36 results about "Gene Circuits" patented technology

The invention provides a dynamic control system for coordinating streptomyces secondary metabolite production and application of the dynamic control system. Firstly, a general artificial promoter PQS (trigger) responding to a quorum sensing system of various streptomycetes is designed, then a stabilizer is constructed based on a bistable gene circuit, instantaneous signal output is converted into stable signal output, dependence of an inducer is successfully eliminated, finally, an orthogonal amplifier is developed based on a streptomycete rare extracellular function sigma factor, and finally, the specific function of the quorum sensing system of various streptomycetes is achieved. And different promoter element libraries containing amplifiers with response from weak to strong and with strength span of about 535 times are constructed. An amplifier and a CRISPRi system are combined to construct a turner, and gene expression inhibition with the strength span of about 200 times is achieved. The combination of the amplifier, the promoter library and the turner is called a multi-effect device, and abundant elements are provided for fine target gene expression. The combination of the trigger, the stabilizer and the multiple-effect device is called as a streptomycete multipath manual control system (SMARTS), and control tools of streptomycete are enriched.

Aspects of the invention is directed to a high-throughput method for screening a gene circuit library. The pace of gene circuit engineering can thereby be increased by expanding the number of circuits tested in each cycle. By measuring large collections of circuits in a single experiment, such an approach could be used for rapidly profiling complex circuit design spaces to identify part compositions with desired quantitative behaviors.

The invention discloses a cell-free cloning reaction system, a kit and application of the cell-free cloning reaction system. The system comprises a cell lysis solution, an inserted DNA fragment and a linear vector, the cell lysis solution is obtained from Escherichia coli which is subjected to induced expression and then subjected to lysis, and a lambda Red system and a ligase system are expressed in the Escherichia coli; the lambda Red system comprises Exo protein, Beta protein and Gam protein; and the ligase system comprises DNA ligase. The optimized lambda Red system and the efficiently expressed DNA ligase system can complete efficient splicing of multi-fragment DNA within 15 minutes at 37 DEG C, still maintain a high positive rate in a multi-fragment assembly system, show an effect superior to Gibson cloning, and expand the applicability of the Gibson clone in complex pathway synthesis and gene line reconstruction.

The invention belongs to the technical field of biological medicines, and particularly discloses a pH and ultrasound double-response type oncolytic microorganism as well as a preparation method and application thereof. On the basis of a tumor targeting platform, a mild thermal response gene loop expression GM-CSF and a surface-coated oncolytic microbial system are integrated, chemotherapeutic drugs are released in a tumor core area, and immunogenic cell death is induced. A thermal response loop is accurately activated through low-intensity focused ultrasound, and engineering bacteria are promoted to express GM-CSF and secrete a large amount of mannose modified OMVs. Due to the nanometer size and mannose targeting of the OMVs, the OMVs are efficiently enriched in lymph nodes, the OMVs are reprogrammed into an immune activation state from an immune tolerance state, and the OMVs and ICD cooperate to promote dendritic cell maturation, tumor antigen presentation and activation of tumor killer T cells, so that a remarkable and powerful treatment effect is achieved in various tumor models, and the application prospect is wide. A new strategy is provided for remodeling the lymph node immune microenvironment and enhancing the anti-tumor immune response.

Tumor-selective expression of therapeutic molecules by bacteria is achieved by one or more AND, NOR, OR, NOT and / or NAND gate genetic circuits. The therapeutic molecules can be proteins, metabolites or catabolites, and may also be immunotherapeutics or immunotherapeutic cytotoxins. Single or multiple expression components may be used. Tumor selective expression of multimeric proteins utilizes multimerization as to complete the genetic circuit. Genetic circuits that are unlinked, function to achieve a combined effect on specificity of delivery of antitumor therapeutic molecules. Compositions and methods to generate silica, PEG and Poly-HPMA coated bacteria are also provided. Compositions and methods for selectively sensing or imaging tumors are also described.

Disclosed herein are nucleic acid constructs that can be used to build genetic circuits for producing antibodies comprising split toxins. Also disclosed herein are methods of producing platelets comprising the antibodies. The platelets produced by the methods disclosed herein can be used to target circulating tumor cells.

