WO2023197883A1 - 原儿茶酸和白藜芦醇调控基因表达生物逻辑运算器及其应用 - Google Patents

原儿茶酸和白藜芦醇调控基因表达生物逻辑运算器及其应用 Download PDF

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WO2023197883A1
WO2023197883A1 PCT/CN2023/085135 CN2023085135W WO2023197883A1 WO 2023197883 A1 WO2023197883 A1 WO 2023197883A1 CN 2023085135 W CN2023085135 W CN 2023085135W WO 2023197883 A1 WO2023197883 A1 WO 2023197883A1
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gene expression
resveratrol
expression
protocatechuic acid
gene
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PCT/CN2023/085135
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English (en)
French (fr)
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叶海峰
尹剑丽
万航
刘兴万
吴嘉丽
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华东师范大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation

Definitions

  • the invention relates to the fields of synthetic biology and gene editing, and specifically to a gene expression biological logic operator regulated by green small molecule substances protocatechuic acid and resveratrol and its application for accurate calculations in vivo and in vitro.
  • biological logic operators are the basis of computer operation. They perform logical operations on various input signals and then output the required information.
  • Boolean logic gates By artificially combining many different Boolean logic gates, complex biological logic arithmetic circuits can be created within cells.
  • the most initially studied biological logic operators were conducted in bacteria. For example, the Voigt CA laboratory constructed 16 two-input logic gates that responded to arabinose and tetracycline. The Collins and Peng Yin research teams jointly completed RNA molecule biological logic operations. The synthesis of devices, etc. Compared with biological logic operators in bacteria, biological logic operators in mammalian cells started late.
  • the CRISPR-dCas9 system provides a platform for site-specific transcriptional regulation of the genome.
  • dCas9 can perform site-specific gene expression regulation, epigenetic modification and other behaviors through fusion with other effector proteins (such as transcription factors, modification factors, etc.).
  • Gene circuits have been constructed to regulate the CRISPR-dCas9 system with single signaling substances such as red light, blue light and rapamycin, but there is still a lack of genetic devices for multiple signaling substances to jointly control the CRISPR-dCas9 system.
  • the present invention innovatively discloses for the first time a biological logic operator controlled by green small molecule substances.
  • protocatechuic acid and resveratrol which have multiple biological effects, are used as double-transfusion products.
  • Input signal substances that is, a dual-input Boolean logic gate control system, using protocatechuic acid (PCA) and resveratrol (RES) as inducers, a logic circuit that can operate correctly in cells and animals.
  • PCA and RES are natural, healthy and safe small molecule compounds.
  • protocatechuic acid is widely distributed and is present in Chinese herbal medicines such as Guangzao and Danshen.
  • the present invention discloses a biological logic operator for regulating exogenous/endogenous gene expression with protocatechuic acid and resveratrol, that is, the biological logic operator can simultaneously activate single genes or multiple genes at the cellular level. Accurate logical operation, which provides a precise control operation method for CRISPR/dCas9-mediated endogenous gene expression.
  • the invention provides a biological logic operator regulated by protocatechuic acid and resveratrol, wherein the biological logic operator includes an exogenous gene expression biological logic operator regulated by protocatechuic acid and resveratrol, A biological logic operator for endogenous gene expression regulated by protocatechuic acid and resveratrol.
  • the logic operations of the biological logic operator include AND gates (AND).
  • A does not include B (A NIMPLY B), which is protocatechuic acid.
  • Acid does not include the resveratrol gate
  • B does not include A
  • A resveratrol does not include the protocatechuic acid gate
  • the NOR gate NOR
  • OR OR gate
  • A represents the input signal of protocatechuic acid
  • B represents the input signal of resveratrol
  • the biological logic operator is a dual-input, single-output operation mode
  • the dual input includes protocatechuic acid and resveratrol.
  • Input signal the single output includes the up-regulated expression of the output signal reporter protein d2EYFP, Luciferase, SEAP or any specific gene or functional protein.
  • the AND gate (AND) of gene expression regulated by protocatechuic acid and resveratrol is a logical operation method that only outputs signals when both signals are input.
  • the AND gate includes two versions of the gene expression module construct.
  • the gene expression module of the AND gate includes three gene expression modules, namely a gene module with a strong promoter driving the expression of the protocatechuic acid-responsive recombinant transcription repressor KRAB-PcaV, and a strong promoter driving the expression of Chenopodium quinoa
  • the gene module is expressed in response to the recombinant transcriptional repressor TtgR-KRAB, and the gene expression module in which protocatechuic acid and resveratrol work together to drive downstream signal output from the promoter P PR1 .
  • the recombinant transcription repressors KRAB-PcaV and TtgR-KRAB bind to P PR1 to block downstream gene expression, and the signal is not output; only when protocatechuic acid and resveratrol signals are input at the same time, recombinant transcription
  • the inhibitors KRAB-PcaV and TtgR-KRAB dissociate from P PR1 , promoting the expression of downstream genes, thereby allowing the signal to be output.
  • P PR1 is composed of a strong promoter and PcaV and TtgR operator binding sites.
  • the PcaV The operon binding site is an operon element OPcaV derived from the protocatechuic acid operon system, and the TtgR operon binding site is an operon element OTRC1 derived from the resveratrol operon system, and the P PR1 can drive the expression of downstream genes.
  • the P PR1 can form different types of protocatechuic acid and resveratrol-regulated promoters according to the type of strong promoter and the different copy numbers of the operators OPcaV and OTRC1 , including: a) SEQ ID The nucleotide sequence of the protocatechuic acid and resveratrol combined inducible promoter shown in NO.1 is P PR1-1 [(O TRC1 ) 2 -P SV40- O PcaV- (O TRC1 ) 2 ]; b) Protocatechuic acid and resveratrol combined inducible promoter nucleotide sequence P PR1-2 [(O TRC1 ) 2 -P SV40- (O PcaV ) 2- (O TRC1 ) shown in SEQ ID NO.2 2 ]; c) Protocatechuic acid and resveratrol combined inducible promoter nucleotide sequence P PR1-3 shown in SEQ ID NO.3 [(O TRC1 )
  • a gene module with a strong promoter driving the expression of the protocatechuic acid transporter PcaK can also be included, that is, the second version of the AND gate construction adds a new gene expression module based on the first version. , that is, a gene module with a strong promoter driving the expression of protocatechuic acid transporter PcaK.
  • the protocatechuic acid transporter PcaK has the function of pumping extracellular protocatechuic acid small molecules into the cell. Therefore, compared with the first version of the AND gate, when dual signals are input simultaneously, the second version of the AND gate signal output requires a lower protocatechuic acid concentration.
  • the AND gate of exogenous gene expression can perform correct logical operations in mouse liver.
  • an AND gate of endogenous gene expression can perform the correct logical operation of endogenous gene activation in mouse liver.
  • the A that regulates gene expression does not include the B gate, that is, protocatechuic acid does not include the resveratrol gate. It is a logic operation method that only has a signal output when the protocatechuic acid input resveratrol signal is not input.
  • the protocatechuic acid gene expression module excluding resveratrol is composed of three gene expression modules, including a gene module with a strong promoter driving the expression of the resveratrol-responsive recombinant transcription activator TtgR-VPR, a strong promoter
  • the promoter drives the gene module for expression of protocatechuic acid in response to the recombinant transcriptional repressor KRAB-PcaV, and the gene expression module for protocatechuic acid and resveratrol to drive downstream signal output from the promoter P PR2 ;
  • the P PR2 It is composed of a weak promoter whose 5' end is immediately adjacent to the TtgR operator binding site and the 3' end is immediately adjacent to the PcaV operator binding site.
  • the resveratrol-responsive recombinant transcription activator TtgR-VPR and the protocatechuic acid-responsive recombinant transcription repressor KRAB-PcaV bind to the operons O TRC1 and O PcaV of the promoter P PR2 respectively, and transcription
  • the combination of the activator TtgR-VPR and P PR2 can drive the weak promoter, but the combination of the recombinant transcriptional repressor KRAB-PcaV and the operator O PcaV ultimately prevents P PR2 from driving the expression of downstream genes.
  • protocatechuic acid Only when protocatechuic acid is imported and resveratrol is not imported, protocatechuic acid promotes the dissociation of the recombinant transcriptional repressor KRAB-PcaV from the promoter P PR2 , and at the same time the binding of the transcriptional activator TtgR-VPR to P PR2 Successfully drives downstream gene expression, enabling signal output.
  • the P PR2 can be composed of different types of protocatechuic acid and resveratrol-regulated promoters according to the type of weak promoter and the different copy numbers of the operators OPcaV and OTRC1 , including: a) SEQ ID NO Protocatechuic acid and resveratrol combined inducible promoter nucleotide sequence P PR2-1 [(O TRC1 ) 2 -P hCMVmin- O PcaV ] shown in .7; b) shown in SEQ ID NO.8 The protocatechuic acid and resveratrol combined inducible promoter nucleotide sequence P PR2-2 [(O TRC1 ) 2 -P hCMVmin- (O PcaV ) 2 ]; c) shown in SEQ ID NO.9 Protocatechuic acid and resveratrol combined inducible promoter nucleotide sequence P PR2-3 [(O TRC1 ) 2 -P hCMVmin- (O P
  • the B gate that regulates gene expression does not include the A gate, that is, resveratrol does not include the protocatechuic acid gate. It is a logical operation method that has a signal output only when the resveratrol input protocatechuic acid signal is not input.
  • the resveratrol excluding protocatechuic acid gate is composed of three gene expression modules, including a constitutive promoter driving the expression of protocatechuic acid-responsive recombinant transcription activator PcaV-VPR, a constitutive promoter
  • the gene module that drives the expression of the recombinant transcriptional repressor TtgR-KRAB in response to resveratrol and the gene expression module that drives the downstream signal output from the protocatechuic acid and resveratrol promoter P PR3 ;
  • the P PR3 is composed of a weak
  • the 5' end of the promoter is immediately adjacent to the PcaV operator binding site and the 3' end is adjacent to the TtgR operator binding site.
  • the protocatechuic acid-responsive recombinant transcription activator PcaV-VPR and the resveratrol-responsive recombinant transcription repressor TtgR-KRAB bind to the operons O TRC1 and O PcaV of the promoter P PR3 respectively, and transcription
  • the combination of the activator PcaV-VPR and P PR3 can drive the weak promoter, but the combination of the recombinant transcriptional repressor TtgR-KRAB and the operator O TRC1 ultimately prevents the expression of downstream genes driven by P PR3 .
  • resveratrol Only when resveratrol is input and protocatechuic acid is not input, resveratrol promotes the dissociation of the recombinant transcription repressor TtgR-KRAB from the promoter P PR3 , and at the same time the binding of the transcription activator PcaV-VPR to P PR3 Successfully drives downstream gene expression, enabling signal output.
  • the P PR3 can be composed of different types of protocatechuic acid and resveratrol-regulated promoters according to the type of weak promoter and the different copy numbers of the operators OPcaV and OTRC1 , including: a) SEQ ID NO .10 shows the protocatechuic acid and resveratrol combined inducible promoter nucleotide sequence P PR3-1 [O PcaV -P hCMVmin- (O TRC1 ) 2 ]; b) SEQ ID NO.11 The protocatechuic acid and resveratrol combined inducible promoter nucleotide sequence P PR3-2 [(O PcaV ) 2 -P hCMVmin- (O TRC1 ) 2 ]; c) shown in SEQ ID NO.12 Protocatechuic acid and resveratrol combined inducible promoter nucleotide sequence P PR3-3 [(O PcaV ) 3 -P hCMVmin- (O TRC1 )
  • the NOR gate (NOR) for regulating gene expression is a logic operation method that has a signal output only when neither protocatechuic acid nor resveratrol is input.
  • the NOR gate is composed of five gene expression modules, including a constitutive promoter driving the expression of protocatechuic acid-responsive recombinant transcription activator PcaV-VPR, and a protocatechuic acid-inducible promoter driving the fusion protein Coh2 - a gene module for VP16 expression, a gene module for a constitutive promoter to drive the expression of the resveratrol-responsive recombinant transcriptional activator TtgR-VPR, a gene module for a resveratrol-inducible promoter to drive the expression of the fusion protein TetR-Docs, and the original Catechin and resveratrol work together to promote P PR4 , a gene expression module that drives downstream signal output; the P PR4 is composed of the 5' end of a weak promoter followed by the
