CN107174655B

CN107174655B - Application of far-red light gene loop expression control system in treatment of diabetes

Info

Publication number: CN107174655B
Application number: CN201610136156.1A
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: 2020-10-30
Anticipated expiration: 2036-03-10
Also published as: CN107174655A

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

Application of far-red light gene loop expression control system in treatment of diabetes

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.2014Aug; 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 induction regulated "caging cage" technology [ Keyes, WM. et al, Trends biotechnol.2003feb; 21(2):53-5 ], far infrared light control heat shock effect induction regulation gene expression system [ Kamei, Y, et al, Nat methods.2009Jan; 79-81 ], a radio-controlled temperature-induced regulation and control gene expression system [ Stanley, SA. et al, science.2012May4; 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. Therefore, it is a constant pursuit of biologists to use light as an inducer to regulate gene expression and thus various metabolic activities of a living body. In our previous work, attempts were made to design and synthesize gene loops for blue light-regulated transgene expression using synthetic biology methods, and blue light was used as a switch to deactivate and regulate the expressed gene [ Ye, H. et al, Science,2011.332(6037): p.1565-8 ]. The Lighton blue light-regulated gene expression switch was reported by the subject group of professor Yanghi university of eastern science and industry [ Wang, X., et al, Nat Methods,2012.9(3): p.266-9 ]. However, the blue light has great limitation on regulating and controlling gene expression in vivo, namely the blue light has low transdermal efficiency, is not easy to pass through skin or abdominal cavity to deactivate target genes, and greatly limits the deep development and clinical application of light regulation and control systems. A subject group of the teaching of Wilfire Weber of Freuberg university in Germany develops a transgenic expression control switch for red light regulation [ Muller K. et al, Nucleic Acids Res,2013,41(7): e77.nat Protoc,2014,9(3):622-32], but the red light control system does not show good gene expression intensity, and no experimental data show that the red light control switch has the effect of regulating gene expression in an animal model mouse. More importantly, the red light control switch needs to add an additional phytochrome phycocyanin (phytoyanobilin) to activate gene expression. Moreover, the pigment is very inconvenient to source, cannot be obtained commercially, needs to be artificially synthesized in a laboratory, has complex synthesis steps, is unstable in synthesized products and has extremely low yield. These disadvantages greatly limit the further applications of the red control system.

Diabetes is one of the most important chronic epidemic diseases that currently threaten human health worldwide. Diabetes is a chronic lifelong disease affecting human health, mainly including blood sugar rise after the disease occurs, and with the aggravation of the disease, the diabetes can involve each important organ and whole body tissue of the body, causing various complications, which can not be cured completely, but can be controlled only. In recent decades, the number of diabetics worldwide has increased at an alarming rate. A multinational union study showed that in 1980-2008, the number of diabetes mellitus worldwide increased from 1.53 to 3.47 million. The diabetes epidemiological survey data of 14 provinces and cities across the country of China is organized by the diabetes society of the Chinese medical society, and the number of people suffering from diabetes of adults in China reaches 9240 thousands, and the incidence rate is 9.7 percent. With the improvement of living standard and life style, the proportion of obesity and overweight is increased year by year, so that the 'back-up military' of diabetes is continuous. Type 1 diabetes is a chronic autoimmune disease, because the pancreatic beta cells secreting insulin in the pancreas suffer from the targeted destruction of autoreactive T cells, resulting in reduced insulin secretion, thereby causing the body to lose the ability to regulate blood sugar, further affecting the body metabolism, and patients usually need to receive insulin therapy for their lifetime. The hallmark of type 2 diabetes is abnormal glucose balance, which is also caused by insulin deficiency resulting from the inactivation of the beta cell function of the pancreatic islets or insulin resistance, i.e. the decreased or lost response of the target organs, tissues for insulin action to its biological effects, and the body needs to secrete more insulin to compensate for the deficiency. This often leads to other serious, life-threatening secondary complications such as cardiovascular disease, renal failure and retinopathy. However, diabetes cannot be treated radically so far and needs to be administered for life. The diabetic needs to take hypoglycemic drugs or inject insulin every day to maintain stable blood sugar, and especially the insulin cannot be injected to controllably release the insulin, so that the risk of hypoglycemia is easily caused.

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.

Wherein the promoter expressing photoreceptors comprises: a) SV 40; b) a CMV; c) hEF1 α; d) mPGK; e) and (4) 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 NLS, a polypeptide as a linking domain, and a polypeptide as a transcription 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 serving as the nuclear localization signal NLS can be in various forms of 1-3 copies, and the amino acid sequence of the polypeptide is selected from the sequence 54.

Wherein, the polypeptide as a connecting functional domain (Linker) can be in various forms with the length of 0-30 amino acids, and the amino acid sequence 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.

Wherein, the protein coded and expressed by the nucleic acid sequence to be transcribed can be any meaningful protein, including a protein used as a reporter gene and/or a nucleic acid sequence used 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 (Luciferase); the nucleic acid sequence of the pharmaceutical protein or small peptide for treating diseases comprises Insulin (Insulin) and glucagon-like peptide (GLP-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.

Second, it carries a processor plasmid that processes the signals transmitted by the photoreceptors. The processor consists of polypeptide BldD as a DNA binding domain and a c-di-GMP binding domain, polypeptide NLS as a nuclear entry signal domain, polypeptide Linker as a connecting domain, polypeptide P65, VP64, VP16, HSF1 and the like as a transcription activation domain. Wherein the BldD protein can be derived from Streptomyces venezuelae [ tschwri n. et al, Cell,2014,158(5):1136-1147 ], or can be synthesized by artificial chemistry, the gene encoding BldD protein of the present invention is synthesized by artificial chemistry, VP16 is herpes simplex virion protein transcriptional activation domain, p65 is NF-bk 65 subunit transcriptional activation domain, HSF1 is heat shock transcription factor transcriptional activation domain [ Konermann s. et al, nature.2015jan 29; 517(7536):583-8.].

