into a 24-well culture plate, wherein the total preparation volume of each well is 50 mu L, the mass ratio of the plasmid to the PEI is 1:3, and culturing is carried out by replacing 500 mu L with DMEM medium containing 10% of FBS after 6 hours of transfection.

And fourthly, after the liquid is changed by illumination for 14-18 hours, dividing the six plates into 6 groups, placing the 6 groups under L ED with the wavelength of 720nm and the illumination intensity of 1mW/cm2, and ultra-remotely setting the illumination time to be 0, 0.1, 0.25, 0.5, 1 and 2 hours (the plates are always stored in the dark when the illumination time is 0 hour) by the smart phone through a local area network WiFi or a 2G/3G/4G network respectively.

And fifthly, detecting the expression of the insulin, photographing the expression condition of the green fluorescent protein of each group after culturing for 48 hours, and measuring the expression quantity of the insulin of each group by using an insulin E L ISA kit.

Experiments show that the gene loop control system for ultra-remote regulation and control of transgenic expression of the smart phone through a local area network WiFi or a 2G/3G/4G network can accurately regulate and control insulin expression in vitro, and the expression level of the gene loop control system is in positive correlation with the illumination intensity. The experimental data are shown in the attached figures 28 and 29 of the specification in detail.

Example 20 treatment of type I diabetes mellitus by Smart Phone precise control of insulin expression in type I diabetes model mice by local area network WiFi or 2G/3G/4G network ultra-remote Gene expression-mediated Gene Loop control System

In the first step, we adopt a multiple low dose streptozotocin (Streptozocin, STZ, purchased from Sigma S0130, 18883-6, 6-4) administration method to induce the model.C 57B L/6J mice 25 (from Chinese academy of sciences), 8 weeks old, male, were injected with sodium citrate buffer solution dissolved with STZ (dose 40-50mg/kg) by intraperitoneal (fasting 12-16h before injection) injection for 5 consecutive days, STZ was dissolved in fresh citric acid buffer solution (0.1 mol/L, pH 4.5) during injection, prepared on ice, and the injection was completed within 30min after light-shielding and dissolution.

The second step of the preparation of the gene loop control system engineered cell for the smartphone to super-remotely modulate gene expression via the local area network WiFi or 2G/3G/4G network and the third step of the preparation of the transplantation tube containing the gene loop control system engineered cell for the smartphone to super-remotely modulate gene expression via the local area network WiFi or 2G/3G/4G network refer to example 16.

The fourth step is that the smartphone is transplanted to the back of the mouse through a transplantation tube of the gene loop control system engineering cells of the local area network WiFi or 2G/3G/4G network ultra-remote transfer gene expression, and the fifth step is illumination, which specifically refers to the embodiment 18.

Sixthly, determining the fasting blood glucose value of the type I diabetic mouse. After 8 hours of transplantation, the mice were fasted (fed with water) for 16 hours, and then blood was taken from the tail, and the fasting blood glucose level was measured. Experimental data show that the expressed insulin has good blood sugar reducing effect. The experimental data are shown in figure 36 of the specification.

The seventh step, carrying on the sugar tolerance experiment, after transplanting 24h, carrying on the sugar tolerance experiment, first, prepare fresh glucose aqueous solution (125mg/ml)10m L, and then according to each mouse's weight (1.25g/kg) calculate the glucose aqueous solution injection volume (intraperitoneal injection volume V is 10 × mouse's weight (g), unit: mu L) then each mouse (starvation 16 hours before measurement) 0 point blood sugar, intraperitoneal injection of the above-mentioned glucose solution, finally, each

mouse

30, 60, 90, 120min blood sugar value, the experiment shows, the diabetes mouse's sugar tolerance has very good improvement, the experimental data is detailed in the description of figure 37.