  • the protocatechuic acid or resveratrol-responsive recombinant transcriptional activator binds to the corresponding inducible promoter and drives the expression of the fusion protein Coh2-VP16 and TetR-Docs. Since Docs and Coh2 are a pair of proteins that can automatically bind, the resulting TetR-Docs-Coh2-VP16 fusion protein can bind to the P PR4 promoter to drive the output of downstream signals. When one of the signals or both signals are not input, the TetR-Docs-Coh2-VP16 fusion protein cannot be formed, resulting in the P PR4 promoter being unable to drive the output of downstream signals.
  • the OR gate (OR) for regulating exogenous gene expression is a logical operation method that outputs a signal as long as protocatechuic acid or resveratrol exists or both exist at the same time.
  • the OR gate is composed of five gene expression modules, including a gene module in which a constitutive promoter drives the expression of protocatechuic acid-responsive recombinant transcriptional suppressor KRAB-PcaV, and a protocatechuic acid-inducible promoter drives the expression of Gal4-VP64.
  • the retinol co-operating promoter P PR5 drives the gene expression module of downstream signal output; the P PR5 is composed of the 5' end of the weak promoter followed by the Gal4 operator binding site. P PR5 is also known as the galactose response. Promoter.
  • protocatechuic acid-responsive recombinant transcription suppressor KRAB-PcaV and resveratrol-responsive recombinant transcription repressor TtgR-KRAB respectively bind to the corresponding repressed promoters, preventing tetracycline-responsive recombinant transcription.
  • Activator Gal4-VP64 expression The promoter P PR5 activated without Gal4-VP64 binding cannot drive the output of downstream signals.
  • Gal4-VP64 is expressed and binds to the promoter P PR5 to drive the output of downstream signals.
  • the above-mentioned protocatechuic acid and resveratrol biological logic operator of the present invention regulates gene expression, including exogenous gene expression, and also includes endogenous gene expression.
  • the present invention also provides a biological logic operator for regulating gene expression that realizes accurate logical operations for simultaneous activation of single genes or multiple genes at the cellular level, which includes a combination of CRISPR/dCas9 system modules and is implemented as CRISPR/dCas9 through combination.
  • a biological logic operator for regulating gene expression that realizes accurate logical operations for simultaneous activation of single genes or multiple genes at the cellular level, which includes a combination of CRISPR/dCas9 system modules and is implemented as CRISPR/dCas9 through combination.
  • the precise regulatory operation provided by mediated endogenous gene expression enables simultaneous activation of single genes or multiple genes at the cellular level.
  • the present invention also provides the endogenous gene expression biological logic operator regulated by protocatechuic acid and resveratrol, which includes two processors.
  • the output signal of processor 1 It is a transcription activator containing the MS2 protein structure; the output signal of processor 2 is the activation of specific endogenous genes.
  • the transcription activator (MS2-TAT) containing the MS2 protein structure is formed by fusing the MS2 protein with different transcription activators, including: MS2-VPR, MS2-VP64-P65, MS2-p65-HSF1, etc.
  • the genetic components of the processor 2 include constitutively expressed dCas9 and gRNA MS2 (containing the binding site of MS2 protein).
  • the transcription activator MS2-TAT (derived from the output signal of processor 1) containing the MS2 protein structure, dCas9 and gRNA MS2 of processor 2 form a transcription complex gRNA MS2- dCas9-MS2-TAT to achieve specificity Target and activate endogenous gene expression.
  • the endogenous gene expression biological logic operator can realize the logic operation of whether multiple endogenous genes are simultaneously activated or not by adding different gRNA MS2 targeting endogenous genes.
  • the present invention also proposes a method for constructing the gene expression biological logic operator, in which the gene expression modules involved in each logic operation are cloned onto a mammalian expression vector to complete the construction of each logic operation.
  • the construction method includes the following steps: gene synthesis of gene expression modules designed by various logical operations; cloning of synthesized gene fragments into mammalian expression vectors through enzyme digestion or seamless gene assembly; screening and sequencing identification to obtain Plasmid containing gene expression module.
  • the present invention also proposes a method for regulating expression using the gene expression biological logic operator.
  • the method is controlled by the processor included in each logical operation; the method includes but is not limited to at least one or more of the following types:
  • Protocatechuic acid does not include resveratrol gate regulatory expression method: only when protocatechuic acid is input and resveratrol is not input, protocatechuic acid does not include protocatechuic acid in the resveratrol gate processor Prompts the recombinant transcriptional repressor KRAB-PcaV to dissociate from the promoter P PR2 , and at the same time, the combination of the transcriptional activator TtgR-VPR and P PR2 successfully drives the expression of downstream genes, allowing the signal to be output;
  • Resveratrol does not include protocatechuic acid gate regulatory expression method: only when resveratrol is input and protocatechuic acid is not input, resveratrol does not include protocatechuic acid gate processor in resveratrol Alcohol promotes the dissociation of the recombinant transcriptional repressor TtgR-KRAB from the promoter P PR3 . At the same time, the combination of the transcriptional activator PcaV-VPR and P PR3 successfully drives the expression of downstream genes, allowing the signal to be output;
  • NOR gate regulated expression method Only when no signal is input, the protocatechuic acid or resveratrol-responsive recombinant transcription activator in the NOR gate processor binds to the corresponding inducible promoter and drives Expression of fusion proteins Coh2-VP16 and TetR-Docs. Since Docs and Coh2 are a pair of proteins that can automatically bind, the resulting TetR-Docs-Coh2-VP16 fusion protein can bind to the P PR4 promoter to drive the output of downstream signals;
  • the method of regulating expression includes CRISPR/dCas9-mediated expression of endogenous genes to achieve accurate logical operations of simultaneous activation of single genes or multiple genes at the cellular level.
  • the present invention also provides a biological computer and or system, equipment or device for regulating gene expression by protocatechuic acid and resveratrol, which includes the above biological logic operator for regulating gene expression by protocatechuic acid and resveratrol.
  • the present invention also provides the application of the gene expression biological logic operator, the gene expression biological computer and/or system, etc. in in vitro and in vitro logic operations.
  • the present invention also provides a eukaryotic expression vector, that is, a eukaryotic expression vector for performing logical operations in the gene expression biological logic operator, the gene expression biological computer and/or the system.
  • the invention also discloses a eukaryotic expression vector for performing operations on exogenous gene expression and endogenous gene expression in animals.
  • the exogenous genes regulated by protocatechuic acid and resveratrol are plasmids pWH75 (P hCMV -TtgR-KRAB-P2A-KRAB-PcaV-T2A-PcaK-pA) and pWH47 [P PR1-5 - firefly luciferase-pA;P PR1-5 ,(O TRC1 ) 2 -P hCMV -(O PcaV ) 2 -(O TRC1 ) 2 ].
  • the eukaryotic expression vector that regulates the expression of endogenous genes regulated by protocatechuic acid and resveratrol and performs correct logical operations in mouse liver is the plasmid pWH75 (P hCMV -TtgR- KRAB-P2A-KRAB-PcaV-T2A-PcaK-pA), pWH43 [P PR1-5 -MS2-p65-HSF1-pA; P PR1-5 , (O TRC1 ) 2 -P hCMV - (O PcaV ) 2 - (O TRC1 ) 2 ] and pJY493 (P U6 -sgRNA1 Ascl1- P U6 -sgRNA2 Ascl1 -P hCMV -dCas9-pA).
  • the invention also provides a regulatory input signal, namely green small molecule substances protocatechuic acid and resveratrol as gene expression regulatory input signals.
  • the present invention also provides applications of protocatechuic acid and resveratrol as regulatory input signals in regulating exogenous gene expression, endogenous gene expression biological logic operators, and in the gene expression biological computers and/or systems. In specific embodiments, it is used as a regulatory input signal in a biological logic operator that regulates mammalian gene expression, including applications in lactation and in mice.
  • the present invention also proposes a nucleotide sequence of a combined inducible promoter of protocatechuic acid and resveratrol.
  • the nucleic acid sequence of the promoter sequence is selected from one or more of the following sequences SEQ ID NO. 1-17 :
  • SEQ ID NO.1 Nucleotide sequence of protocatechuic acid and resveratrol combined inducible promoter P PR1-1 ;
  • SEQ ID NO.2 Nucleotide sequence of protocatechuic acid and resveratrol combined inducible promoter P PR1-2 ;
  • SEQ ID NO.3 Nucleotide sequence of protocatechuic acid and resveratrol combined inducible promoter P PR1-3 ;
  • SEQ ID NO.4 Nucleotide sequence of protocatechuic acid and resveratrol combined inducible promoter P PR1-4 ;
  • SEQ ID NO.5 Nucleotide sequence of protocatechuic acid and resveratrol combined inducible promoter P PR1-5 ;
  • SEQ ID NO.6 Nucleotide sequence of protocatechuic acid and resveratrol combined inducible promoter P PR1-6 ;
  • SEQ ID NO.7 Nucleotide sequence of protocatechuic acid and resveratrol combined inducible promoter P PR2-1 ;
  • SEQ ID NO.8 Nucleotide sequence of protocatechuic acid and resveratrol combined inducible promoter P PR2-2 ;
  • SEQ ID NO.9 Nucleotide sequence of protocatechuic acid and resveratrol combined inducible promoter P PR2-3 ;
  • SEQ ID NO.10 Nucleotide sequence of protocatechuic acid and resveratrol combined inducible promoter P PR3-1 ;
  • SEQ ID NO.11 Nucleotide sequence of protocatechuic acid and resveratrol combined inducible promoter P PR3-2 ;
  • SEQ ID NO.12 Nucleotide sequence of protocatechuic acid and resveratrol combined inducible promoter P PR3-3 ;
  • SEQ ID NO.13 Nucleotide sequence of protocatechuic acid and resveratrol combined inducible promoter P PR3-4 ;
  • SEQ ID NO. 14 Nucleotide sequence of protocatechuic acid and resveratrol combined inducible promoter P PR3-5 .
  • the gene expression biological logic operator, gene expression biological computer and/or system, eukaryotic expression vector, plasmid, construction method, and control expression method of the present invention include but It is not limited to mammalian gene expression, including uploading to mammalian cells and mice, or in vitro applications.
  • the beneficial effects of the present invention include: for the first time, a mammalian biological logic operator regulated by protocatechuic acid and resveratrol is artificially designed and synthesized, including a biological logic operator that regulates exogenous gene expression and endogenous gene expression.
  • Biological logic operators can be uploaded to mammalian cells and mice to perform correct logic operations.
  • This invention innovatively proposes for the first time the combination of mammalian synthetic biology, biological logic operators, and gene epigenetic modification technology mediated by the CRISPR-dCas9 system.
  • the present invention further combines and utilizes the assembly of CRISPR/dCas9 system modules, so that the biological logic operator of endogenous gene expression can perform accurate logic operations of simultaneous activation of single genes or multiple genes at the cellular level, which is a CRISPR/dCas9-mediated Endogenous gene expression provides a precise regulatory algorithm.
  • the biological logic operator of the present invention uses protocatechuic acid and resveratrol as regulatory input signals, and has various biological effects such as antioxidant and anti-cancer. Compared with the existing technology that uses antibiotics as input signals, it is less effective in clinical applications. More healthy and safer.
  • the present invention also provides a new mammalian computer controlled by green small molecules, and provides a precise control calculation method for CRISPR/dCas9-mediated endogenous gene expression.
  • Figure 1 is an AND gate optimization data diagram of protocatechuic acid and resveratrol regulating exogenous gene expression according to the present invention.
  • Figure 2 is a graph showing optimization data of the protocatechuic acid-excluding resveratrol gate in regulating exogenous gene expression by protocatechuic acid and resveratrol according to the present invention.
  • Figure 3 is a diagram showing the optimization data of protocatechuic acid and resveratrol regulating the expression of exogenous genes according to the present invention. Resveratrol does not include protocatechuic acid.
  • Figure 4 is a schematic diagram, a value-added table and a fluorescence calculation result of an AND gate for regulating exogenous gene expression by protocatechuic acid and resveratrol according to the present invention.
  • Figure 5 is a schematic diagram, value-added table and fluorescence calculation results of protocatechuic acid excluding resveratrol gate in regulating exogenous gene expression with protocatechuic acid and resveratrol according to the present invention.
  • Figure 6 is a schematic diagram, a value-added table and a fluorescence calculation result of protocatechuic acid and resveratrol regulating the expression of exogenous genes according to the present invention.
  • Resveratrol does not include protocatechuic acid gate.