NLS can be 1-3 copies and the like, is directly connected to the N end of BldD, P65, VP64, HSF1 and the like serving as transcription activation domains can be respectively connected to the N end or the C end of BldD in a two-by-two combination or three-by-three series connection mode, and the components are connected by polypeptide Linker (0-30 amino acids are different) serving as a connection function. The specific construction can have the following forms: NLS-BldD-VP16, VP64-Linker-NLS-BldD, VP64-Linker-NLS-BldD-Linker-VP64, NLS-BldD-Linker-VP64, P65-Linker-VP64-Linker-NLS-BldD, P65-HSF1-Linker-NLS-BldD, NLS-BldD-Linker-P65-HSF1, and VP64-NLS-BldD-Linker-P65-HSF1, and the like.

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 (SV40PolyA), bovine auxin gene PolyA (BGHpolyA), 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 protein of interest, and can be reporter gene secreted alkaline phosphatase (SEAP), enhanced green fluorescent protein (EGFP, EYFP), Luciferase (Luciferase) and the like; functional proteins for treating diseases, such as Insulin (Insulin), glucagon-like peptide (GLP-1), etc., can also be used. Different desired proteins can also be linked 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, etc.

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.

Wherein, the far-red light source device can generate a light source of 600-900nm wavelength far-red light, such as 600-900nm LED, infrared therapeutic device, laser lamp, etc. In a specific embodiment, the 720nm far-red light LED is adopted in the in-vitro experiment, so that the ultra-remote control of the smart phone, the regulation and control of gene expression in different illumination time, the regulation and control of gene expression in different illumination intensity and the like are realized. The in vivo experiment is realized by controlling red light physiotherapy instrument or 720nmLED in vivo transplantation, and realizing ultra-remote control of the smart phone, regulation and control gene expression in different illumination time, regulation and control gene expression in different illumination intensity and the like through radio power supply. The intelligent control system for controlling the far-red light source by the intelligent mobile phone through the local area network WiFi or the 2G/3G/4G network has three ways to realize the control of the far-red light source, which is that: the mobile phone and the far-red light source are accessed to the same local area network WiFi, and the mobile phone controls the far-red light source through the accessed local area network WiFi; or the mobile phone controls the far-red light source through a 2G/3G/4G network in an ultra-remote mode and controls the far-red light source through a manual key in an emergency.

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_FRLThe reporter-encoding gene (e.g., SEAP,EGFP, Luciferase, Insulin, GLP-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 smart phone regulates and controls certain far-red light irradiation conditions including light source selection and irradiation intensity, time and irradiation method selection through a local area network WiFi or a 2G/3G/4G network in an ultra-remote mode. Wherein the light source comprises an LED lamp, a far-red light physiotherapy instrument and the like; irradiation intensity of 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 and expressing the engineering cell transplantation carrier by remotely regulating and controlling a far-red light source to ensure that an effector P in the transplantation carrier_FRLExpression of reporter-encoding genes (e.g. SEAP, EGFP, Luciferase, Insulin, GLP-1, etc.). e) And detecting the expression condition of the target gene 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-expression of reporter encoding genes (e.g. SEAP, EGFP, Luciferase, Insulin, GLP-1, etc.);

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 smart phone regulates and controls certain far-infrared light irradiation conditions including light source selection and irradiation intensity, time and irradiation method selection through a local area network WiFi or a 2G/3G/4G network in an ultra-remote mode. Wherein the light source comprises an LED lamp, a far-red light physiotherapy instrument and the like; irradiation intensity of 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 the far-red light gene loop expression control system or the gene loopA transplantation carrier of a remote 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-expression of reporter encoding genes (e.g. SEAP, EGFP, Luciferase, Insulin, GLP-1, etc.); f) and detecting the expression condition of the target gene 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-expression of reporter encoding genes (e.g. SEAP, EGFP, Luciferase, Insulin, GLP-1, etc.);

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.;

methods for modulating gene expression in mice include selection of and control of light sources. The light source comprises an LED lamp, a physiotherapy instrument and a laser lamp. The illumination method comprises the selection of illumination time, illumination intensity and illumination frequency.

The invention also provides application of the far-red light gene loop expression control system or the gene loop remote regulation and control system in preparation of diabetes treatment medicines. The diabetes mellitus 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 diabetes. The present invention provides new methods and strategies for treating diabetes. The system can modulate the expression of insulin and/or glucagon-like peptide, GLP-1. The expression construction of the Insulin comprises SEAP-2A-Insulin, EGFP-2A-Insulin and EGFP-2A-SEAP-2A-Insulin. The expression of the glucagon-like peptide GLP-1 comprises GLP-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 LED 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. 9 is a layout diagram of 24 far infrared LED 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 LED circuits of the intelligent control system for controlling the far infrared light source by the smart phone of the present invention through the local area network WiFi or 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 present invention demonstrating that the intelligent mobile phone can express all meaningful proteins by controlling the far-red light gene loop expression control system via WiFi or 2G/3G/4G network ultra-remote control, with the expression of SEAP, Luciferase, EGFP and GLP1 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 a smart phone of the present invention in a type I diabetic mouse by using a WiFi or 2G/3G/4G network to remotely regulate the far-red light gene loop expression control system to precisely regulate GLP-1 expression for the treatment of type II diabetes.

FIG. 39 shows sugar tolerance experimental results of the smart phone of the present invention for treating type II diabetes by precisely regulating GLP-1 expression in a type I diabetes model mouse through a local area network WiFi or 2G/3G/4G network ultra-remote regulation far-red light gene loop expression control system.

FIG. 40 shows the results of insulin resistance experiments in which the smartphone regulates GLP-1 expression precisely in a type I diabetic mouse to treat type II diabetes by using the WiFi or 2G/3G/4G network ultra-remote regulation far-red light gene loop expression control system of the invention through a local area network.