Example 21 Smart Phone Gene Loop control System for ultra remote transfer of Gene expression Via local area network WiFi or 2G/3G/4G network expression in vitro precise control of glucagon (G L P-1)

In the second step, cells are seeded. Good HEK-293T cells in growth state were seeded in 6 different 24-well plates with 6x10 per well after 0.25% pancreatin⁴Cells were plated and 500 μ L DMEM medium containing 10% FBS was added.

And thirdly, transfecting, namely uniformly mixing 0.1 mu g of photoreceptor pWS189, 0.1 mu g of processor pGY32, 0.1 mu g of effector pWS212 and PEI transfection reagent with serum-free DMEM within 16 to 24 hours of inoculating the cells, standing at room temperature for 15min, and uniformly dripping into a 24-well culture plate, wherein the total preparation volume of each well is 50 mu L, the mass ratio of the plasmid to the PEI is 1:3, and culturing is carried out by replacing 500 mu L with DMEM medium containing 10% of FBS after 6 hours of transfection.

And fifthly, measuring the amount of G L P1-Fc expressed in vitro by the gene loop control system for the smartphone to regulate the transgenic expression through WiFi of a local area network or 2G/3G/4G network, and measuring the expression amount of G L P1-Fc of each group by using an Active G L P-1E L ISA kit after culturing for 48 hours.

Experiments show that the gene loop control system for ultra-remote regulation and control of the transgenic expression of the smart phone through a local area network WiFi or a 2G/3G/4G network can accurately regulate and control the expression of the glucagon in vitro, and the expression level of the gene loop control system is in positive correlation with the illumination intensity. The experimental data are shown in figure 27 of the specification.

Example 22, Smart Phone precise control of G L P-1 expression in type I diabetic mice by local area network WiFi or 2G/3G/4G network ultra-remote control transgenic expression Gene Loop control System

In the second step, type II diabetes model mice db/db mice 20 (from Chinese academy of sciences), 8 weeks old, female, divided into four groups. The groups are respectively a non-transplanted non-illumination group, a non-transplanted illumination group, a transplanted non-illumination group and a transplanted illumination group.

The third step is the far-red light engineering cell preparation process and the fourth step is the transplantation tube preparation process of the gene loop control system engineering cell containing the smartphone for ultra-remote regulation and control of transgene expression through the local area network WiFi or 2G/3G/4G network, which is specifically referred to the example 16.

The fourth step is that the smartphone is transplanted to the back of the mouse through a transplantation tube of the gene loop control system engineering cells of the local area network WiFi or 2G/3G/4G network ultra-remote regulation and control transgene expression, and the fifth step is illumination, which specifically refers to the embodiment 18.

Sixthly, determining the fasting blood glucose value of the type I diabetic mouse. After 8 hours of transplantation, the mice were fasted (fed with water) for 16 hours, and then blood was taken from the tail, and the fasting blood glucose level was measured. The experimental data show that the expressed glucagon has good blood sugar reducing effect. The experimental data are shown in the specification and figure 38.

mouse

30, 60, 90, 120min blood sugar value, the experiment shows, the diabetes mouse's sugar tolerance has very good improvement, the experimental data is detailed in the instruction figure 39.

The eighth step, insulin resistance experiment is carried out, the insulin resistance experiment is carried out 24h after transplantation, firstly, fresh insulin solution (0.1U/ml) is prepared, then the injection volume of insulin is calculated according to the weight (1.1U/kg) of each mouse (the intraperitoneal injection volume V is 1.1 × mouse weight (g), unit is mu L), then 0-point blood sugar of each mouse is measured (hunger is 4 hours before measurement), the insulin solution is injected in an abdominal cavity, and finally, the blood sugar value of each mouse is measured for 30, 60, 90 and 120 min.

And ninthly, determining the expression of G L P-1 in the II type diabetic mouse by the gene loop control system for the smartphone to regulate the transgenic expression through the local area network WiFi or the 2G/3G/4G network, taking blood through the inner canthus 48h after transplantation, and detecting the content of G L P-1 (activivie) in serum by using a G L P-1(7-36) activiE L ISA kit.