  • Figure 7 is a schematic diagram and value-added NOR gate for regulating exogenous gene expression with protocatechuic acid and resveratrol according to the present invention. Table and fluorescence calculation results.
  • Figure 8 is an OR gate schematic diagram, a value-added table and fluorescence calculation results for regulating exogenous gene expression by protocatechuic acid and resveratrol according to the present invention.
  • Figure 9 is a schematic diagram of the design of a biological logic operator for regulating endogenous gene expression with protocatechuic acid and resveratrol according to the present invention.
  • Figure 10 is a diagram showing the calculation results of the biological logic operator targeting endogenous gene RHOXF2 using protocatechuic acid and resveratrol according to the present invention to regulate endogenous gene expression.
  • Figure 11 is a diagram showing the calculation results of the biological logic operator of the present invention for regulating endogenous gene expression by protocatechuic acid and resveratrol while simultaneously targeting endogenous genes ASCL1, MIAT and RHOXF2.
  • Figure 12 is a diagram showing the logical operation results of the AND gate of the present invention in which protocatechuic acid and resveratrol regulate exogenous gene expression in mouse liver.
  • Figure 13 is a diagram showing the logical operation results of the AND gate of protocatechuic acid and resveratrol regulating endogenous gene expression in mouse liver according to the present invention.
  • Example 1 Construction of biological logic operator for exogenous gene expression regulated by protocatechuic acid and resveratrol
  • This example includes the construction method of the plasmid vector involved in the biological logic operator for exogenous gene expression regulated by protocatechuic acid and resveratrol. The detailed design plan and steps are shown in Table 1.
  • Example 2 Optimizing the AND gate of protocatechuic acid and resveratrol in regulating exogenous gene expression.
  • the first step is plasmid construction.
  • the plasmid construction in this example is detailed in Table 1.
  • the second step is cell seeding.
  • HEK-293T cells were seeded in a 24-well plate at a density of 5 ⁇ 10 4 cells per well, and 500 ⁇ l of DMEM medium containing 10% FBS was added to each well.
  • the third step is plasmid transfection.
  • 100ng pJY29 P hEF1 ⁇ -KRAB-PcaV-pA
  • 100ng pLF24 P hCMV -TtgR-KRAB-pA
  • 100ng pPR1 P PR1-1 -SEAP-pA; P PR1-1 : SEQ ID NO.1
  • 100ng pPR2 P PR1-2 -SEAP-pA; P PR1-2 : SEQ ID NO.2
  • 100ng pPR3 P PR1-3 -SEAP-pA; P PR1-3 : SEQ ID NO.3
  • 100ng pPR4 P PR1-4 -SEAP-pA; P PR1-4 : SEQ ID NO.4
  • 100ng pPR5 P PR1-5 -SEAP-pA; P PR1-5 : SEQ ID NO.5
  • 100ng pPR6 P PR1-6 -SEAP-pA; P PR1-6 : SEQ ID NO.
  • each group of plasmids mentioned above was 300ng, premixed with the transfection reagent polyethylenimine PEI (mass ratio of plasmid to PEI 1:3), and dissolved in 50 ⁇ l of serum-free and antibiotic-free DMEM. After resting for 15 minutes, dropwise add DNA-PEI premix to the cells in each well.
  • the fourth step is to add the inducer. After 6 hours of transfection, the medium was replaced with fresh medium and different combinations of inducers were added.
  • the inducer combinations involved in each logic gate are divided into 4 types, including (1) no inducer, (2) only 400 ⁇ M protocatechuic acid inducer, (3) only 20 ⁇ M resveratrol Alcohol inducer, (4) simultaneously add two inducers, 400 ⁇ M protocatechuic acid and 20 ⁇ M resveratrol.
  • the cell supernatant was collected and the expression of SEAP was detected.
  • Example 3 Optimizing protocatechuic acid and resveratrol for regulating exogenous gene expression.
  • Protocatechuic acid does not include the resveratrol phylum.
  • the first step is plasmid construction.
  • the plasmid construction in this example is detailed in Table 1.
  • the second step is cell seeding. (The specific steps are the same as Embodiment 2 of the present invention)
  • the third step is plasmid transfection. 120ng pJY19 (P CAG -KRAB-PcaV-pA), 25ng pLF119 (P SV40 -PcaV-VPR-pA) and 25ng pPR7 (P PR2-1 -SEAP-pA; P PR2-1 : SEQ ID NO.7 ), 25ng pPR8 (P PR2-2 -SEAP-pA; P PR2-2 : SEQ ID NO.8), 25ng pPR9 (P PR2-3 -SEAP-pA; P PR2-3 : SEQ ID NO.9) in total transfected into HEK-293T cells.
  • each set of plasmids mentioned above is 170ng, premixed with the transfection reagent PEI (mass ratio of plasmid to PEI 1:3), and dissolved in 50 ⁇ l of serum-free and antibiotic-free DMEM. After resting for 15 minutes, dropwise add DNA-PEI premix to the cells in each well.
  • the fourth step is to add the inducer. (The specific steps are the same as Embodiment 2 of the present invention)
  • the cell supernatant was collected and the expression of SEAP was detected.
  • Example 4 Optimizing protocatechuic acid and resveratrol to regulate exogenous gene expression. Resveratrol does not include the protocatechuic acid gate.
  • the first step is plasmid construction.
  • the plasmid construction in this example is detailed in Table 1.
  • the second step is cell seeding. (The specific steps are the same as Embodiment 2 of the present invention)
  • the third step is plasmid transfection.
  • Mix 100ng pLF24(P hCMV -TtgR-KRAB-pA), 20ng pLF215 (P SV40 -PcaV-VPR-pA) were respectively compared with 25ng pPR10 (P PR3-1 -SEAP-pA; P PR3-1 : SEQ ID NO.10), 25ng pPR11 (P PR3-2 -SEAP-pA; P PR3 -2 : SEQ ID NO.11), 25ng pPR12 (P PR3-3 -SEAP-pA; P PR3-3 : SEQ ID NO.12), 25ng pPR13 (P PR3-4 -SEAP-pA; P PR3-4 : SEQ ID NO.13), 25ng pPR14 (P PR3-5 -SEAP-pA; P PR3-5 : SEQ ID NO.14) was co-transfected into HEK-293T cells.
  • each set of plasmids mentioned above is 145ng, premixed with the transfection reagent PEI (mass ratio of plasmid to PEI 1:3), and dissolved in 50 ⁇ l of serum-free and antibiotic-free DMEM. After resting for 15 minutes, dropwise add DNA-PEI premix to the cells in each well.
  • the fourth step is to add the inducer. (The specific steps are the same as Embodiment 2 of the present invention)
  • the cell supernatant was collected and the expression of SEAP was detected.
  • Example 5 verifying the calculation results of the exogenous gene expression biological logic operator regulated by protocatechuic acid and resveratrol in HEK-293T cells, that is, uploading plasmid elements related to five logic gates to HEK-293T. Logical operations are performed in cells.
  • the first step is plasmid construction.
  • the plasmid construction in this example is detailed in Table 1.
  • the second step is cell seeding. (The specific steps are the same as Embodiment 2 of the present invention)
  • the third step is plasmid transfection.
  • the transfection system in this example is divided into 5 groups based on five logic gates.
  • the amount of transfected plasmid and plasmid elements are shown in Table 2.
  • the total amount of each group of plasmids mentioned above is 300ng, premixed with the transfection reagent PEI (mass ratio of plasmid to PEI 1:3), and dissolved in 50 ⁇ l of serum-free and antibiotic-free DMEM. After resting for 15 minutes, dropwise add DNA-PEI premix to the cells in each well.
  • the fourth step is to add the inducer. (The specific steps are the same as Embodiment 2 of the present invention)
  • the fluorescence images of each group were taken, and the fluorescence expression was quantitatively analyzed using flow cytometry.
  • Example 6 verifying the calculation results of the endogenous gene expression biological logic operator (Figure 6) regulated by protocatechuic acid and resveratrol in HEK-293T cells, that is, uploading plasmid elements related to five logic gates respectively to HEK-293T cells for logical operations.
  • the first step is plasmid construction.
  • the plasmid construction in this example is detailed in Table 1.
  • the second step is cell seeding. (The specific steps are the same as Embodiment 2 of the present invention)
  • the third step is plasmid transfection.
  • the transfection system in this example can be divided into 5 groups.
  • the amount of transfected plasmid and plasmid components are shown in Table 3.
  • the total amount of each group of plasmids mentioned above is 450ng, premixed with the transfection reagent PEI (mass ratio of plasmid to PEI 1:3), and dissolved in 50 ⁇ l of serum-free and antibiotic-free DMEM. After resting for 15 minutes, dropwise add DNA-PEI premix to the cells in each well.
  • the fourth step is to add the inducer. (The specific steps are the same as Embodiment 2 of the present invention)
  • the fifth step is to collect cells from each group after induction for 24 hours, and use RT-qPCR to detect the mRNA level of the endogenous gene RHOXF2.
  • the specific steps include: (1) using Trizol method to extract total RNA; (2) using inversion The kit reverses RNA into cDNA; (3) Quantitative analysis of genes. The quantitative system was placed in a real-time PCR instrument for PCR reaction, and the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was selected as the internal reference gene. (4) Analyze and calculate the relative quantitative value of the gene.
  • GPDH human glyceraldehyde-3-phosphate dehydrogenase
  • Example 7 Research on endogenous gene expression regulated by protocatechuic acid and resveratrol using a biological logic operator to simultaneously perform logic operations on multiple target genes.
  • the first step is plasmid construction.
  • the plasmid construction in this example is detailed in Table 1.
  • the second step is cell seeding. (The specific steps are the same as Embodiment 2 of the present invention)
  • the third step is plasmid transfection.
  • the transfection system in this example can be divided into 5 groups. The amount of transfected plasmids and plasmid components are shown in Table 4. The total amount of plasmids in each group is 500ng, and the transfection reagent PEI (mass ratio of plasmid to PEI is 1:3) is used. Premix and dissolve in 50 ⁇ l of serum-free and antibiotic-free DMEM. After resting for 15 minutes, dropwise add DNA-PEI premix to the cells in each well.
  • the fourth step is to add the inducer. (The specific steps are the same as Embodiment 2 of the present invention)
  • Example 8 Research on the logical operation of exogenous gene expression and gate regulated by protocatechuic acid and resveratrol in animals.
  • the first step is plasmid construction.
  • the plasmid construction in this example is detailed in Table 1.
  • the plasmid is delivered to the mouse liver.
  • the related plasmids of the AND gate are pWH75 (P hCMV -TtgR-KRAB-P2A-KRAB-PcaV-T2A-PcaK-pA) and pWH47 [P PR1-5 -firefly luciferase-pA; P PR1-5 , (O TRC1 ) 2 -P hCMV -(O PcaV ) 2 -(O TRC1 ) 2 ], and the plasmid was delivered into the mouse liver via tail vein injection.
  • mice were divided into four groups.
  • the first group of mice was a no-signal input group, that is, only solvent was injected;
  • the second group was a protocatechuic acid signal input group, that is, protocatechuic acid was injected intraperitoneally, 3 times a day.
  • the third group is the resveratrol signal input group, that is, intraperitoneal injection of resveratrol twice a day (the total daily dose of resveratrol is 150 mg/kg );
  • the fourth group is the dual signal input group of protocatechuic acid and resveratrol, that is, intraperitoneal injection of protocatechuic acid (total amount: 750 mg/kg) and resveratrol (total amount: 150 mg/kg).
  • the fourth step is in vivo imaging of mice. Thirty-six hours after the completion of plasmid delivery through the tail vein of mice, the mice were anesthetized and intraperitoneally injected with luciferase substrate, and 5 minutes later, the mice were placed in an in vivo imager for imaging.
  • Example 9 Research on the logical operation of endogenous gene expression and gate regulated by protocatechuic acid and resveratrol in animals.
  • the first step is plasmid construction.
  • the plasmid construction in this example is detailed in Table 1.
  • the plasmid is delivered to the mouse liver.
  • the related plasmids of the AND gate are pWH75 (P hCMV -TtgR-KRAB-P2A-KRAB-PcaV-T2A-PcaK-pA), pWH43 [P PR1-5 -MS2-p65-HSF1-pA; P PR1-5 , (O TRC1 ) 2 -P hCMV -(O PcaV ) 2 -(O TRC1 ) 2 ] and pJY493 (P U6 -sgRNA1 Ascl1 -P U6 -sgRNA2 Ascl1 -P hCMV -dCas9-pA), via tail vein injection Plasmids were delivered into mouse livers.
  • mice were divided into four groups.
  • the mice in the first group were the group without signal input, that is, only the solvent was injected;
  • the second group is the protocatechuic acid signal input group, that is, intraperitoneal injection of protocatechuic acid, 3 times a day (the total daily dose of protocatechuic acid is 750mg/kg);
  • the third group is the resveratrol signal input group, that is, intraperitoneal injection Resveratrol was injected twice a day (the total dose of resveratrol per day was 150 mg/kg);
  • the fourth group was the dual signal input group of protocatechuic acid and resveratrol, that is, intraperitoneal injection of protocatechuic acid (total dose of resveratrol).
  • the amount is 750mg/kg) and resveratrol (the total amount is 150mg/kg).
  • the fourth step is to use the RT-qPCR method to detect the mRNA level of the endogenous gene Ascl1 in the mouse liver.
  • the specific steps include: (1) After the mouse is euthanized, the mouse liver is removed and ground; (2) Trizol's method is used Extract total RNA; (2) reverse RNA into cDNA using a reversal kit; (3) quantitative analysis of genes. The quantitative system was placed in a real-time PCR instrument for PCR reaction, and the mouse glyceraldehyde-3-phosphate dehydrogenase (Gadph) gene was selected as the internal reference gene. (4) Analyze and calculate the relative quantitative value of the gene.
  • Table 2 Plasmid names and transfection tables of exogenous gene expression computers regulated by protocatechuic acid and resveratrol
  • Table 4 Plasmid names and transfection tables for endogenous gene expression regulated by protocatechuic acid and resveratrol. The computer simultaneously performs logical operations on three target genes.