FIG. 41 shows that the smartphone regulates the amount of glucagon expressed by GLP-1 expression therapy for type II diabetes mellitus in a type I diabetic model mouse by a local area network 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. The dimensions 62mm x 44mm x 18mm

7. Weight 45g

Far infrared LED lamp

The used far infrared LED lamp beads are purchased from a hundred-light photoelectricity, the design of the far infrared LED lamp circuit board is completed by a microelectronic engineer in a white space, and the processing is completed by Shenzhen Jie Multi-upper science and technology Limited. 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 were synthesized by Kingzhi Biotech, Inc. The expression plasmids constructed in the examples of the present invention were subjected to sequence determination, which was accomplished by Jinzhi Biotechnology, Inc. Phanta Max Super-Fidelity DNA polymerase used in the examples of the present invention was purchased from Biotech, Inc. of Nanjing Novowed Toxa. The endonuclease and the T4DNA ligase were purchased from TaKaRa. Homologous recombinases were purchased from Heyu Biotechnology (Shanghai) Ltd. Phanta Max Super-Fidelity DNA polymerase was purchased with the corresponding polymerase buffer and dNTPs. The endonuclease, T4DNA ligase and homologous recombinase were purchased with the corresponding buffer. Yeast extract (Yeast extract), tryptone (Trypton), agar powder, and ampicillin (Amp) were obtained from Shanghai Biotech, Inc. DNA Marker DL5000, DNA Marker DL2000 (Bao bioengineering Co., Ltd.); nucleic acid dye EB (guangdong national ao biotechnology); a plasmid small extraction kit (Tiangen Biochemical technology (Beijing) Co., Ltd.); the DNA gel recovery kit and the PCR product purification kit are purchased from Shikoku Biotechnology GmbH; the other reagents such as absolute ethyl alcohol, NaCl and the like mentioned in the examples are all domestic analytical pure products.

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 ultrapure 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 ultrapure 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 alpha strain on a flat plate without antibiotics, and performing inverted culture at 37 ℃ for 12-16 h;

picking a single colony in a 2mL LB shake tube without any antibiotic, and carrying out shaking culture at 37 ℃ and 180rpm overnight;

2. absorbing 1mL of bacterial liquid, transferring the bacterial liquid into a 250mL triangular flask LB 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 between 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 4mL of ice-cold 0.1M CaCl were added₂And 0.1mL of precooled sterilized pure glycerol, and suspending and precipitating;

7. subpackaging the suspension into PCR tubes (the PCR tubes are preferably pre-cooled on ice) at a rate of 100 mu L/tube, and storing 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 LB liquid culture medium (without antibiotics), mixing well, and performing shake culture at 37 deg.C for 40-60 min;

4. transferring the bacterial liquid into a centrifugal tube with the volume of 1.5mL, 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 LB solid culture medium containing Amp, and inverting in a 37 ℃ 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, the regulation method of the gene loop remote regulation and control system is illustrated by using 720nm wavelength LED (L720- __ AU, epitex, Japan) and infrared therapeutic equipment (CQ-61, Chongqing space rocket electronics Co., Ltd., China) as examples, but the protection scope of the invention is not limited.

The LEDs used in the experiment are purchased from epitex corporation of Japan; the infrared therapy apparatus was purchased from Chongqing space rocket electronics technologies, Inc. of China. And other accessories are domestic conventional consumables.

720nm wavelength LED, providing a low power far-red light emitter. 4X 6LED board, its connected mode is: according to the arrangement of the cell culture plate (24 wells), each LED corresponds to the center of each well, and each LED is connected in parallel. And adjusting the illumination time according to the experiment requirement, and carrying out the experiment according to the illumination intensity.

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), 15mL and 50mL centrifuge tubes for cell culture were purchased from Thermo Fisher Scientific, USA (Labserv); modified Eagle Medium, fetal bovine serum, penicillin and streptomycin solutions used were purchased from Gibico, USA; PEI used for transfection was purchased from Polysciences; cell culture chambers were purchased from Thermo Fisher Scientific, usa. The other consumables are common domestic consumables.

And (5) culturing the cells. Human embryonic kidney cells (HEK-293T, ATCC: CRL-11268), continuous expression of RTP1, RTP2, REEP1 and G_αoλφHEK-293(Hana3A), telomerase immortalized human mesenchymal stem cells (hMSC-TERT, ATCC: CRL-3220), human embryonic kidney epithelial cells (HEK-293A), human cervical cancer cells (HeLa, ATCC: CCL-2), murine neuroma blast cells (Neuro-2A, ATCC: CCL-131) were cultured in modified Eagle medium to which 10% (v/v) fetal bovine serum and 1% (v/v) penicillin and streptomycin solution were added. All types of cells were cultured in an incubator containing 5% carbon dioxide concentration at 37 ℃.

And (4) transfection. All cell lines were transfected using the procedure for optimized PEI (Wieland M, Methods 56(3): 351). Simply, 6 × 10 is planted in each well of a 24-well plate with a culture system of 500 μ L⁴After culturing for 16h, the optimal proportion of DNA and PEI are mixed and dissolved in 50 μ L of culture medium according to the mass ratio of 3:1(PEI: DNA) for 6 h.

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 200uL of cell culture supernatant into a centrifuge tube (Note: generally over 150uL because some volume is lost by subsequent heating)

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 80uL (diluted by itself according to 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 1 activity unit (1U) by reacting with substrate disodium p-nitrophenylphosphate (PNPP-Na2) at 37 ℃ and pH 9.8 within 1min to produce 1mol/L p-nitrophenol. The p-nitrophenol has bright yellow, and at the wavelength of 405nm, the p-nitrophenol with different concentrations corresponds to different light absorption values. The calculation method comprises the following steps: the slope 256.8 of the curve formed by the OD values measured at different time points in the reaction process of the sample and the substrate is the enzyme activity in U/L.

Detection of reporter Gene Luciferase (Luciferase)

The dual-luciferase reporter gene detection kit used was purchased from biotool, usa. The detailed operation steps are described in the specification of a double-luciferase reporter gene detection kit (Cat: B17001, Lot71005, Bitool, Houston, TX, USA).