TABLE 1 plasmid construction Table

The primers, cleavage sites and the seamless cloned fragments are underlined.

Abbreviations:

P_hCMVhuman cytomegalovirus promoter; p_SV40Simian vacuolar virus promoter; p_hCMVmin3GCytomegalovirus CMV minimal promoter mutant; p_CAGThe method comprises the steps of artificially constructing a cytomegalovirus early enhancer and chicken β -actin promoter combination promoter, EGFP, enhanced green fluorescent protein, SEAP, human exocrine alkaline phosphatase, G L P-1, glucagon-like peptide 1, VP64, herpes virus transcription activation domain tetramer, BldD, a far-red light processor, 2A, an independent oligopeptide of which the carbon end contains a D (V/I) EXNPGP domain, pA, a polyadenylation signal, Bphs, artificially synthesized bacterial photosensitive diguanylate cyclase, YhjH, c-di-GMP degrading enzyme, Bpho, pigment synthetase, PCR and polymerase chain reaction.

In the sequence table:

sequence 1: artificially synthesized bacterial photosensitive diguanylate cyclase (Bphs) nucleotide sequence

Sequence 2: photopigment synthetase (Bpho) nucleotide sequences

And (3) sequence: self-cleaving 2A peptide nucleotide sequences

And (3) sequence 4: nucleotide sequence of c-di-GMP degrading enzyme (YhjH)

And (5) sequence: nucleotide sequence of simian vacuolating virus promoter (SV40)

Sequence 6 human EF1 α (hEF1 α) promoter nucleotide sequence

And (3) sequence 7: mouse-derived PGK (mPGK) promoter nucleotide sequence

Sequence 8, artificially constructing nucleotide sequence of cytomegalovirus early enhancer and chicken β -actin promoter combined promoter (CAG)

Sequence 9: cytomegalovirus promoter CMV nucleotide sequence

Sequence 10: far-red light processor (BldD) nucleotide sequence

Sequence 11: herpes simplex virus particle protein transcription activation domain (VP16) nucleotide sequence

Sequence 12: nucleic acid sequence of core sequence tetramer (VP64) of transcription activation domain of herpes simplex virus particle protein

Sequence 13: nucleotide sequence of NF-Kv B p65 subunit transcription activation domain

Sequence 14: nucleotide sequence of transcription activation domain of heat shock transcription factor (HSF1)

Sequence 15: nucleotide sequence of TATA Box

Sequence 16: cytomegalovirus minimal promoter CMV_minNucleotide sequence of (A)

Sequence 17: cytomegalovirus minimal promoter CMV_minMutant CMV_min3GNucleotide sequence of (A)

Sequence 18: BldD binding site (bldM) nucleotide sequence

Sequence 19: BldD binding site (whiG) nucleotide sequence

Sequence 20: far-red light effect device promoter P_FRL0(1×bldM-h_CMV_min) Nucleotide sequence

Sequence 21: far-red light effect device promoter P_FRL1(2×bldM-h_CMV_min) Nucleotide sequence

Sequence 22: far-red light effectPromoter P of reactor_FRL2(3×bldM-h_CMV_min) Nucleotide sequence

Sequence 23: far-red light effect device promoter P_FRL3(4×bldM-h_CMV_min) Nucleotide sequence

Sequence 24: far-red light effect device promoter P_FRL4(5×bldM-h_CMV_min) Nucleotide sequence

Sequence 25: far-red light effect device promoter P_FRL5(1×whiG-h_CMV_min) Nucleotide sequence

Sequence 26: far-red light effect device promoter P_FRL6(2×whiG-h_CMV_min) Nucleotide sequence

Sequence 27: far-red light effect device promoter P_FRL7(3×whiG-h_CMV_min) Nucleotide sequence