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Abstract

本发明公开了一种原儿茶酸与白藜芦醇调控基因表达的生物逻辑运算器，其逻辑运算包含以下逻辑门，分别为与门，蕴含非门，或非门以及或门，包括外源基因表达、内源基因表达；所述生物逻辑运算器为双输入、单输出的运算模式。本发明还公开了通过组装CRISPR/dCas9系统模块的原儿茶酸与白藜芦醇调控内源基因表达的生物逻辑运算器，其能够在细胞水平上进行单基因或多基因同时激活的准确逻辑运算。本发明还公开了外源基因表达以及内源基因表达与门在动物体内进行运算的真核表达载体。本发明提供了新型绿色小分子调控的哺乳动物计算机，且为CRISPR/dCas9介导的内源基因表达提供了精准调控运算方式。

Description

原儿茶酸和白藜芦醇调控基因表达生物逻辑运算器及其应用 技术领域
本发明涉及合成生物学、基因编辑领域，具体涉及绿色小分子物质原儿茶酸和白藜芦醇调控的基因表达生物逻辑运算器及其在体内、外进行准确运算的应用。
背景技术
合成生物学作为全新的研究领域，其核心是设计组装各种分子组件(如DNA,RNA,多肽和蛋白质等)，以获取具有特定功能的生物模块，从而实现对细胞乃至生物体的功能改造。近年来，合成生物学取得了蓬勃发展，基因线路的设计不再局限于单一信号的输入输出，更趋于多层次和复杂化，以推进精准医学的发展。
生物逻辑运算器的构建是基因线路走向设计复杂化的一大代表。其中，逻辑门是计算机的运行基础，它通过对输入的各种信号进行逻辑运算，然后输出所需信息的基础装置。通过对多种不同的布尔逻辑门进行人工组合，可以在细胞内创造出复杂的生物逻辑运算器电路。最初研究较多的生物逻辑运算器是在细菌中进行，例如Voigt CA实验室构建了16种由阿拉伯糖和四环素响应的双输入逻辑门，Collins和Peng Yin研究团队共同完成了RNA分子生物逻辑运算器的合成等。相对于细菌中的生物逻辑运算器，哺乳动物细胞中的生物逻辑运算器起步较晚，如Martin Fussenegger研究团队在2012年首次在哺乳动物细胞中合成了多种由根皮素和红霉素响应的双输入逻辑运算以及在2018年成功构建了三输入-两输出的全加器。而如上所述目前合成的哺乳动物生物逻辑运算器一般都采用了抗生素作为输入信号物，但抗生素的毒副作用会影响该类生物逻辑运算器在医学上的进一步应用。
CRISPR-dCas9系统提供了一个基因组定点转录调控平台，dCas9通过与其他效应蛋白的融合(如转录因子、修饰因子等)可进行定点基因表达调控，表观遗传修饰等行为。现已构建出红光，蓝光和雷帕霉素等单个信号物质调控CRISPR-dCas9系统的基因线路，但目前仍缺乏多个信号物质共同控制CRISPR-dCas9系统的基因装置。
发明内容
本发明首次创新公开了一种由绿色小分子物质调控的生物逻辑运算器。本发明公开的具体实施方案中，以具有多种生物功效的原儿茶酸和白藜芦醇作为双输 入信号物质，即双输入布尔逻辑门调控系统，以原儿茶酸(PCA)和白藜芦醇(RES)为诱导物，能够在细胞内和动物体内正确运行的逻辑电路，其优势在于PCA和RES均是天然的健康安全的小分子化合物，其中原儿茶酸分布广泛，存在于广枣、单参等中草药中，同时也是花青素和茶多酚的主要代谢产物；白藜芦醇是一种天然酚类化合物，广泛存在于葡萄、花生、蓝莓等植物中，已有多篇研究报道其具有抗肿瘤、抗炎等功效。同时，本发明公开了一种原儿茶酸与白藜芦醇调控外源/内源基因表达的生物逻辑运算器，即该生物逻辑运算器能够在细胞水平上进行单基因或多基因同时激活的准确逻辑运算，这为CRISPR/dCas9介导的内源基因表达提供了一种精准的调控运算方式。
本发明提供了一种原儿茶酸和白藜芦醇调控的生物逻辑运算器，其中所述生物逻辑运算器包括原儿茶酸和白藜芦醇调控的外源基因表达生物逻辑运算器、原儿茶酸和白藜芦醇调控的内源基因表达生物逻辑运算器，所述生物逻辑运算器的逻辑运算均包括与门(AND)，A不包括B(A NIMPLY B)即原儿茶酸不包括白藜芦醇门，B不包括A(B NIMPLY A)即白藜芦醇不包括原儿茶酸门，或非门(NOR)以及或门(OR)五种逻辑运算；其中，A代表原儿茶酸输入信号，B代表白藜芦醇输入信号；所述生物逻辑运算器为双输入、单输出的运算模式；所述双输入包括原儿茶酸和白藜芦醇两种输入信号；所述单输出包括输出信号报告蛋白d2EYFP、Luciferase、SEAP或任何一种特定基因或功能蛋白质的上调表达。
其中，所述原儿茶酸和白藜芦醇调控的基因表达与门(AND)是一种只有当信号都输入时才有信号输出的逻辑运算方式。所述与门包括两个版本的基因表达模块构建。第一个版本中，所述与门的基因表达模块包括三个基因表达模块，分别为强启动子驱动原儿茶酸响应重组转录抑制子KRAB-PcaV表达的基因模块，强启动子驱动白藜芦醇响应重组转录抑制子TtgR-KRAB表达的基因模块，以及原儿茶酸和白藜芦醇共同作用启动子PPR1驱动下游信号输出的基因表达模块。当信号都不输入时，重组转录抑制子KRAB-PcaV和TtgR-KRAB结合至PPR1上阻碍下游基因表达，信号不输出；只有当原儿茶酸和白藜芦醇信号同时输入时，重组转录抑制子KRAB-PcaV和TtgR-KRAB从PPR1上解离下来，促使下游基因表达，从而使信号得以输出。
其中，PPR1是由强启动子和PcaV、TtgR操纵子结合位点构成。所述PcaV 操纵子结合位点是来源于原儿茶酸操纵子系统的操纵子元件OPcaV，所述TtgR操纵子结合位点是来源于白藜芦醇操纵子系统的操纵子元件OTRC1，所述的PPR1可驱动下游基因的表达。
其中，所述PPR1可根据强启动子的种类和操纵子OPcaV、OTRC1的不同拷贝数可组成不同类型的原儿茶酸和白藜芦醇调控型启动子，包括：a)SEQ ID NO.1所示的原儿茶酸和白藜芦醇联合诱导型启动子核苷酸序列PPR1-1[(OTRC1)2-PSV40-OPcaV-(OTRC1)2]；b)SEQ ID NO.2所示的原儿茶酸和白藜芦醇联合诱导型启动子核苷酸序列PPR1-2[(OTRC1)2-PSV40-(OPcaV)2-(OTRC1)2]；c)SEQ ID NO.3所示的原儿茶酸和白藜芦醇联合诱导型启动子核苷酸序列PPR1-3[(OTRC1)2-PSV40-(OPcaV)3-(OTRC1)2]；d)SEQ ID NO.4所示的原儿茶酸和白藜芦醇联合诱导型启动子核苷酸序列PPR1-4[(OTRC1)2-PhCMV-OPcaV-(OTRC1)2]；e)SEQ ID NO.5所示的原儿茶酸和白藜芦醇联合诱导型启动子核苷酸序列PPR1-5[(OTRC1)2-PhCMV-(OPcaV)2-(OTRC1)2]；f)SEQ ID NO.6所示的原儿茶酸和白藜芦醇联合诱导型启动子核苷酸序列PPR1-6[(OTRC1)2-PhCMV-(OPcaV)3-(OTRC1)2]等。
进一步地，还可以包括强启动子驱动原儿茶酸转运蛋白PcaK表达的基因模块，即所述与门构建的第二个版本是在第一个版本的基础上增加了一个新的基因表达模块，即强启动子驱动原儿茶酸转运蛋白PcaK表达的基因模块。所述原儿茶酸转运蛋白PcaK具有将细胞外原儿茶酸小分子泵入胞内的功能。因此，与第一版本的与门相比，在双信号同时输入时，第二版本与门信号输出所需的原儿茶酸浓度更低。
在一些具体实施方案中，外源基因表达的与门可以在小鼠肝脏内进行正确的逻辑运算。在一些具体实施方案中，内源基因表达的与门可以在小鼠肝脏内进行内源基因激活的正确逻辑运算。
所述调控基因表达的A不包括B门即原儿茶酸不包括白藜芦醇门，是一种只有原儿茶酸输入白藜芦醇信号不输入时才有信号输出的逻辑运算方式。所述原儿茶酸不包括白藜芦醇门的基因表达模块是由三个基因表达模块组成，其中包括强启动子驱动白藜芦醇响应重组转录激活子TtgR-VPR表达的基因模块，强启动子驱动原儿茶酸响应重组转录抑制子KRAB-PcaV表达的基因模块，以及原儿茶酸和白藜芦醇共同作用启动子PPR2驱动下游信号输出的基因表达模块；所述PPR2 是由弱启动子的5’端紧接着TtgR操纵子结合位点以及3’端紧接着PcaV操纵子结合位点构成。当信号都不输入时，白藜芦醇响应重组转录激活子TtgR-VPR和原儿茶酸响应重组转录抑制子KRAB-PcaV分别结合至启动子PPR2的操纵子OTRC1、OPcaV上，转录激活子TtgR-VPR与PPR2的结合能驱动弱启动子，但重组转录抑制子KRAB-PcaV与操纵子OPcaV的结合最终阻碍了PPR2驱动下游基因的表达。只有当原儿茶酸输入且白藜芦醇不输入时，原儿茶酸促使重组转录抑制子KRAB-PcaV从启动子PPR2上解离下来，同时转录激活子TtgR-VPR与PPR2的结合成功驱动下游基因表达，使信号得以输出。