Preparation method of sodium alginate-PLL-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 as follows: the diameter of the 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 20mL/min, 500 microcapsules are formed per second, and the size of each microcapsule is 300-350 mu m; the number of cells encapsulated by each microcapsule is 200-250.

Preparation method of hollow fiber membrane transplanting tube

Hollow fiber membrane graft tube for experiment (

Implant Membrane) purchased from Spectrum laboratories, Inc., USA. The preparation method of the hollow fiber membrane transplanting tube is detailed in

Implant Membrane product Specification.

Method for detecting Insulin (Insulin)

The Insulin detection kit (Mouse Insulin ELISA kit) used for the experiments was purchased from Mercodia, Sweden, and the detailed determination method is described in the product instruction.

Method for detecting glucagon (GLP-1)

Glucagon detection kits for the assay (Millipore Corporation, Billerica, MA01821 USA, Cat. No. EGLP-35K, Lot. 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 LED lamps are taken as an example of a far infrared light source, and a manufacturing method of an intelligent control system for controlling the far infrared light source by an intelligent mobile phone through a local area network WiFi or a 2G/3G/4G network in an ultra-remote mode is explained, 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 3 in 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 5 in the specification in detail.

And fourthly, realizing the ultra-remote intelligent controller with the function of multi-path output. In the invention, the manufacturing method of the ultra-remote intelligent controller with the multi-path output function comprises the following steps: the output ports of 6 independent switching power supplies are respectively connected with 6 input ports of the intelligent controller, and then the corresponding 6 output ports of the intelligent controller are connected with the input ports of the far infrared LEDs to serve as direct current power supply ports of the far infrared LEDs. The power supply of the whole system is completed by an AC/DC power adapter, a fuse with the 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 function of protecting the multi-output normally works in a safe current range, and a system wiring diagram is shown in figure 6 in the specification in detail. When installing the APP software supporting with the intelligent control ware on the smart mobile phone, when smart mobile phone and intelligent control ware configuration insert LAN wiFi or 2G 3G 4G network resource jointly, the accessible operation smart mobile phone APP software control switches the opening and closing of 6 ways of independent switching power supplies to can set for the time of opening and closing of 6 ways of independent switching power supplies, thereby the different operating condition of far-infrared light source of super remote control, like different illumination intensity, different illumination time etc.. The physical diagram of the ultra-remote intelligent controller with the function of multi-path output is shown in detail in the attached figure 7.

And fifthly, realizing the far infrared LED lamp. The 24 far infrared LEDs are respectively connected with the resistors in series and then are connected in parallel for power supply. This far-red light source's printed circuit board has designed 6 ways power supply ends, and wherein 6 ways power supply end every department adds a voltage clamp diode, effectively protects the LED circuit to add a total accuse self-lock switch in whole power supply, thereby realize the breaking of manual control lamp plate circuit. The 24 far infrared LED circuit design diagrams are shown in the attached figure 8 in detail. In the PCB circuit layout, in order to guarantee the distribution uniformity of illumination, 24 LED lamps are uniformly placed at equal intervals, and the row-to-row spacing is 2cm according to the layout of 4 rows and 6 columns. The layout of 24 far infrared LED circuits is shown in detail in figure 9; the 24 far infrared LED circuits are shown in detail in figure 10.

In conclusion, after the APP client, the remote intelligent controller, the multi-output switching power supply and the far infrared LED lamp are integrated and connected, the intelligent control system of the intelligent mobile phone for controlling the far infrared light source through the local area network WiFi or the 2G/3G/4G network is formed. The overall system is shown in detail in fig. 11.

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.

In the second step, cells are seeded. Good HEK-293T cells in growth state were seeded in 7 24-well plates with 6X10 cells per well after digestion with 0.25% pancreatin⁴Cells were cultured and 500. mu.L of DMEM medium containing 10% FBS was added.

And thirdly, transfection. Within 16 to 24 hours of cell inoculation, 0.1 μ g of photoreceptor pWS189, PEI transfection reagent and serum-free DMEM were mixed well, left to stand at room temperature for 15min and then added dropwise to 24-well culture plates. Wherein the total preparation volume of each hole is 50 mu L, and the mass ratio of the plasmid to the PEI is 1: 3. After transfection for 6 hours, 500. mu.L of DMEM medium containing 10% FBS was added and cultured.

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/cm²Under the LED (the specific connection mode refers to the experimental material and method) for 1 h.

And fifthly, collecting cells. After one hour of illumination, cells were harvested at 0, 5, 10, 15, 30, 60min (corresponding to

numbers

2, 3,4, 5, 6, 7, respectively; number 1 24 well plate was always in dark condition). The medium in the 24-well plate was removed, 200. mu.L of PBS was added, and cells were exfoliated by repeatedly pipetting hole by hole in sequence with a pipette. It was transferred to a new 2mL centrifuge tube and centrifuged at 1000 Xg for 5min to collect the cells.

And sixthly, cracking the cells. Adding 200 μ L lysis buffer into the collected cells, standing at-80 deg.C for 15min, quickly taking out, standing at 37 deg.C for 5min, repeating for 3 times, shaking and mixing. Centrifuging at 5000 Xg for 5min, and collecting supernatant.

And a seventh step of measuring the amount of c-di-GMP. The expression level of each group of c-di-GMP was measured by using a c-di-GMP ELISA kit. The experimental data are shown in figure 12 in the specification.

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:

In the second step, cells are seeded. Good HEK-293T cells in growth state were seeded in 2 24-well plates after digestion with 0.25% pancreatin per wellPore seed of 6X10⁴Cells were cultured and 500. mu.L of DMEM medium containing 10% FBS was added.