The invention discloses a saccharomyces cerevisiae pyruvic acid response biosensor as well as a construction method and application thereof. The biosensor comprises a pyruvic acid activation type gene loop or a pyruvic acid inhibition type gene loop or a bifunctional gene loop. According to the invention, a pyruvic acid activation type gene loop is constructed in saccharomyces cerevisiae through pyruvic acid transcription factor PdhR structural design and PdhR expression level optimization, and the dynamic multiple is 3.4. According to the method, a pyruvic acid activated promoter is used for inducing XylR expression by utilizing the performance of a xylose transcription inhibition factor XylR inversion gene loop, so that transcription of a promoter embedded with an XylR binding site is inhibited, a pyruvic acid inhibition type gene loop is constructed, and the dynamic multiple is 3.1 times. And a difunctional pyruvic acid gene loop with orthogonality is further constructed. The pyruvic acid response loop constructed by the invention takes glucose as an activating agent, and can more widely regulate and control the complex metabolic pathway of the saccharomyces cerevisiae.

The present disclosure generally relates to cancer therapies and, in some embodiments, to using genetic circuits designed for targeted cancer therapies. The genetic circuit, in some embodiments, utilizes a suitable vector capable of delivering DNA sequences comprising functional units comprising a transcriptional promoter, target sequences and a transcriptional terminator. Together, the genetic circuit, in some embodiments, may trigger apoptosis — a target protein activity where cell death occurs — in cancer cells, and protect non-cancer cells from apoptosis. In some embodiments, the genetic circuit may direct target protein activity in cancer cells, while not directing protein activity in non-cancer cells. However, in the presence of certain stimuli, the inhibition is relieved, which may lead to the induction of target protein activity in cancer cells. In addition, some embodiments are generally directed to vectors comprising a first sequence encoding at least a portion of Mdm2 and a promoter, and a second sequence encoding a promoter and a fusion protein comprising at least a portion of p53 and iCasp-9 (or another protein capable of achieving a therapeutic objective), optionally connected by a linker sequence capable of allowing each protein subunit of the fusion protein to be independently active but subject to the same proteolytic pathways as p53 in cancer and non-cancer cells. Other aspects are generally directed to methods of making or using such compositions, kits including such compositions, or the like.

The invention provides a core-shell hydrogel microbead sensing platform as well as a preparation method and application thereof, and belongs to the technical field of synthetic biology and environmental monitoring. The invention provides an engineering sensing microorganism. The engineering sensing microorganism comprises a recombinant vector, the recombinant vector contains a gene expression cassette with target substance response, transformation and report functions; the gene expression cassette comprises a coding gene of a transcription factor ChpR, a coding gene of a fluorescent reporter protein GFP and a coding gene of a chlorpyrifos degrading enzyme, and the gene expression cassette is assembled into a sensing gene line and then introduced into microorganisms to construct the whole-cell biosensor for detecting chlorpyrifos and a metabolite 3, 5, 6-trichloro-2-pyridinol thereof. According to the invention, the chlorpyrifos degradation conversion module is assembled and optimized, and the performance of the sensor is obviously improved. The core-shell hydrogel microbead prepared by the invention can ensure the biological safety and improve the stability of a sensor, and is suitable for high-sensitivity detection of trace chlorpyrifos residues in fruits, vegetables and environmental samples.

The invention belongs to the technical field of biomedical engineering and synthetic biology, and particularly discloses a biofilm dynamic regulation and control system and method based on microfluidics and optogenetics, the system comprises a microfluidic chip, a detection module and an optogenetics regulation and control module, the detection module is electrically connected with the microfluidic chip through a multi-model detection interface, and the optogenetics regulation and control module is electrically connected with the detection module through a multi-model detection interface. The detection module comprises a laser confocal microscope, a [mu] CT three-dimensional reconstruction instrument and a DESI-MSI mass spectrum imager; the photogenetic regulation and control module comprises pseudomonas fluorescens, and the genome of the pseudomonas fluorescens is integrated with a red light response type PhyB-PIF3 gene loop. The biofilm dynamic regulation and control system and the biofilm dynamic regulation and control method based on microfluidics and photogenetics are used for accurately regulating and controlling the physicochemical characteristics of the biofilm and quorum sensing (QS) signal molecule expression, and can monitor the thickness of the biofilm, porosity distribution QS signal molecules and the diffusion efficiency of an antibacterial agent in real time and in situ.