其中，所述PPR2可根据弱启动子的种类和操纵子OPcaV、OTRC1的不同拷贝数可组成不同类型的原儿茶酸和白藜芦醇调控型启动子包括：a)SEQ ID NO.7所示的原儿茶酸和白藜芦醇联合诱导型启动子核苷酸序列PPR2-1[(OTRC1)2-PhCMVmin-OPcaV]；b)SEQ ID NO.8所示的原儿茶酸和白藜芦醇联合诱导型启动子核苷酸序列PPR2-2[(OTRC1)2-PhCMVmin-(OPcaV)2]；c)SEQ ID NO.9所示的原儿茶酸和白藜芦醇联合诱导型启动子核苷酸序列PPR2-3[(OTRC1)2-PhCMVmin-(OPcaV)3]等。
所述调控基因表达的B不包括A门即白藜芦醇不包括原儿茶酸门，是一种只有白藜芦醇输入原儿茶酸信号不输入时才有信号输出的逻辑运算方式。所述白藜芦醇不包括原儿茶酸门是由三个基因表达模块组成，其中包括组成型启动子驱动原儿茶酸响应重组转录激活子PcaV-VPR表达的基因模块，组成型启动子驱动白藜芦醇响应重组转录抑制子TtgR-KRAB表达的基因模块，以及原儿茶酸和白藜芦醇共同作用启动子PPR3驱动下游信号输出的基因表达模块；所述PPR3是由弱启动子的5’端紧接着PcaV操纵子结合位点以及3’端紧接着TtgR操纵子结合位点构成。当信号都不输入时，原儿茶酸响应重组转录激活子PcaV-VPR和白藜芦醇响应重组转录抑制子TtgR-KRAB分别结合至启动子PPR3的操纵子OTRC1、OPcaV上，转录激活子PcaV-VPR与PPR3的结合能驱动弱启动子，但重组转录抑制子TtgR-KRAB与操纵子OTRC1的结合最终阻碍了PPR3驱动下游基因的表达。只有当白藜芦醇输入且原儿茶酸不输入时，白藜芦醇促使重组转录抑制子TtgR-KRAB从启动子PPR3上解离下来，同时转录激活子PcaV-VPR与PPR3的结合成功驱动下游基因表达，使信号得以输出。
其中，所述PPR3可根据弱启动子的种类和操纵子OPcaV、OTRC1的不同拷贝数可组成不同类型的原儿茶酸和白藜芦醇调控型启动子包括：a)SEQ ID NO.10所示的原儿茶酸和白藜芦醇联合诱导型启动子核苷酸序列PPR3-1[OPcaV-PhCMVmin-(OTRC1)2]；b)SEQ ID NO.11所示的原儿茶酸和白藜芦醇联合诱导型启动子核苷酸序列PPR3-2[(OPcaV)2-PhCMVmin-(OTRC1)2]；c)SEQ ID NO.12所示的原儿茶酸和白藜芦醇联合诱导型启动子核苷酸序列PPR3-3[(OPcaV)3-PhCMVmin-(OTRC1)2]；d)SEQ ID NO.13所示的原儿茶酸和白藜芦醇联合诱导型启动子核苷酸序列PPR3-4[(OPcaV)4-PhCMVmin-(OTRC1)2]；e)SEQ ID NO.14所示的原儿茶酸和白藜芦醇联合诱导型启动子核苷酸序列PPR3-5[(OPcaV)5-PhCMVmin-(OTRC1)2]等。
所述调控基因表达的或非门(NOR)，是一种只有当原儿茶酸和白藜芦醇都不输入时才有信号输出的逻辑运算方式。所述或非门是由五个基因表达模块组成，其中包括组成型启动子驱动原儿茶酸响应重组转录激活子PcaV-VPR表达的基因模块，原儿茶酸诱导型启动子驱动融合蛋白Coh2-VP16表达的基因模块，组成型启动子驱动白藜芦醇响应重组转录激活子TtgR-VPR表达的基因模块，白藜芦醇诱导型启动子驱动融合蛋白TetR-Docs表达的基因模块，以及原儿茶酸和白藜芦醇共同作用启动子PPR4驱动下游信号输出的基因表达模块；所述PPR4是由弱启动子的5’端紧接着TetR操纵子结合位点构成，PPR4又被称为四环素响应的启动子。当信号都不输入时，原儿茶酸或白藜芦醇响应的重组转录激活子结合至相对应的诱导型启动子上，并驱动表达融合蛋白Coh2-VP16和TetR-Docs。由于Docs和Coh2是一对能够自动结合的蛋白，所形成的TetR-Docs-Coh2-VP16融合蛋白能够结合至PPR4启动子上驱动下游信号的输出。当其中一个信号不输入或两个信号都不输入时，TetR-Docs-Coh2-VP16融合蛋白无法形成导致PPR4启动子不能驱动下游信号的输出。
所述调控外源基因表达的或门(OR)，是一种只要原儿茶酸或白藜芦醇存在或两者同时存在时即有信号输出的逻辑运算方式。所述或门是由五个基因表达模块组成，其中包括组成型启动子驱动原儿茶酸响应重组转录抑制子KRAB-PcaV表达的基因模块，原儿茶酸诱导型启动子驱动Gal4-VP64表达的基因模块，组成型启动子驱动白藜芦醇响应重组转录抑制子TtgR-KRAB表达的基因模块，白藜芦醇诱导型启动子驱动融合蛋白Gal4-VP64表达的基因模块，以及原儿茶酸和白藜 芦醇共同作用启动子PPR5驱动下游信号输出的基因表达模块；所述PPR5是由弱启动子的5’端紧接着Gal4操纵子结合位点构成，PPR5又被称为半乳糖响应的启动子。当信号都不输入时，原儿茶酸响应重组转录抑制子KRAB-PcaV和白藜芦醇响应重组转录抑制子TtgR-KRAB分别结合至相对应的抑制型启动子上，阻碍四环素响应的重组转录激活子Gal4-VP64表达。无Gal4-VP64结合激活的启动子PPR5不能驱动下游信号的输出。当其中一个信号输入或两个信号都输入时，Gal4-VP64得以表达并结合至启动子PPR5上驱动下游信号的输出。
本发明上述原儿茶酸和白藜芦醇调控基因表达生物逻辑运算器包括外源基因表达，还包括内源基因表达。
进一步地，本发明还提供了实现在细胞水平上进行单基因或多基因同时激活的准确逻辑运算的调控基因表达生物逻辑运算器，其包括结合CRISPR/dCas9系统模块，通过结合实现为CRISPR/dCas9介导的内源基因表达提供的精准调控运算，实现在细胞水平上进行单基因或多基因同时激活。
进一步地，本发明还提供了所述原儿茶酸和白藜芦醇调控的内源基因表达生物逻辑运算器，其包括两个处理器，在具体实施方案中，其中处理器1的输出信号为包含MS2蛋白结构的转录激活因子；处理器2的输出信号为特定内源基因的激活。所述包含MS2蛋白结构的转录激活因子(MS2-TAT)是由MS2蛋白与不同的转录激活子融合形成，包括：MS2-VPR、MS2-VP64-P65、MS2-p65-HSF1等。所述处理器2的基因组成元件包含组成型表达dCas9和gRNAMS2(含有MS2蛋白的结合位点)。其中，所述包含MS2蛋白结构的转录激活因子MS2-TAT(来源于处理器1的输出信号)与处理器2的dCas9、gRNAMS2组成转录复合物gRNAMS2-dCas9-MS2-TAT，实现特异性地靶向和激活内源基因的表达。进一步地，所述内源基因表达生物逻辑运算器可以通过添加不同靶向内源基因的gRNAMS2可实现多个内源基因同时激活与否的逻辑运算。
本发明还提出了所述基因表达生物逻辑运算器的构建方法，为各个逻辑运算所涉及的基因表达模块克隆至哺乳动物表达载体上，以完成各个逻辑运算的构建。所述构建方法包括以下步骤：基因合成各个逻辑运算所设计的基因表达模块；通过酶切酶连或基因无缝组装的方法将合成的基因片段克隆至哺乳动物表达载体上；筛选测序鉴定以获得包含基因表达模块的质粒。
本发明还提出了利用所述基因表达生物逻辑运算器的调控表达方法，所述方 法由各个逻辑运算中所包含的处理器进行调控；所述方法包括但不限于以下至少一种或几种类型：
1)与门调控表达方法：只有当原儿茶酸和白藜芦醇信号同时输入时，与门处理器中的重组转录抑制子KRAB-PcaV和TtgR-KRAB从PPR1上解离下来，促使下游基因表达，从而使信号得以输出；
2)原儿茶酸不包括白藜芦醇门调控表达方法：只有当原儿茶酸输入且白藜芦醇不输入时，原儿茶酸不包括白藜芦醇门处理器中原儿茶酸促使重组转录抑制子KRAB-PcaV从启动子PPR2上解离下来，同时转录激活子TtgR-VPR与PPR2的结合成功驱动下游基因表达，使信号得以输出；
3)白藜芦醇不包括原儿茶酸门调控表达方法：只有当白藜芦醇输入且原儿茶酸不输入时，白藜芦醇不包括原儿茶酸门处理器中白藜芦醇促使重组转录抑制子TtgR-KRAB从启动子PPR3上解离下来，同时转录激活子PcaV-VPR与PPR3的结合成功驱动下游基因表达，使信号得以输出；
4)或非门调控表达方法：只有当信号都不输入时，或非门处理器中原儿茶酸或白藜芦醇响应的重组转录激活子结合至相对应的诱导型启动子上，并驱动表达融合蛋白Coh2-VP16和TetR-Docs。由于Docs和Coh2是一对能够自动结合的蛋白，所形成的TetR-Docs-Coh2-VP16融合蛋白能够结合至PPR4启动子上驱动下游信号的输出；
5)或门调控表达方法：当其中一个信号输入或两个信号都输入时，Gal4-VP64得以表达并结合至启动子PPR5上驱动下游信号的输出。
进一步地，所述调控表达方法包括CRISPR/dCas9介导的内源基因表达，实现在细胞水平上进行单基因或多基因同时激活的准确逻辑运算。
本发明还提供了原儿茶酸和白藜芦醇调控基因表达生物计算机和或系统、设备或装置，其包含以上所述原儿茶酸和白藜芦醇调控基因表达生物逻辑运算器。本发明还提供了所述基因表达生物逻辑运算器、所述基因表达生物计算机和/或系统等在体外、体外的逻辑运算中的应用。