And thirdly, transfection. The 2 24-well plates were divided into a dark group and an illuminated group, each group being divided into 1-8 sub-groups. In the inoculation of cells 16 to 24h, 0.3. mu.g of pcDNA3.1(+) in the 1 st 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 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 pWS189, 0.1. mu.g of pGXY 34 and 0.1. mu.g of pcDNA3.1(+) in the 6 th group, 0.1. mu.g of pGY32, 0.1. mu.1. g of pWS2 and PEI 0.1. mu.1. g of pcDNA3.1. mu.1. g of pGDNA3.1 (+) in the 7 th group, and 0.1. mu.1. mu.8. mu.1. mu.g of pGWS 3.1. Wherein the total preparation volume of each hole is 50 mu L, and the mass ratio of the plasmid to the PEI is 1: 3. After transfection for 6 hours, 500. mu.L of DMEM medium containing 10% FBS was added and cultured.

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/cm²The LED (the specific connection mode refers to the material and the method) is illuminated for 4 hours, and the dark group is always 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, transfection. Within 16 to 24h of seeding the cells, 2 24-well plates were divided into dark and light groups, each divided into 1-4 groups. Within 16 to 24h of inoculating cells, 0.1. mu.g of pWS46 in group 1, 0.1. mu.g of pWS48 in group 2, 0.1. mu.g of pWS1 in group 3, and 0.1. mu.g of pWS50 in group 4 were mixed with 0.1. mu.g of pGY32, 0.1. mu.g of pXY34, and a PEI transfection reagent in serum-free DMEM, and the mixture was dropped into a 24-well culture plate after standing at room temperature for 15 min. Wherein the total preparation volume of each hole is 50 mu L, and the mass ratio of the plasmid to the PEI is 1: 3. After transfection for 6 hours, 500. mu.L of DMEM medium containing 10% FBS was added and cultured.

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, transfection. Within 16 to 24h of seeding the cells, 2 24-well plates were divided into dark and light groups, each divided into 1-5 groups. Within 16 to 24h of seeding cells, 0.1. mu.g of pWS189 in group 1, 0.1. mu.g of pWS50 in group 2, 0.1. mu.g of pWS51 in group 3, 0.1. mu.g of pWS55 in group 4, 0.1. mu.g of pWS59 in group 5, and 0.1. mu.g of processor pGY32, 0.1. mu.g of effector pXY34, PEI transfection reagent were mixed with serum-free DMEM, and the mixture was dropped into 24-well culture plates after standing at room temperature for 15 min. Wherein the total preparation volume of each hole is 50 mu L, and the mass ratio of the plasmid to the PEI is 1: 3. After transfection for 6 hours, 500. mu.L of DMEM medium containing 10% FBS was added and cultured.

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).

And thirdly, transfection. Within 16 to 24h of seeding the cells, 2 24-well plates were divided into dark and light groups, each divided into 1-8 groups. Within 16 to 24h of seeding cells, 0.1. mu.g of pWS200 in group 1, 0.1. mu.g of pXY34 in group 2, 0.1. mu.g of pXY35 in group 3, 0.1. mu.g of pXY36 in group 4, 0.1. mu.g of pGY28 in group 5, 0.1. mu.g of pGY32 in group 6, 0.1. mu.g of pGY33 in group 7, 0.1. mu.g of pGY34 in group 8, and 0.01. mu.g of red light receptor pWS189, 0.1. mu.g of effector pXY24, PEI transfection reagent were mixed with serum-free DMEM in group 8, and uniformly added to 24-well plates after standing for 15min at room temperature. Wherein the total preparation volume of each hole is 50 mu L, and the mass ratio of the plasmid to the PEI is 1: 3. After transfection for 6 hours, 500. mu.L of DMEM medium containing 10% FBS was added and cultured.

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).

And thirdly, transfection. Within 16 to 24h of seeding the cells, 2 24-well plates were divided into dark and light groups, each divided into 1-10 groups. Within 16 to 24h of inoculation of cells, 0.1. mu.g of pXY19 in group 1, 0.1. mu.g of pXY20 in group 2, 0.1. mu.g of pXY21 in group 3, 0.1. mu.g of pXY22 in group 4, 0.1. mu.g of pXY23 in group 5, 0.1. mu.g of pXY16 in group 6, 0.1. mu.g of pXY17 in group 7, 0.1. mu.g of pXY18 in group 8, 0.1. mu.g of pXY31 in group 9, 0.1. mu.g of pXY32 in group 10 and 0.1. mu.g of far-red light receptor pWS189, 0.1. mu.g of processor pGY32, PEI transfection reagent was mixed with serum-free DMEM, and added to 24-well plates uniformly after standing for 15min at room temperature. Wherein the total preparation volume of each hole is 50 mu L, and the mass ratio of the plasmid to the PEI is 1: 3. After transfection for 6 hours, 500. mu.L of DMEM medium containing 10% FBS was added and cultured.

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).

And thirdly, transfection. Within 16 to 24h of seeding the cells, 2 24-well plates were divided into dark and light groups, each divided into 1-5 groups. Within 16 to 24h of seeding cells, 0.1. mu.g of pXY33 in group 1, 0.1. mu.g of pXY28 in group 2, 0.1. mu.g of pXY34 in group 3, 0.1. mu.g of pXY39 in group 4, and 0.1. mu.g of pXY40 in group 5, and 0.1. mu.g of far-red light receptor pWS189, 0.1. mu.g of processor pGY32, PEI transfection reagent were mixed with serum-free DMEM, and were dropped into 24-well culture plates uniformly after standing at room temperature for 15 min. Wherein the total preparation volume of each hole is 50 mu L, and the mass ratio of the plasmid to the PEI is 1: 3. After transfection for 6 hours, 500. mu.L of DMEM medium containing 10% FBS was added and cultured.

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).

And thirdly, transfection. Within 16 to 24h of seeding the cells, 2 24-well plates were divided into dark and light groups, each divided into 1-5 groups. Within 16 to 24h of seeding cells, 0.1. mu.g pGY36 in group 1, 0.1g pGY37 in group 2, 0.1. mu.g pGY38 in group 3, 0.1. mu.g pGY39 in group 4, 0.1. mu.g pGY40 in group 5, and 0.1. mu.g of far-red light receptor pWS189, 0.1. mu.g processor pGY32, PEI transfection reagent were mixed with serum-free DMEM, and were dropped into 24-well plates after standing at room temperature for 15 min. Wherein the total preparation volume of each hole is 50 mu L, and the mass ratio of the plasmid to the PEI is 1: 3. After transfection for 6 hours, 500. mu.L of DMEM medium containing 10% FBS was added and cultured.