Sequence 28: far-red light effect device promoter P_FRL8(4×whiG-h_CMV_min) Nucleotide sequence

Sequence 29: far-red light effect device promoter P_FRL9(5×whiG-h_CMV_min) Nucleotide sequence

Sequence 30: far-red light effect device promoter P_FRL10(SV40PolyA-1×whiG-h_CMV_min) Nucleotide sequence

Sequence 31: far-red light effect device promoter P_FRL11(SV40PolyA-2×whiG-h_CMV_min) Nucleotide sequence

Sequence 32: far-red light effect device promoter P_FRL12(SV40PolyA-3×whiG-h_CMV_min) Nucleotide sequence

Sequence 33: far-red light effect device promoter P_FRL13(SV40PolyA-4×whiG-h_CMV_min) Nucleotide sequence

Sequence 34: far-red light effect device promoter P_FRL14(SV40PolyA-5×whiG-h_CMV_min) Nucleotide sequence

Sequence 35: far-red light effect device promoter P_FRL15(1×whiG-h_CMV_min3G) Nucleotide sequence

Sequence 36: far-red light effect device promoter P_FRL16(2×whiG-h_CMV_min3G) Nucleotide sequence

Sequence 37: far-red light effect device promoter P_FRL17(3×whiG-h_CMV_min3G) Nucleotide sequence

Sequence 38: far-red light effect device promoter P_FRL18(4×whiG-h_CMV_min3G) Nucleotide sequence

Sequence 39: far-red light effect device promoter P_FRL19(4×whiG-h_CMV_min3G) Nucleotide sequence

Sequence 40: nucleotide sequence of secretory alkaline phosphatase (SEAP) of sequence to be transcribed expressed by far-red light effector

Sequence 41: nucleotide sequence of green fluorescent protein (EGFP) to be transcribed expressed by far-red light effector

42 nucleotide sequence of luciferase (L uciferase) to be transcribed expressed by far-red light effector

Sequence 43 nucleotide sequence of glucagon-like peptide-1 (G L P-1-Fc) to be transcribed expressed by far-red light effector

Sequence 44: nucleotide sequence of nucleotide sequence (EGFP-2A-Insulin) of sequence to be transcribed expressed by far-red light effector

Sequence 45: artificially synthesized bacterial photosensitive diguanylate cyclase (Bphs) amino acid sequence

Sequence 46: photopigment synthetase (Bpho) amino acid sequence

Sequence 47: self-cleaving 2A peptide nucleotide sequences

Sequence 48: c-di-GMP degrading enzyme (YhjH) amino acid sequence

Sequence 49: far-red light processor (BldD) amino acid sequence

Sequence 50: herpes simplex virus particle protein transcription activation domain (VP16) amino acid sequence

Sequence 51: nucleic acid sequence of core sequence tetramer (VP64) of transcription activation domain of herpes simplex virus particle protein

Sequence 52: NF-Kv B p65 subunit transcription activation domain amino acid sequence

Sequence 53: heat shock transcription factor transcriptional activation domain (HSF1) amino acid sequence

Sequence 54 amino acid sequence of a Nuclear localization Signal (N L S)

Sequence 55 amino acid sequence of connecting functional peptide (L inker)

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Claims

1. A gene loop remote regulation system, comprising: a far-red light gene loop expression control system;

the control device is used for sending a remote control instruction; and

a far-red light source device;

wherein,

the far-red light gene loop expression control system comprises:

a photoreceptor that senses a far-red light source;

a processor for processing the signals transmitted by the photoreceptors; and

an effector responsive to signals communicated by said processor;

the control device remotely regulates and controls the far-red light source device by sending a control instruction, and regulates and controls the far-red light gene loop expression control system by the far-red light sent by the far-red light source device to realize the regulation and control of transgenic expression;

the photoreceptors include photosensitive diguanylate cyclase BphS, which converts GTP to c-di-GMP under far-red light conditions;