本发明还提供了真核表达载体，即提供了所述基因表达生物逻辑运算器、所述基因表达生物计算机和/或系统中进行逻辑运算的真核表达载体。
本发明还公开了外源基因表达以及内源基因表达与门在动物体内进行运算的真核表达载体。在具体实施方案中，所述原儿茶酸和白藜芦醇调控的外源基因 表达与门在小鼠肝脏内进行正确逻辑运算的真核表达载体，即为质粒pWH75(PhCMV-TtgR-KRAB-P2A-KRAB-PcaV-T2A-PcaK-pA)和pWH47[PPR1-5-firefly luciferase-pA；PPR1-5,(OTRC1)2-PhCMV-(OPcaV)2-(OTRC1)2]。
在具体实施方案中，所述原儿茶酸和白藜芦醇调控的内源基因表达与门在小鼠肝脏内进行正确逻辑运算的真核表达载体，即为质粒pWH75(PhCMV-TtgR-KRAB-P2A-KRAB-PcaV-T2A-PcaK-pA)、pWH43[PPR1-5-MS2-p65-HSF1-pA；PPR1-5,(OTRC1)2-PhCMV-(OPcaV)2-(OTRC1)2]以及pJY493(PU6-sgRNA1Ascl1-PU6-sgRNA2Ascl1-PhCMV-dCas9-pA)。
本发明还提供了一种调控输入信号，即绿色小分子物质原儿茶酸、白藜芦醇作为基因表达调控输入信号。本发明还提供了在原儿茶酸、白藜芦醇调控外源基因表达、内源基因表达生物逻辑运算器、在所述基因表达生物计算机和/或系统中作为调控输入信号的应用。在具体实施方案中，其在调控哺乳动物基因表达生物逻辑运算器中作为调控输入信号，包括在哺乳动行、在小鼠体内的应用。
本发明还提出了原儿茶酸和白藜芦醇联合诱导型启动子的核苷酸序列，所述启动子序列的核酸序列选自以下序列SEQ ID NO.1-17之一种或几种：
SEQ ID NO.1：原儿茶酸和白藜芦醇联合诱导型启动子PPR1-1的核苷酸序列；
SEQ ID NO.2：原儿茶酸和白藜芦醇联合诱导型启动子PPR1-2的核苷酸序列；
SEQ ID NO.3：原儿茶酸和白藜芦醇联合诱导型启动子PPR1-3的核苷酸序列；
SEQ ID NO.4：原儿茶酸和白藜芦醇联合诱导型启动子PPR1-4的核苷酸序列；
SEQ ID NO.5：原儿茶酸和白藜芦醇联合诱导型启动子PPR1-5的核苷酸序列；
SEQ ID NO.6：原儿茶酸和白藜芦醇联合诱导型启动子PPR1-6的核苷酸序列；
SEQ ID NO.7：原儿茶酸和白藜芦醇联合诱导型启动子PPR2-1的核苷酸序列；
SEQ ID NO.8：原儿茶酸和白藜芦醇联合诱导型启动子PPR2-2的核苷酸序列；
SEQ ID NO.9：原儿茶酸和白藜芦醇联合诱导型启动子PPR2-3的核苷酸序列；
SEQ ID NO.10：原儿茶酸和白藜芦醇联合诱导型启动子PPR3-1的核苷酸序列；
SEQ ID NO.11：原儿茶酸和白藜芦醇联合诱导型启动子PPR3-2的核苷酸序列；
SEQ ID NO.12：原儿茶酸和白藜芦醇联合诱导型启动子PPR3-3的核苷酸序列；
SEQ ID NO.13：原儿茶酸和白藜芦醇联合诱导型启动子PPR3-4的核苷酸序列；
SEQ ID NO.14：原儿茶酸和白藜芦醇联合诱导型启动子PPR3-5的核苷酸序列。
在一些具体实施方案中，本发明所述基因表达生物逻辑运算器，基因表达生物计算机和/或系统，真核表达载体，质粒，所述构建方法，调控表达方法，所述应用中，包括但不限于哺乳动物基因表达，包括上载至哺乳动物细胞及小鼠体内，或体外的应用。
本发明有益效果包括：首次人工设计、合成一种原儿茶酸和白藜芦醇调控的哺乳动物生物逻辑运算器，包括调控外源基因表达和内源基因表达的生物逻辑运算器，所述生物逻辑运算器均可上载至哺乳动物细胞和小鼠体内进行正确的逻辑运算。本发明首次创新提出了将哺乳动物合成生物学、生物逻辑运算器以及CRISPR-dCas9系统介导的基因表观修饰技术相结合。本发明进一步结合利用CRISPR/dCas9系统模块的组装，使所述内源基因表达的生物逻辑运算器能够在细胞水平上进行单基因或多基因同时激活的准确逻辑运算，为CRISPR/dCas9介导的内源基因表达提供了一种精准的调控运算方式。本发明生物逻辑运算器是以原儿茶酸和白藜芦醇作为调控输入信号，具有抗氧化、抗癌等多种生物功效，相较于现有技术中以抗生素作为输入信号在临床应用上更加健康安全。本发明还提供了新型绿色小分子调控的哺乳动物计算机，且为CRISPR/dCas9介导的内源基因表达提供了精准调控运算方式。
附图说明
图1为本发明原儿茶酸和白藜芦醇调控外源基因表达的与门优化数据图。
图2为本发明原儿茶酸和白藜芦醇调控外源基因表达的原儿茶酸不包括白藜芦醇门优化数据图。
图3为本发明原儿茶酸和白藜芦醇调控外源基因表达的白藜芦醇不包括原儿茶酸门优化数据图。
图4为本发明原儿茶酸和白藜芦醇调控外源基因表达的与门的原理图、增值表以及荧光运算结果。
图5为本发明原儿茶酸和白藜芦醇调控外源基因表达的原儿茶酸不包括白藜芦醇门的原理图、增值表以及荧光运算结果。
图6为本发明原儿茶酸和白藜芦醇调控外源基因表达的白藜芦醇不包括原儿茶酸门的原理图、增值表以及荧光运算结果。
图7为本发明原儿茶酸和白藜芦醇调控外源基因表达的或非门原理图、增值 表以及荧光运算结果。
图8为本发明原儿茶酸和白藜芦醇调控外源基因表达的或门原理图、增值表以及荧光运算结果。
图9为本发明原儿茶酸和白藜芦醇调控内源基因表达的生物逻辑运算器设计原理图。
图10为本发明原儿茶酸和白藜芦醇调控内源基因表达的生物逻辑运算器靶向内源基因RHOXF2的运算结果图。
图11为本发明原儿茶酸和白藜芦醇调控内源基因表达的生物逻辑运算器同时靶向内源基因ASCL1、MIAT以及RHOXF2的运算结果图。
图12为本发明原儿茶酸和白藜芦醇调控外源基因表达的与门在小鼠肝脏中的逻辑运算结果图。
图13为本发明原儿茶酸和白藜芦醇调控内源基因表达的与门在小鼠肝脏中的逻辑运算结果图。
具体实施方式
结合以下具体实施例和附图，对本发明作进一步的详细说明。实施本发明的过程、条件、实验方法等，除以下专门提及的内容之外，均为本领域的普遍知识和公知常识，本发明没有特别限制内容。
实施例1，原儿茶酸和白藜芦醇调控的外源基因表达生物逻辑运算器的构建
本实施例中包含了原儿茶酸和白藜芦醇调控的外源基因表达生物逻辑运算器所涉及质粒载体的构建方法，详细设计方案及步骤见表1。
实施例2，优化原儿茶酸和白藜芦醇调控外源基因表达的与门。
第一步，质粒构建。本实例中的质粒构建详见表1。
第二步，细胞接种。转染前一天将HEK-293T细胞以每孔5×104个的细胞量接种于24孔板中，每孔加入500μl含10％FBS的DMEM培养基。
第三步，质粒转染。将100ng pJY29(PhEF1α-KRAB-PcaV-pA),100ng pLF24(PhCMV-TtgR-KRAB-pA)分别与100ng pPR1(PPR1-1-SEAP-pA；PPR1-1:SEQ ID NO.1)，100ng pPR2(PPR1-2-SEAP-pA；PPR1-2:SEQ ID NO.2)，100ng pPR3(PPR1-3-SEAP-pA；PPR1-3:SEQ ID NO.3)，100ng pPR4(PPR1-4-SEAP-pA；PPR1-4:SEQ ID NO.4)，100ng pPR5(PPR1-5-SEAP-pA；PPR1-5:SEQ ID NO.5)，100ng pPR6 (PPR1-6-SEAP-pA；PPR1-6:SEQ ID NO.6)共转染进HEK-293T细胞中。上述每组质粒按总量为300ng，与转染试剂聚乙烯亚胺PEI(质粒与PEI质量比1:3)进行预混，溶于50μl无血清无抗生素的DMEM中。静止15分钟后，将DNA-PEI预混液滴加至每孔细胞中。
第四步，加入诱导剂。转染6小时后，更换新鲜的培养基同时加入不同组合的诱导物。其中每个逻辑门所涉及的诱导物组合分为4种，包括(1)不加任何诱导物，(2)只加400μM的原儿茶酸诱导物，(3)只加20μM的白藜芦醇诱导物，(4)同时加入400μM的原儿茶酸和20μM的白藜芦醇两种诱导物。
第五步，诱导48小时后，收集细胞上清液并检测SEAP的表达量。
优化数据(图1)显示与门的最佳质粒组合为pJY29,pLF24和pPR5。
实施例3，优化原儿茶酸和白藜芦醇调控外源基因表达的原儿茶酸不包括白藜芦醇门。
第一步，质粒构建。本实例中的质粒构建详见表1。
第二步，细胞接种。(具体步骤同本发明实施例2)
第三步，质粒转染。将120ng pJY19(PCAG-KRAB-PcaV-pA),25ng pLF119(PSV40-PcaV-VPR-pA)分别与25ng pPR7(PPR2-1-SEAP-pA；PPR2-1:SEQ ID NO.7)，25ng pPR8(PPR2-2-SEAP-pA；PPR2-2:SEQ ID NO.8)，25ng pPR9(PPR2-3-SEAP-pA；PPR2-3:SEQ ID NO.9)共转染进HEK-293T细胞中。上述每组质粒按总量为170ng，与转染试剂PEI(质粒与PEI质量比1:3)进行预混，溶于50μl无血清无抗生素的DMEM中。静止15分钟后，将DNA-PEI预混液滴加至每孔细胞中。