The invention belongs to the technical field of edible mushroom genetic engineering, and relates to a method for constructing a mushroom genetic transformation system by using a G418 resistance selection marker, which comprises the following steps: taking a mushroom strain Shenxiang 215 cultured in a PDA (potato dextrose agar) culture medium as a receptor to obtain hypha blocks; the method comprises the following steps: transferring a pLgnpt plasmid into agrobacterium tumefaciens GV3101; agrobacterium tumefaciens is inoculated into an IM liquid culture medium containing acetosyringone and MES, hypha blocks are subjected to ultrasonic treatment and co-incubation with the hypha blocks, then the hypha blocks are transferred to an IM solid culture medium laid with filter paper, and dark culture is carried out; and transferring the hyphae subjected to dark culture to a PDA resistant plate culture medium containing G418 and Cef for resistance screening to obtain the shiitake transformant. The method not only verifies the applicability of the novel selection marker in flammulina velutipes, but also constructs a multi-marker technology platform parallel to the existing hygromycin system, and lays a foundation for subsequent complex operations such as multi-gene superposition editing, gene loop construction and the like.

Provided herein are stoichiometric Gas Vesicle Expression Systems (GVES), sets of polynucleotide constructs, and related vectors, cells, compositions, and methods configured for robust expression of Gas Vesicle Gene Clusters (GVGCs) in mammalian cells, particularly primary and immune cells. The GVES comprises distinct gene modules for the primary structural protein (gvpA / B) and assembly factors (AF1, AF2), operably configured to achieve a stoichiometric expression ratio, quantified by Dosage Index (DI), wherein the gvpA / B module is expressed at least 2-fold higher than the AF1 and AF2 modules.

The present invention discloses the construction and application of a screening system for lactose or its analog biosensors in Bacillus subtilis. The present invention designs a "dual-screening" biosensor repressor screening gene circuit based on the lactose operon and applicable to Bacillus subtilis: the DNA binding activity of the repressor is screened through a negative screening lethal circuit; the allosteric activation activity is screened through a positive screening fluorescence signal; and finally, a biosensor with the recognition activity of the target lactose analog is obtained. Using this screening platform, novel biosensors responsive to substances such as lactose, 2'-fucosyllactose, and 3-fucosyllactose can be obtained. Moreover, it has been verified that the biosensors constructed according to the screening results of the present invention have high detection sensitivity and specificity.

The invention discloses an application of a gene line DgCsp2-DrWhy in regulation and control of a rice tillering phenotype. The invention provides a method for preparing a transgenic plant with increased effective tillering number and / or plant height, which comprises the following step: introducing a DgCsp2 gene and a Drwhy gene into a receptor plant to obtain the transgenic plant with increased effective tillering number and / or plant height. The DgCsp2 gene is a gene for coding a protein as shown in SEQ ID NO: 1. And the Drwhy gene is a gene for coding a protein as shown in SEQ ID NO: 3. The method disclosed by the invention has application and popularization values for cultivating new plant germplasm, particularly new rice planting with increased tillering quantity and plant height.

Provided herein are genetic circuits and encoded RNA transcripts that produce an output molecule in response to an RNA cleavage event that removes a degradation signal. In some embodiments, the genetic circuits described herein may be used for detecting RNA cleaver activities (e.g., in a cell). Methods of using the genetic circuits described herein in diagnostic or therapeutic applications are also provided.

Methods of detecting a target analyte in subsurface soil can providing a bacterial consortium embodying a genetic circuit. Such a consortium can include a sensor bacterium, a signal propagation bacterium, and a display bacterium, which are provided at a soil surface. The sensor bacterium and signal propagation can be allowed to migrate into subsurface soil below the soil surface. The sensor bacterium can be exposed to the target analyte in the subsurface soil. The sensor bacterium can produce a signal molecule in response to the presence of the target analyte. The signal propagation bacterium can amplify the signal molecule by producing additional signal molecules in response to the presence of the signal molecule. The display bacterium can produce an observable signal in response to presence of the signal molecule.

The application discloses a bacteria with limited reproduction capacity, a method for controlling bacterial reproduction and application, and belongs to the technical field of biotechnology and cell immunotherapy. The bacterial DNA comprises an unnatural amino acid orthogonal translation module, a suicide module and a rescue module. Based on the unnatural amino acid (ncAA) orthogonal translation system, the orthogonal genetic barrier theory is used, gene circuit design and element modularization are used, bacterial reproduction generations are accurately controlled, bacterial growth is effectively controlled, and the bacteria are prevented from escaping, and the bacteria have wide applicability. The bacteria and the method have wide application prospects in the development of attenuated live vaccines.

The invention relates to a T7 RNA polymerase mutant with K1F promoter recognition activity and application of the T7 RNA polymerase mutant in construction of gene circuit logic gate design. The T7 RNA polymerase mutant provided by the invention has RNA polymerase activity based on a K1F promoter, can recognize the corresponding K1F promoter, and keeps orthogonality with a T7 RNAP promoter on the full-length division polymerase variant and on the division polymerase variant with proximity dependency. Compared with the existing K1F RNA polymerase, the K1F RNA polymerase has the advantages of high specificity, enhanced RNA transcriptional activity and the like, the performance is obviously improved, the requirements of current gene circuit multi-path orthogonal regulation and control are met, and an effective candidate enzyme tool is provided for RNA research and application.