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:

In the second step, cells are seeded. Neuro-2A cells, HeLa cells, hMSC-TERT cells, Hana3A cells, HEK-293A cells and HEK-293T cells in good growth were digested with 0.25% trypsin and seeded in 2 (4 wells per plate) different 24-well plates, 6X10 per well⁴Cells were cultured and 500. mu.L of DMEM medium containing 10% FBS was added.

And thirdly, transfection. Within 16 to 24 hours of cell inoculation, 0.1. mu.g of pWS189, 0.1. mu.g of pGY32, 0.1. mu.g of pXY34, PEI transfection reagent and serum-free DMEM were mixed well, left to stand at room temperature for 15min and then added dropwise to a 24-well culture plate. Wherein the total preparation volume of each hole is 50 mu L, and the mass ratio of the plasmid to the PEI is 1: 3. After transfection for 6 hours, 500. mu.L of DMEM medium containing 10% FBS was added and cultured.

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:

In the second step, cells are seeded. Good HEK-293T cells in growth state were seeded in 12 24-well plates with 6X10 cells per well after digestion with 0.25% pancreatin⁴Cells were cultured and 500. mu.L of DMEM medium containing 10% FBS was added.

And thirdly, transfection. Within 16 to 24 hours of cell inoculation, 0.1. mu.g of photoreceptor pWS189, 0.1. mu.g of pGY32, 0.1. mu.g of pXY34, PEI transfection reagent and serum-free DMEM were mixed well, left to stand at room temperature for 15min and then added dropwise to a 24-well culture plate. Wherein the total preparation volume of each hole is 50 mu L, and the mass ratio of the plasmid to the PEI is 1: 3. After transfection for 6 hours, 500. mu.L of DMEM medium containing 10% FBS was added and cultured.

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/cm²Under the LED (concretely)The connection is with reference to materials and methods). The smart phone 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:

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/cm²Under the LED (the specific connection mode refers to materials and methods). The illumination time is 4h (wherein the illumination intensity is 0, the lamp is always in the darkCultured).

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 can express all meaningful proteins, and in the embodiment, the gene loop control system for regulating and controlling the transgene expression through far-red light is exemplified by expression reporter gene secreted alkaline phosphatase (SEAP), Enhanced Green Fluorescent Protein (EGFP) and glucagon-like peptide (GLP-1). But are not intended to limit the scope of the present invention. The method comprises the following specific steps:

In the second step, cells are seeded. The good HEK-293T cells in the growth state were digested with 0.25% trypsin and seeded in different 24-well plates, 6X10 cells per well⁴Cells were cultured and 500. mu.L of DMEM medium containing 10% FBS was added.

And thirdly, transfection. Within 16 to 24h of seeding the cells, 0.1. mu.g of pWS189, 0.1. mu.g of pGY32, 0.1. mu.g of pXY 34; 0.1. mu.g of pWS189, 0.1. mu.g of pGY32, 0.1. mu.g of pGY 44; 0.1 mu g of pWS189, 0.1 mu g of pGY32 and 0.1 mu g of pWS212 are respectively mixed with PEI transfection reagent and serum-free DMEM uniformly, and the mixture is placed for 15min at room temperature and then uniformly dropped into a 24-well culture plate. Wherein the total preparation volume of each hole is 50 mu L, and the mass ratio of the plasmid to the PEI is 1: 3. After transfection for 6 hours, 500. mu.L of DMEM medium containing 10% FBS was added and cultured.

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/cm²The LED (the specific connection mode refers to the material and the method) is illuminated for 4 hours, and the dark group is always cultured in the dark.

And fifthly, detecting the reporter gene. Measuring the amount of SEAP at 24h, 48h and 72h respectively; taking fluorescence pictures at 0h, 6h, 12h, 24h and 48h respectively; detecting the amount of luciferase by using a dual-luciferase reporter gene detection kit at 24, 48 and 72 hours; and measuring the expression quantity of the GLP-1 under different illumination time by using an ELISA 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:

In the second step, cells are seeded. Will be in good growth stateHEK-293T cells were digested with 0.25% trypsin and plated in separate 24-well plates, 6X10 cells per well⁴Cells were cultured and 500. mu.L of DMEM medium containing 10% FBS was added.

And thirdly, transfection. Within 16 to 24 hours of cell inoculation, 0.1. mu.g of pWS189, 0.1. mu.g of pGY32, 0.1. mu.g of pWS213 and PEI transfection reagent were mixed with serum-free DMEM, and the mixture was allowed to stand at room temperature for 15min and then added dropwise to a 24-well plate. Wherein the total preparation volume of each hole is 50 mu L, and the mass ratio of the plasmid to the PEI is 1: 3. After transfection for 6 hours, 500. mu.L of DMEM medium containing 10% FBS was added and cultured.

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

And fifthly, detecting the reporter gene. Taking fluorescent protein images of different illumination times at 48 h; the expression quantity of the Insulin under different illumination times is measured by using an ELISA 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:

In the second step, cells are seeded. After digesting the good HEK-293T cells in the growth state with 0.25% pancreatinPlanting in 10cm cell culture dish, each dish is 4 × 10⁶Cells were plated and 10mL of DMEM medium containing 10% FBS was added.