第四步，加入诱导剂。(具体步骤同本发明实施例2)
第五步，诱导48小时后，收集细胞上清液并检测SEAP的表达量。
优化数据(图2)显示原儿茶酸不包括白藜芦醇门的最佳质粒组合为pJY19,pLF119和pPR8。
实施例4，优化原儿茶酸和白藜芦醇调控外源基因表达的白藜芦醇不包括原儿茶酸门。
第一步，质粒构建。本实例中的质粒构建详见表1。
第二步，细胞接种。(具体步骤同本发明实施例2)
第三步，质粒转染。将100ng pLF24(PhCMV-TtgR-KRAB-pA),20ng pLF215 (PSV40-PcaV-VPR-pA)分别与25ng pPR10(PPR3-1-SEAP-pA；PPR3-1:SEQ ID NO.10)，25ng pPR11(PPR3-2-SEAP-pA；PPR3-2:SEQ ID NO.11)，25ng pPR12(PPR3-3-SEAP-pA；PPR3-3:SEQ ID NO.12)，25ng pPR13(PPR3-4-SEAP-pA；PPR3-4:SEQ ID NO.13)，25ng pPR14(PPR3-5-SEAP-pA；PPR3-5:SEQ ID NO.14)共转染进HEK-293T细胞中。上述每组质粒按总量为145ng，与转染试剂PEI(质粒与PEI质量比1:3)进行预混，溶于50μl无血清无抗生素的DMEM中。静止15分钟后，将DNA-PEI预混液滴加至每孔细胞中。
第四步，加入诱导剂。(具体步骤同本发明实施例2)
第五步，诱导48小时后，收集细胞上清液并检测SEAP的表达量。
优化数据(图3)显示白藜芦醇不包括原儿茶酸门的最佳质粒组合为pLF24,pLF215和pPR14。
实施例5，验证原儿茶酸和白藜芦醇调控的外源基因表达生物逻辑运算器在HEK-293T细胞中的运算结果，即分别将五种逻辑门相关的质粒元件上载至HEK-293T细胞中进行逻辑运算。
第一步，质粒构建。本实例中的质粒构建详见表1。
第二步，细胞接种。(具体步骤同本发明实施例2)
第三步，质粒转染。本实施例的转染体系根据五个逻辑门分为5组，转染的质粒量以及质粒元件见表2。将上述每组质粒按总量为300ng，与转染试剂PEI(质粒与PEI质量比1:3)进行预混，溶于50μl无血清无抗生素的DMEM中。静止15分钟后，将DNA-PEI预混液滴加至每孔细胞中。
第四步，加入诱导剂。(具体步骤同本发明实施例2)
第五步，诱导24小时后，拍摄各组荧光图，同时用流式细胞仪定量分析荧光表达情况。
荧光图和流式数据分析表明在上述逻辑门中，d2EYFP的不同输出结果都符合相应的输入组合，即表明逻辑门与门(图4)、原儿茶酸不包括白藜芦醇门(图5)、白藜芦醇不包括原儿茶酸门(图6)、或非门(图7)以及或门(图8)在哺乳动物HEK-293T中都可以进行正确的逻辑运算。
实施例6，验证原儿茶酸和白藜芦醇调控的内源基因表达生物逻辑运算器(图6)在HEK-293T细胞中的运算结果，即分别将五种逻辑门相关的质粒元件上载 至HEK-293T细胞中进行逻辑运算。
第一步，质粒构建。本实例中的质粒构建详见表1。
第二步，细胞接种。(具体步骤同本发明实施例2)
第三步，质粒转染。本实施例的转染体系可分为5组，转染的质粒量以及质粒元件见表3。将上述每组质粒按总量为450ng，与转染试剂PEI(质粒与PEI质量比1:3)进行预混，溶于50μl无血清无抗生素的DMEM中。静止15分钟后，将DNA-PEI预混液滴加至每孔细胞中。
第四步，加入诱导剂。(具体步骤同本发明实施例2)
第五步，诱导24小时后，收集各组细胞，利用RT-qPCR法检测内源基因RHOXF2的mRNA水平，具体步骤包括：(1)采用Trizol的方法提取总的RNA；(2)利用反转试剂盒将RNA反转成cDNA；(3)基因的定量分析。将定量体系置于实时PCR仪器进行PCR反应，内参基因选择人甘油醛-3-磷酸脱氢酶(GAPDH)基因。(4)分析并计算基因的相对定量值。
qPCR数据分析表明在上述逻辑门中，靶基因的不同输出结果都符合相应的输入组合，即表明上述内源基因激活生物逻辑运算器，即逻辑门与门(图10A)、原儿茶酸不包括白藜芦醇门(图10B)、白藜芦醇不包括原儿茶酸门(图10C)、或非门(图10D)以及或门(图10E)在哺乳动物HEK-293T中都可以进行正确的逻辑运算。
实施例7，原儿茶酸和白藜芦醇调控的内源基因表达生物逻辑运算器同时对多个靶基因进行逻辑运算的探究。
第一步，质粒构建。本实例中的质粒构建详见表1。
第二步，细胞接种。(具体步骤同本发明实施例2)
第三步，质粒转染。本实施例的转染体系可分为5组，转染的质粒量以及质粒元件见表4，每组质粒按总量为500ng，与转染试剂PEI(质粒与PEI质量比1:3)进行预混，溶于50μl无血清无抗生素的DMEM中。静止15分钟后，将DNA-PEI预混液滴加至每孔细胞中。
第四步，加入诱导剂。(具体步骤同本发明实施例2)
第五步，诱导24小时后，收集各组细胞，利用RT-qPCR法检测内源基因ASCL1、RHOXF2和MIAT的mRNA水平。(具体步骤同本发明实施例3)
qPCR数据分析表明在上述逻辑门中，靶基因的不同输出结果都符合相应的 输入组合，即表明上述内源基因激活生物逻辑运算器能同时对靶基因ASCL1、RHOXF2和MIAT激活表达进行正确逻辑运算(图11)。
实施例8，原儿茶酸和白藜芦醇调控的外源基因表达与门在动物体内的逻辑运算探究。
第一步，质粒构建。本实例中的质粒构建详见表1。
第二步，质粒递送至小鼠肝脏。
将与门的相关质粒分别为pWH75(PhCMV-TtgR-KRAB-P2A-KRAB-PcaV-T2A-PcaK-pA)和pWH47[PPR1-5-firefly luciferase-pA；PPR1-5,(OTRC1)2-PhCMV-(OPcaV)2-(OTRC1)2]，通过尾静脉注射的方式将质粒递送至小鼠肝脏内部。
第三步，将小鼠分为四组，第一组小鼠为信号不输入组，即只注射溶剂；第二组为原儿茶酸信号输入组，即腹腔注射原儿茶酸，一天3次(原儿茶酸一天总量为750mg/kg)；第三组为白藜芦醇信号输入组，即腹腔注射白藜芦醇，一天2次(白藜芦醇一天总量为150mg/kg)；第四组为原儿茶酸和白藜芦醇双信号输入组，即腹腔注射原儿茶酸(总量为750mg/kg)以及白藜芦醇(总量为150mg/kg)。
第四步，小鼠活体成像。小鼠尾静脉递送质粒完成36小时后，小鼠麻醉后腹腔注射luciferase底物，5分钟后将小鼠置于活体成像仪中进行成像。
小鼠活体成像数据分析表明外源基因表达的逻辑与门能在小鼠肝脏内进行正确逻辑运算，即只有小鼠体内同时存在原儿茶酸和白藜芦醇这两种输入信号时，肝脏内的Luciferase表达远高于不输入信号以及只输入单一信号组(图12)。
实施例9，原儿茶酸和白藜芦醇调控的内源基因表达与门在动物体内的逻辑运算探究。
第一步，质粒构建。本实例中的质粒构建详见表1。
第二步，质粒递送至小鼠肝脏。
将与门的相关质粒分别为pWH75(PhCMV-TtgR-KRAB-P2A-KRAB-PcaV-T2A-PcaK-pA)、pWH43[PPR1-5-MS2-p65-HSF1-pA；PPR1-5,(OTRC1)2-PhCMV-(OPcaV)2-(OTRC1)2]以及pJY493(PU6-sgRNA1Ascl1-PU6-sgRNA2Ascl1-PhCMV-dCas9-pA)，通过尾静脉注射的方式将质粒递送至小鼠肝脏内部。
第三步，将小鼠分为四组，第一组小鼠为信号不输入组，即只注射溶剂；第 二组为原儿茶酸信号输入组，即腹腔注射原儿茶酸，一天3次(原儿茶酸一天总量为750mg/kg)；第三组为白藜芦醇信号输入组，即腹腔注射白藜芦醇，一天2次(白藜芦醇一天总量为150mg/kg)；第四组为原儿茶酸和白藜芦醇双信号输入组，即腹腔注射原儿茶酸(总量为750mg/kg)以及白藜芦醇(总量为150mg/kg)。
第四步，利用RT-qPCR法检测小鼠肝脏中内源基因Ascl1的mRNA水平，具体步骤包括：(1)小鼠安乐死之后，取出小鼠肝脏，进行碾磨；(2)采用Trizol的方法提取总的RNA；(2)利用反转试剂盒将RNA反转成cDNA；(3)基因的定量分析。将定量体系置于实时PCR仪器进行PCR反应，内参基因选择鼠甘油醛-3-磷酸脱氢酶(Gadph)基因。(4)分析并计算基因的相对定量值。
qPCR数据分析表明内源基因表达的逻辑与门能在小鼠肝脏内进行正确逻辑运算，即只有小鼠体内同时存在原儿茶酸和白藜芦醇这两种输入信号时，肝脏内的Ascl1mRNA水平远高于不输入信号以及只输入单一信号组(图13)。
表1质粒构建表