This invention relates to the field of gene editing technology, specifically to a gene circuit-based specific gene expression system and module, a pharmaceutical composition, and its applications. The system includes a first vector and a second vector. The first vector includes a first expression cassette containing a cell-specific promoter, a coding sequence encoding a transcriptionally activated fusion protein, and a regulatory region sequence downstream of the coding sequence. The regulatory region sequence is configured to form a response element in the 3' untranslated region of the fusion protein's mRNA after transcription. This response element binds to a specific long non-coding RNA within the silenced cell, leading to the degradation of the fusion protein's mRNA. The second vector includes a second expression cassette containing an associated promoter that can be activated by the transcriptionally activated fusion protein, and a target gene downstream of the associated promoter. Advantages: This ensures that the target protein is expressed only in target cells and not in cancer cells, avoiding adverse effects on non-target cells or tissues, and reducing treatment risks and side effects.

This invention discloses the construction and application of a highly robust quorum sensing gene circuit. The quorum sensing gene circuit exhibits more stable output performance under different fermentation pH conditions, demonstrating high robustness. The response promoters PHU3 and PEL of the quorum sensing gene circuit possess good quorum sensing performance unaffected by pH crosstalk and exhibit good specificity. Using this quorum sensing gene circuit to express an acid-resistant module can improve the acid tolerance and fermentation production capacity of the strain in acidic environments. This invention provides a quorum sensing gene circuit with greater industrial application value.

The application discloses a gene expression stabilization method and system based on sRNA feedback and non-periodic sampling regulation, relates to the technical field of gene expression optimization and regulation, and comprises the following steps: firstly, an sRNA-mediated feedback control gene circuit is designed and constructed; secondly, a state space model describing system dynamics is established, and minimization of the steady-state expression deviation of a target protein before and after ribosome disturbance is taken as an optimization target; through real-time calculation of a stability evaluation index, a non-periodic sampling strategy is used to dynamically judge a regulation triggering time; finally, when the triggering condition is met, the ribosome disturbance level is estimated in real time, and the optimal ribosome binding site strength adjustment value is calculated through solving an optimization problem and is implemented. The application can adaptively maintain the stability of target protein expression in a culture environment with dynamic competition of ribosome resources at a low intervention frequency, and significantly improves the robustness and output consistency of a synthetic biology system.

The present invention belongs to the fields of biomedical engineering and synthetic biology, and specifically discloses a system and method for dynamically regulating biofilms based on microfluidics and optogenetics. The system and method comprise a microfluidic chip, a detection module, and an optogenetics control module. The detection module is electrically connected to the microfluidic chip via a multi-model detection interface, and the detection module includes a laser confocal microscope, a micro-CT three-dimensional reconstruction system, and a DESI-MSI mass spectrometer. The optogenetics control module includes Pseudomonas fluorescens, whose genome incorporates a red-light-responsive PhyB-PIF3 gene circuit. The present invention utilizes the aforementioned system and method for dynamically regulating biofilms based on microfluidics and optogenetics to precisely control the physicochemical properties of biofilms and the expression of quorum sensing (QS) signaling molecules. The system also enables real-time, in situ monitoring of biofilm thickness, porosity distribution, QS signaling molecules, and antimicrobial agent diffusion efficiency.

The invention provides a microbial production cell for the synthesis of a product, further comprising a charge-dependent genetic circuit whose expression confers a selective growth and / or survival advantage to those cells that synthesize the product, while limiting the proliferation of unproductive or non-productive escape cells.

The application discloses an exonuclease fusing an NTPase domain and application thereof, and belongs to the technical field of bioengineering. The exonuclease is a Ppl protein, which comprises a PHP domain at the N terminal and an NTPase domain at the C terminal, wherein the PHP domain has 3'-5' ssDNA exonuclease activity. The NTPase domain of the application regulates the exonuclease activity of the PHP domain through conformational change. Under the condition of high NTP concentration, the NTPase domain inhibits the activity of the PHP domain; when the NTP concentration decreases, the inhibition is released, and the exonuclease activity is significantly activated. The activated PHP domain shows specific cleavage activity to DNA substrates with 3'-hydroxyl overhang. The unique 'NTP concentration sensing and enzyme activity regulation' characteristics make it a core module for developing biological sensors, gene circuits and controllable nucleic acid tools.