the processor comprises polypeptides serving as a DNA binding domain and a c-di-GMP binding domain, a polypeptide serving as a nuclear localization signal N L S, a polypeptide serving as a connecting domain and a polypeptide serving as a transcription regulation domain, wherein the polypeptides serving as the DNA binding domain and the c-di-GMP binding domain are proteins capable of binding with a specific DNA sequence after being bound with the c-di-GMP, and comprise BldD proteins, and the amino acid sequences of the BldD proteins are selected from sequences 49;

the effector comprises P_FRL-a reporter; the effector comprises a promoter sequence and a nucleic acid sequence of a protein to be transcribed; wherein the promoter sequence comprises a processor BldD protein-binding DNA sequence and a weak promoter that promotes gene expression, the processor BldD protein-binding DNA sequence being a DNA sequence specifically recognized and bound by a polypeptide of a DNA-binding domain and a c-di-GMP-binding domain, being a partial sequence of a bldM and a whiG promoter region, wherein the nucleotide sequence of bldM is selected from the group consisting of sequence 18 and the nucleotide sequence of whiG is selected from the group consisting of sequence 19.

2. The remote gene loop regulation system of claim 1 wherein the photoreceptor photosensitive diguanylate cyclase BphS is prepared by fusing amino acids 1-511 of BphG protein with amino acid 175-343 of Slr1143 protein and mutating arginine 587 of fusion protein to alanine R587A in the far-red gene loop expression control system.

3. The gene loop remote regulation system of claim 1, wherein the photoreceptor is constructed in a form of a far-red light gene loop expression control system comprising:

a) artificially synthesized bacterial photosensitive diguanylate cyclase BphS coding gene BphS;

b) the artificially synthesized bacterial photosensitive diguanylate cyclase BphS coding gene is connected with the c-di-GMP degrading enzyme YhjH coding gene through a 2A sequence to form BphS-2A-YhjH;

c) the artificially synthesized bacterial photosensitive diguanylate cyclase BphS coding gene is connected with a photosensitive pigment synthetase BphO coding gene through a 2A sequence, namely BphS-2A-BphO;

d) the artificially synthesized bacterial photosensitive diguanylate cyclase BphS coding gene is connected with a photosensitive pigment synthetase BphO coding gene through a 2A sequence, and is connected with a c-di-GMP degrading enzyme YhjH coding gene through a 2A sequence to form BphS-2A-BphO-2A-YhjH;

wherein the 2A sequence can be replaced by an internal ribosome entry site sequence IRES;

the photosensitive pigment synthetase BphO has the function of synthesizing photosensitive pigment biliverdin;

the degrading enzyme YhjH of the c-di-GMP has the function of degrading the c-di-GMP into pGpGpGpG.

4. The remote gene loop regulation system of claim 3 wherein the amino acid sequence of BphS, BphO, YhjH in the far-red gene loop expression control system is selected from the group consisting of sequences 45, 46, and 48.

5. The remote gene loop control system as claimed in claim 1, wherein the promoter for expressing photoreceptors in the far-red gene loop expression control system comprises a) SV40, b) CMV, c) hEF1 α, d) mPGK, and e) CAG.

6. The gene loop remote control system according to claim 1, wherein the amino acid sequence of the polypeptide as the nuclear localization signal N L S in the far-red light gene loop expression control system is selected from the group consisting of sequence 54.

7. The gene loop remote regulation system of claim 1, wherein the amino acid sequence of the polypeptide as the linking domain in the far-red gene loop expression control system is selected from the group consisting of SEQ ID NO 55.

8. The remote gene loop regulation system of claim 1 wherein the amino acid sequence of the polypeptide as the transcriptional regulatory domain in the far-red gene loop expression control system is selected from the group consisting of sequences 50, 51, 52, and 53.

9. The remote gene loop regulation system of claim 1, wherein the polypeptide that is a transcriptional regulatory domain is placed N-terminal or C-terminal to the DNA binding domain and the C-di-GMP binding domain of the polypeptide BldD in the far-red gene loop expression control system.