表2原儿茶酸和白藜芦醇调控的外源基因表达计算机的质粒名称以及转染量表
表3原儿茶酸和白藜芦醇调控的内源基因表达计算机的质粒名称以及转染量表

表4原儿茶酸和白藜芦醇调控的内源基因表达计算机同时对三个靶基因进行逻辑运算的质粒名称以及转染量表
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求书为保护范围。

Claims (20)

  1. 一种原儿茶酸和白藜芦醇调控的基因表达生物逻辑运算器，其特征在于，所述生物逻辑运算器的逻辑运算包括与门(AND)，蕴含非门(A NIMPLY B和B NIMPLY A)，或非门(NOR)，和或门(OR)；其中，所述A代表原儿茶酸输入信号，所述B代表白藜芦醇输入信号；其中，所述基因表达包括外源基因表达、细胞内源基因表达；所述生物逻辑运算器为双输入、单输出的运算模式；所述双输入包括原儿茶酸和白藜芦醇两种输入信号；所述单输出包括输出信号报告蛋白d2EYFP、Luciferase、SEAP或任何一种特定基因或功能蛋白质的上调表达。
  2. 如权利要求1所述的基因表达生物逻辑运算器，其特征在于，所述与门的基因表达模块包括：强启动子驱动原儿茶酸响应重组转录抑制子KRAB-PcaV表达的基因模块，强启动子驱动白藜芦醇响应重组转录抑制子TtgR-KRAB表达的基因模块，以及原儿茶酸和白藜芦醇共同作用启动子PPR1驱动下游信号输出的基因表达模块；所述PPR1是由强启动子和PcaV、TtgR操纵子结合位点构成；和/或，进一步包括强启动子驱动原儿茶酸转运蛋白PcaK表达的基因模块。
  3. 如权利要求1所述的基因表达生物逻辑运算器，其特征在于，所述A不包括B门(A NIMPLY B)的基因表达模块包括：强启动子驱动白藜芦醇响应重组转录激活子TtgR-VPR表达的基因模块，强启动子驱动原儿茶酸响应重组转录抑制子KRAB-PcaV表达的基因模块，以及原儿茶酸和白藜芦醇共同作用启动子PPR2驱动下游信号输出的基因表达模块；其中，所述PPR2是由弱启动子的5’端紧接着TtgR操纵子结合位点以及3’端紧接着PcaV操纵子结合位点构成。
  4. 如权利要求1所述的基因表达生物逻辑运算器，其特征在于，所述B不包括A门(B NIMPLY A)的基因表达模块包括：组成型启动子驱动原儿茶酸响应重组转录激活子PcaV-VPR表达的基因模块，组成型启动子驱动白藜芦醇响应重组转录抑制子TtgR-KRAB表达的基因模块，以及原儿茶酸和白藜芦醇共同作用启动子PPR3驱动下游信号输出的基因表达模块；其中，所述PPR3是由弱启动子的5’端紧接着PcaV操纵子结合位点以及3’端紧接着TtgR操纵子结合位点构成。
  5. 如权利要求1所述的基因表达生物逻辑运算器，其特征在于，所述或非门的基因表达模块包括：组成型启动子驱动原儿茶酸响应重组转录激活子PcaV-VPR表达的基因模块，原儿茶酸诱导型启动子驱动融合蛋白Coh2-VP16表达的基因模块，组成型启动子驱动白藜芦醇响应重组转录激活子TtgR-VPR表达的基因模块，白藜芦醇诱导型启动子驱动融合蛋白TetR-Docs表达的基因模块，以及原儿 茶酸和白藜芦醇共同作用启动子PPR4驱动下游信号输出的基因表达模块；其中，所述PPR4是由弱启动子的5’端紧接着TetR操纵子结合位点构成。
  6. 如权利要求1所述的基因表达生物逻辑运算器，其特征在于，所述或门的基因表达模块包括：组成型启动子驱动原儿茶酸响应重组转录抑制子KRAB-PcaV表达的基因模块，原儿茶酸诱导型启动子驱动Gal4-VP64表达的基因模块，组成型启动子驱动白藜芦醇响应重组转录抑制子TtgR-KRAB表达的基因模块，白藜芦醇诱导型启动子驱动融合蛋白Gal4-VP64表达的基因模块，以及原儿茶酸和白藜芦醇共同作用启动子PPR5驱动下游信号输出的基因表达模块；其中，所述PPR5是由弱启动子的5’端紧接着Gal4操纵子结合位点构成。
  7. 如权利要求1所述的基因表达生物逻辑运算器，其特征在于，还包括结合CRISPR/dCas9系统模块，其精准调控CRISPR/dCas9介导的内源基因表达。
  8. 如权利要求1所述的基因表达生物逻辑运算器，其特征在于，所述内源基因表达生物逻辑运算器包括处理器1和处理器2，所述处理器1的输出信号为包含MS2蛋白结构的转录激活因子；所述处理器2的输出信号为内源基因的激活与否。
  9. 如权利要求8所述的基因表达生物逻辑运算器，其特征在于，所述处理器2的基因组成元件包含组成型表达dCas9和gRNAMS2，其中，所述gRNAMS2含有MS2蛋白的结合位点；所述基因表达生物逻辑运算器通过添加不同靶向内源基因的gRNAMS2可实现多个内源基因同时激活与否的逻辑运算。
  10. 如权利要求1-9之任一项所述基因表达生物逻辑运算器的构建方法，其特征在于，所述构建方法为各个逻辑运算所涉及的基因表达模块克隆至表达载体上，以完成各个逻辑运算的构建；所述构建方法包括以下步骤：基因合成各个逻辑运算所设计的基因表达模块；通过酶切酶连或基因无缝组装的方法将合成的基因片段克隆至表达载体上；筛选测序鉴定以获得包含基因表达模块的质粒。
  11. 利用如权利要求1-9之任一项所述基因表达生物逻辑运算器的调控表达方法，其特征在于，所述方法由各个逻辑运算中所包含的处理器进行调控；所述方法为以下至少一种或几种：
    与门调控表达方法：只有当原儿茶酸和白藜芦醇信号同时输入时，与门处理器中的重组转录抑制子KRAB-PcaV和TtgR-KRAB从PPR1上解离下来，促使下游基因表达，从而使信号得以输出；
    原儿茶酸不包括白藜芦醇门调控表达方法：只有当原儿茶酸输入且白藜芦醇不输入时，原儿茶酸不包括白藜芦醇门处理器中原儿茶酸促使重组转录抑制子KRAB-PcaV从启动子PPR2上解离下来，同时转录激活子TtgR-VPR与PPR2的结合成功驱动下游基因表达，使信号得以输出；
    白藜芦醇不包括原儿茶酸门调控表达方法：只有当白藜芦醇输入且原儿茶酸不输入时，白藜芦醇不包括原儿茶酸门处理器中白藜芦醇促使重组转录抑制子TtgR-KRAB从启动子PPR3上解离下来，同时转录激活子PcaV-VPR与PPR3的结合成功驱动下游基因表达，使信号得以输出；
    或非门调控表达方法：只有当信号都不输入时，或非门处理器中原儿茶酸或白藜芦醇响应的重组转录激活子结合至相对应的诱导型启动子上，并驱动表达融合蛋白Coh2-VP16和TetR-Docs。由于Docs和Coh2是一对能够自动结合的蛋白，所形成的TetR-Docs-Coh2-VP16融合蛋白能够结合至PPR4启动子上驱动下游信号的输出；
    或门调控表达方法：当其中一个信号输入或两个信号都输入时，Gal4-VP64得以表达并结合至启动子PPR5上驱动下游信号的输出。
  12. 如权利要求11所述基因表达生物逻辑运算器的调控表达方法，其特征在于，所述方法包括CRISPR/dCas9介导的内源基因表达，实现在细胞水平上进行单基因或多基因同时激活的准确逻辑运算。
  13. 一种小分子调控的基因表达生物计算机和/或系统，其特征在于，其包含如权利要求1-9之任一项所述的基因表达生物逻辑运算器。
  14. 如权利要求1-9之任一项所述的基因表达生物逻辑运算器、如权利要求13所述的基因表达生物计算机和/或系统在体内、体外逻辑运算中的应用。
  15. 一种真核表达载体，其特征在于，其提供如权利要求1-9之任一项所述基因表达生物逻辑运算器、如权利要求13所述的基因表达生物计算机和/或系统进行逻辑运算。
  16. 如权利要求15所述的真核表达载体，其特征在于，其包括：
    外源基因表达与门在小鼠肝脏内进行的正确逻辑运算的真核表达载体，即质粒pWH75(PhCMV-TtgR-KRAB-P2A-KRAB-PcaV-T2A-PcaK-pA)、质粒pWH47[PPR1-5-firefly luciferase-pA；PPR1-5,(OTRC1)2-PhCMV-(OPcaV)2-(OTRC1)2]；和/或，
    内源基因表达与门在小鼠肝脏内进行正确逻辑运算的真核表达载体，即质粒 pWH75(PhCMV-TtgR-KRAB-P2A-KRAB-PcaV-T2A-PcaK-pA)、质粒pWH43[PPR1-5-MS2-p65-HSF1-pA；PPR1-5,(OTRC1)2-PhCMV-(OPcaV)2-(OTRC1)2]、质粒pJY493(PU6-sgRNA1Ascl1-PU6-sgRNA2Ascl1-PhCMV-dCas9-pA)。
  17. 一种基因表达调控输入信号，其特征在于，原儿茶酸、白藜芦醇在如权利要求1-9之任一项所述的基因表达生物逻辑运算器、如权利要求13所述的基因表达生物计算机和/或系统中作为调控输入信号。
  18. 原儿茶酸、白藜芦醇在如权利要求1-9之任一项所述的基因表达生物逻辑运算器、如权利要求13所述的基因表达生物计算机和/或系统中作为调控输入信号的应用。
  19. 一种序列，其特征在于，所述序列为原儿茶酸和白藜芦醇联合诱导型启动子的核苷酸序列，所述启动子序列的核酸序列选自序列1-14之一种或几种。
  20. 如权利要求1-9之任一项所述的基因表达生物逻辑运算器、如权利要求10所述的构建方法、如权利要求11-12的调控表达方法、如权利要求13所述的基因表达生物计算机和/或系统、如权利要求14、18所述的应用、15-16所述的真核表达载体，其特征在于，其包括哺乳动物基因表达，包括在上载至哺乳动物细胞、小鼠体内。
PCT/CN2023/085135 2022-04-11 2023-03-30 原儿茶酸和白藜芦醇调控基因表达生物逻辑运算器及其应用 WO2023197883A1 (zh)

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CN109456992A (zh) * 2017-09-06 2019-03-12 华东师范大学 原儿茶酸调控的多功能基因表达平台及其应用
CN113444746A (zh) * 2021-06-17 2021-09-28 华东师范大学 白藜芦醇调控转基因表达的控制系统及其构建和应用

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CN109456992A (zh) * 2017-09-06 2019-03-12 华东师范大学 原儿茶酸调控的多功能基因表达平台及其应用
CN113444746A (zh) * 2021-06-17 2021-09-28 华东师范大学 白藜芦醇调控转基因表达的控制系统及其构建和应用

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