The invention discloses a gene loop for gene activation and inhibition control in escherichia coli and bacillus subtilis, and belongs to the technical field of gene engineering. The gene loop is composed of two parts: (1) an activation module: integrating T7RNAP controlled by a dehydrated tetracycline inducible promoter Pet and a repressor protein expression cassette corresponding to the inducible promoter on a genome to obtain a chassis strain stably expressing T7RNAP; the module is used for driving a promoter PT7t to efficiently express a gene needing to be activated; (2) an inhibition module: integrating dCpf1 controlled by Pet on the basis of a chassis strain; meanwhile, Pet is used for expressing a crRNA array, and when dCpf1 and crRNA exist at the same time, transcription inhibition of the gene can be achieved. The gene loop constructed by the invention can simultaneously control activation and inhibition of genes, realizes high-efficiency expression and complete inhibition of different genes, shows an excellent effect in production of lycopene, and has important significance in construction of escherichia coli and bacillus subtilis cell factories and research of synthetic biology.

The present disclosure generally relates to a gene circuit comprising a response element engineered from NR4A1 that increases the activation of chimeric antigen receptors by the delivery of bioactive molecules. In particular, the present disclosure provides a nucleic acid construct comprising a response element operably linked to a nucleic acid sequence of interest that responds to the activation of CAR-T cells. The present disclosure also relates to vectors and cells comprising the gene circuit and the nucleic acid construct. Also provided are methods of inducing an immune response, methods of treating a subject, and methods for delivering a bioactive molecule by activated T cells having a chimeric antigen receptor and a response element construct of the present disclosure.

The invention discloses a method for reversely regulating and controlling a vanillic acid gene circuit control system, the vanillic acid gene circuit control system comprises a vanillic acid trans-transcription factor, a vanillic acid operon and optional Va, and the method comprises the following steps: carrying out amino acid mutation on the vanillic acid trans-transcription factor, therefore, reverse regulation and control of the vanillic acid gene circuit control system can be realized; wherein the amino acid mutation sites comprise the 97th site, the 127th site, the 137th site and the 183th site. According to the vanillic acid trans-transcription factor, a Va dissociable vanillic acid operon is not added, and a Va combinable vanillic acid operon is added. Therefore, by means of the method, the vanillic acid gene circuit control system can be reversely regulated and controlled, the system can be kept in a closed state without adding Va, the system can be kept in an activated state with adding Va, higher practicability and safety are achieved, a new method and a new strategy are provided for a clinical cell treatment scheme, and good clinical application prospects are achieved.

UO2F2 biosensors, and related U-sensing and / or F-sensing genetic molecular components, genetic circuits, compositions, methods and systems are described, which in several embodiments can be used to detect and / or neutralize uranium and in particular bioavailable UO2F2.

Provided are construction and use of a biosensor selection system for lactose or an analogue thereof in Bacillus subtilis. A lactose operon-based "dual-selection" genetic circuit used for selecting repressor proteins for biosensors and applicable to Bacillus subtilis is designed; the selection of repressor protein DNA binding activity is realized by means of a negative-selection lethal circuit; the allosteric activation-based activity screening is realized by means of a positive selection fluorescence signal; and finally, a biosensor having target lactose analogue recognition activity is obtained. A novel biosensor responding to substances such as lactose, 2'-fucosyllactose, and 3-fucosyllactose can be obtained by using the selection platform, and upon verification, the biosensor constructed on the basis of the selection result has high detection sensitivity and specificity.

The invention discloses an expression vector of an AND gate positive feedback gene loop as well as a construction method and synthetic biology application thereof. The expression vector comprises an ignition element P1, a first terminator Tnos and a second terminator Tnos, wherein the ignition element P1 comprises a heat shock promoter PGmhsp17.6L, a nucleotide sequence for coding a transcription factor XVE and a first terminator Tnos; and a power element P2 comprising a chemically induced promoter PLexA35S, a nucleic acid sequence encoding a transcription factor XVE II, and a second terminator T35S. Through cooperation of P1 and P2, a dual induction control positive feedback loop is constructed: when heat shock and a chemical inducer exist at the same time, the loop is started, and rapid accumulation of transcription factors is realized through self-activation of P2, so that downstream gene expression is driven at high intensity. The design effectively overcomes the problems of insufficient expression quantity of a single induction system and heavy burden of constitutive expression metabolism, avoids cytotoxicity, and can be universally applied to synthesis of various secondary metabolites.