10. The gene loop remote control system according to claim 1, wherein the polypeptides of different functional domains in the processor are linked directly or via a linker peptide in the far-red gene loop expression control system.

11. The gene loop remote control system according to claim 1, wherein the sequences of the bldM and whiG promoter regions in the far-red gene loop expression control system are 1 to 10 copies.

12. The remote gene loop regulation system of claim 1 wherein the weak promoter that promotes gene expression in the far-red light gene loop expression control system comprises tatabex, cytomegalovirus CMV minimal promoter and its mutant CMVmin 3G.

13. The remote gene loop control system of claim 1, wherein the nucleic acid sequence of said promoter sequence in said far-red gene loop expression control system is selected from the group consisting of sequences 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39.

14. The gene loop remote control system as claimed in claim 1, wherein in the far-red light gene loop expression control system, the protein to be transcribed and encoded and expressed by the nucleic acid sequence comprises a protein serving as a reporter gene and/or a nucleic acid sequence serving as a drug protein or a small peptide for treating diseases; wherein, the protein as the reporter gene comprises nucleic acid sequences of secreted alkaline phosphatase, enhanced green fluorescent protein and luciferase; nucleic acid sequences of insulin, glucagon-like peptides are included as pharmaceutical proteins or small peptides for the treatment of disease.

15. The remote gene loop regulation system of claim 14 wherein the far-red gene loop expression control system is one in which the protein can simultaneously express multiple proteins via 2A sequences on a single expression vector, including SEAP-2A-Insulin, EGFP-SEAP-2A-Insulin; wherein the 2A sequence may be replaced by an internal ribosome entry site sequence IRES.

16. The gene loop remote control system according to claim 1, wherein the control device sends remote control commands to control different operating states of the far-red light source device through a local area network WiFi or a 2G/3G/4G network.

17. The gene loop remote control system according to claim 16, wherein the different operation states include turning on or off of the far-red light source, an illumination intensity, an illumination time or an illumination method that can be adjusted as desired.

18. The remote gene loop regulation system of claim 17 wherein the illumination intensity is from 0 to 5mW/cm²(ii) a The irradiation time is 0-72 h; the irradiation method comprises pulse irradiation, continuous irradiation, direct irradiation or irradiation for spatially controlling the gene expression level of cells at different positions by using a projection card with hollow depiction.

19. The gene loop remote control system according to claim 1, wherein the control device comprises a smartphone App and/or a smart remote controller.

20. The gene loop remote control system of claim 19, wherein the intelligent remote controller comprises a single-output and/or multi-output intelligent remote controller.

21. The remote gene loop regulating system as claimed in claim 1, wherein the far-red light source device is a light source device capable of generating far-red light with a wavelength of 600-900 nm.

22. A kit comprising a gene loop remote control system according to any one of claims 1 to 21.

23. A method of regulating gene expression in a host cell using the remote gene loop regulation system of any one of claims 1 to 21, comprising the steps of:

a) constructing the far-red light gene loop expression control system of any one of claims 1-21 in a eukaryotic plasmid expression vector;

b) transfected into said host cell;

c) and inducing and regulating the host cell by remotely regulating and controlling far-red light irradiation conditions to realize the expression of the effector coding gene.

24. A method for regulating expression of a transgene in a transplantation vector using the remote gene loop regulation system of any one of claims 1 to 21, comprising the steps of:

a) preparing a eukaryotic plasmid expression vector containing the far-red light gene loop expression control system of any one of claims 1-21;

b) preparing an engineered cell comprising the far-red light gene loop expression control system of any one of claims 1-21;

c) preparing an engineered cell transplantation vector comprising the far-red light gene loop expression control system of any one of claims 1-21;

d) the far-red light source is remotely regulated to perform induced expression on the engineered cell transplantation vector.