Thirdly, within 16-24 h of inoculating cells, 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, standing at room temperature for 15min, and uniformly dripping into a 10cm cell culture dish. Wherein the total preparation volume of each dish is 2mL, and the mass ratio of the plasmid to the PEI is 1: 3. After 6h of transfection, 10mL of DMEM medium containing 10% FBS was added 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 in accordance with the procedure of example 14, and the microcapsules prepared were cultured in a 6-well plate while irradiating with an infrared ray therapeutic apparatus at an 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:

In the second step, cells are seeded. Good HEK-293T cells in growth state were seeded in 24-well cell culture plates with 6X10 cells per well after digestion with 0.25% trypsin⁴Cells were plated and 500. mu.L of DMEM medium containing 10% FBS was added.

Thirdly, within 16 to 24 hours of inoculating the cells, 0.1 mu g of pWS189, 0.1 mu g of pGY32, 0.1 mu g of pXY34 and PEI transfection reagent are mixed uniformly with serum-free DMEM, and the mixture is placed for 15min at room temperature and then uniformly dripped into a 24-well cell culture plate. Wherein the total preparation volume of each hole is 50 mu L, and the mass ratio of the plasmid to the PEI is 1: 3. After 6h of transfection, 10mL of DMEM medium containing 10% FBS was added 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 system with smart phone through local area network WiFi or 2G/3G/4G networkExterior line therapeutic equipment (10 mW/cm)²) 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

In the second step, cells are seeded. Good HEK-293T cells in growth state were seeded in 6 24-well plates after digestion with 0.25% pancreatin, 6X 104 cells per well, and 500. mu.L of DMEM medium containing 10% FBS was added.

And thirdly, transfection. Within 16 to 24 hours of cell seeding, 0.1. mu.g of photoreceptor pWS189, 0.1. mu.g of processor pGY32, 0.1. mu.g of effector pWS213, PEI transfection reagent and serum-free DMEM were mixed well, left to stand at room temperature for 15min and then added dropwise to 24-well culture plates. Wherein the total preparation volume of each hole is 50 mu L, and the mass ratio of the plasmid to the PEI is 1: 3. After transfection for 6 hours, 500. mu.L of DMEM medium containing 10% FBS was added and cultured.

And fourthly, illuminating. After the liquid is changed for 14-18 hours, the six plates are divided into 6 groups, the six plates are placed under an LED with the wavelength of 720nm and the illumination intensity of 1mW/cm2, and the smart phone sets 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 plates are illuminated for 0 hour) through a local area network WiFi or a 2G/3G/4G network respectively in an ultra-remote mode.

And fifthly, detecting the expression of insulin. After 48 hours of culture, the expression of green fluorescent protein in each group was photographed, and the amount of insulin expression in each group was measured by using an insulin ELISA 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

Firstly, constructing a type I diabetes mouse model. We used multiple low dose streptozotocin (Streptozocin, STZ, from Sigma S0130, 18883-6, 6-4) dosing induction model. 25C 57BL/6J mice (from Chinese academy of sciences), 8 weeks old, male, were injected intraperitoneally (fasted for 12-16h before injection) for 5 consecutive days with sodium citrate buffer dissolved with STZ (at a dose of 40-50 mg/kg). STZ was dissolved in fresh citrate buffer (0.1mol/L, pH 4.5) at the time of injection and prepared on ice, with care taken to avoid light. After dissolution, the injection is completed within 30 min.

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.

Seventh, a sugar tolerance test is performed. 24h after transplantation, sugar tolerance experiments were performed. First, 10mL of a fresh glucose aqueous solution (125mg/mL) was prepared, and the injection volume of the glucose aqueous solution was calculated from the body weight of each mouse (1.25g/kg) (i.p. injection volume V ═ 10 × body weight (g) of the mouse, unit: μ L). Then, the blood glucose at 0 point of each mouse (starved for 16 hours before measurement) was measured, and the above-mentioned glucose solution was intraperitoneally injected. Finally, the blood glucose values were measured 30, 60, 90, 120min per mouse. Experiments show that the glucose tolerance of diabetic mice is well improved. The experimental data are shown in figure 37 of the specification.

Example 21 Smart Phone Gene Loop control System for ultra remote Regulation of Gene expression Via local area network WiFi or 2G/3G/4G network to regulate glucagon (GLP-1) expression in vitro

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 cultured and 500. mu.L of DMEM medium containing 10% FBS was added.

And thirdly, transfection. Within 16 to 24h of cell inoculation, 0.1. mu.g of photoreceptor pWS189, 0.1. mu.g of processor pGY32, 0.1. mu.g of effector pWS212, PEI transfection reagent and serum-free DMEM were mixed well, left to stand at room temperature for 15min and then added dropwise to 24-well culture plates. Wherein the total preparation volume of each hole is 50 mu L, and the mass ratio of the plasmid to the PEI is 1: 3. After transfection for 6 hours, 500. mu.L of DMEM medium containing 10% FBS was added and cultured.

And fifthly, measuring the amount of GLP1-Fc expressed in vitro by the gene loop control system for the smartphone to regulate and control the transgenic expression through the WiFi of the local area network or the 2G/3G/4G network. After 48 hours of culture, the expression level of GLP1-Fc in each group was measured by an Active GLP-1ELISA kit.

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, the smartphone accurately regulates GLP-1 expression in type I diabetic mice to treat type II diabetes mellitus by the gene loop control system for ultra-remote regulation of transgene expression through local area network WiFi or 2G/3G/4G network

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.

Seventh, a sugar tolerance test is performed. 24h after transplantation, sugar tolerance experiments were performed. First, 10mL of a fresh glucose aqueous solution (125mg/mL) was prepared, and the injection volume of the glucose aqueous solution was calculated from the body weight of each mouse (1.25g/kg) (i.p. injection volume V ═ 10 × body weight (g) of the mouse, unit: μ L). Then, the blood glucose at 0 point of each mouse (starved for 16 hours before measurement) was measured, and the above-mentioned glucose solution was intraperitoneally injected. Finally, the blood glucose values were measured 30, 60, 90, 120min per mouse. Experiments show that the glucose tolerance of diabetic mice is well improved. The experimental data are shown in figure 39 of the specification.

And eighthly, performing an insulin resistance experiment. 24h after transplantation, insulin resistance experiments were performed. First, a fresh insulin solution (0.1U/ml) was prepared, and the injection volume of insulin was calculated from the body weight of each mouse (1.1U/kg) (i.p. injection volume V ═ 1.1 × body weight (g) of mouse, unit: μ L). Then, 0-point blood glucose was measured for each mouse (starved for 4 hours before measurement), and the above insulin solution was intraperitoneally injected. Finally, the blood glucose values were measured 30, 60, 90, 120min per mouse. Experiments show that the insulin resistance of diabetic mice is well improved. The experimental data are shown in the attached figure 40 of the specification in detail.

And ninthly, measuring the expression of GLP-1 in the type II diabetic mouse by the gene loop control system for the smartphone to regulate and control the transgenic expression through WiFi (wireless fidelity) of a local area network or 2G/3G/4G network in an ultra-remote way. Blood was collected at the inner canthus 48h after transplantation, and the GLP-1(activie) content in serum was measured using GLP-1(7-36) activivieELISA kit. Experiments show that the intelligent mobile phone can accurately regulate the expression of the glucagon in vivo through a gene loop control system for ultra-remote regulation and control of transgenic expression through a local area network WiFi or a 2G/3G/4G network. The experimental data are shown in figure 41 of the specification in detail.

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_CAGArtificially constructing a cytomegalovirus early enhancer and chicken beta-actin promoter combined promoter; EGFP, enhanced green fluorescent protein; SEAP, human exocrine alkaline phosphatase; GLP-1, glucagon-like peptide 1; VP64, herpes virus transcriptional activation domain tetramer; BldD, far-red processor; 2A, an independent oligopeptide of which the carbon terminal contains a D (V/I) EXNPGP structural domain; pA, polyadenylation signal; bphs, artificially synthesized bacterial photosensitive diguanylate cyclase; YhjH, c-di-GMP degrading enzyme; bpho, pigment synthase; PCR, 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)

And (3) sequence 6: nucleotide sequence of human EF1 alpha (hEF1 alpha) promoter

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

And (2) sequence 8: artificially constructed nucleotide sequence of cytomegalovirus early enhancer and chicken beta-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 effect device promoter P_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

Sequence 42: nucleotide sequence of sequence Luciferase (Luciferase) to be transcribed expressed by far-red light effector

Sequence 43: nucleotide sequence of glucagon-like peptide-1 (GLP-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 Nuclear Localization Signal (NLS)

Sequence 55: amino acid sequence of connecting functional peptide (Linker)

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Claims

1. The application of a far-red light gene loop expression control system or a eukaryotic expression vector containing the far-red light gene loop expression control system in the preparation of diabetes treatment medicines; the diabetes comprises type I diabetes and/or type II diabetes; 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 photoreceptors include photosensitive diguanylate cyclase BphS, which converts GTP to c-di-GMP under far-red light conditions;

the processor includes: a polypeptide as a DNA binding domain and a c-di-GMP binding domain, a polypeptide as a nuclear localization signal NLS, a polypeptide as a linking domain, and a polypeptide as a transcriptional regulatory domain; the polypeptide serving as the DNA binding domain and the c-di-GMP binding domain is a protein which can be combined with a specific DNA sequence after being combined with the c-di-GMP, and comprises a BldD protein, wherein the amino acid sequence of the BldD protein is selected from a sequence 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 use as claimed in 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 the fusion protein to alanine R587A.

3. The use of claim 1, wherein said photoreceptor is constructed in a form 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 use of claim 3, wherein the amino acid sequence of BphS, BphO, YhjH is selected from the group consisting of sequences 45, 46, 48.

5. The use of claim 1, wherein the promoter expressing photoreceptors comprises: a) SV 40; b) a CMV; c) hEF1 α; d) mPGK; e) and (4) CAG.

6. The use according to claim 1, wherein the polypeptide acting as nuclear localization signal NLS has an amino acid sequence selected from the group consisting of SEQ ID NO 54.

7. The use according to claim 1, wherein said polypeptide as a linking domain has an amino acid sequence selected from the group consisting of SEQ ID NO 55.

8. The use according to claim 1, wherein the polypeptide acting as a transcriptional regulatory domain has an amino acid sequence selected from the group consisting of seq id nos 50, 51, 52, and 53.

9. Use according to claim 1, wherein the polypeptide acting as a transcriptional regulatory domain is placed N-terminal or C-terminal to the polypeptide BldD of the DNA binding domain and the C-di-GMP binding domain.

10. The use of claim 1, wherein the polypeptides of different domains in the processor are linked directly or via a linker peptide.

11. Use according to claim 1, wherein the partial sequence of the bldM and whiG promoter regions is in 1-10 copies.

12. The use of claim 1, wherein the weak promoter that promotes gene expression comprises tatabex, cytomegalovirus CMV minimal promoter and its mutant CMVmin 3G.

13. The use of claim 1, 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.

14. The use according to claim 1, wherein the nucleic acid sequence to be transcribed encodes an expressed protein comprising a protein as a reporter gene and/or a protein or small peptide as a drug for the treatment of a disease; wherein the protein serving as a reporter gene comprises secreted alkaline phosphatase, enhanced green fluorescent protein and luciferase; pharmaceutical proteins or small peptides include insulin, glucagon-like peptides as therapeutics for diseases.

15. The use of claim 14, wherein the protein is capable of expressing multiple proteins simultaneously via the 2A sequence on one expression vector; wherein the 2A sequence may be replaced by an internal ribosome entry site sequence IRES.

16. The use of claim 15, wherein the plurality of proteins comprises SEAP-2A-Insulin, EGFP-SEAP-2A-Insulin.

17. The use of claim 1, wherein the system modulates the expression of insulin and/or glucagon-like peptide, GLP-1.

18. The use of claim 17, wherein the expression construct for Insulin comprises SEAP-2A-Insulin, EGFP-2A-SEAP-2A-Insulin; the expression of the glucagon-like peptide GLP-1 comprises GLP-1-Fc.