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AND gate gene circuit and method for acquiring AND gate gene circuit

A technology of gene circuits and coding genes, applied in the field of genetic engineering, can solve problems such as low execution efficiency, mutual interference, and inability to work normally

Pending Publication Date: 2022-02-22
珠海中科先进技术研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, with the increasing complexity of promoters, when multiple promoters are used in combination, they often interfere with each other and fail to work properly. However, SpCas9 or SaCas9 are based on DNA level editing, and their gene fragments are all larger than 3000bp
In clinical gene therapy, because the fragment is too long, the therapeutic vector AAV virus must be assembled separately, and the execution efficiency is low

Method used

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  • AND gate gene circuit and method for acquiring AND gate gene circuit
  • AND gate gene circuit and method for acquiring AND gate gene circuit
  • AND gate gene circuit and method for acquiring AND gate gene circuit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0079] Material

[0080] cell:

[0081] HEK293T cell line was provided by Shanghai Institute of Cell Biology, Chinese Academy of Sciences.

[0082] Drugs and reagents:

[0083] Roseville-1640 (RPMI-1640) medium and fetal bovine serum (FBS) were purchased from GIBCO, USA.

[0084] Doxycycline (doxycycline hydrochloride), specification: 10mM / mL, batch number: ID0390-10mM*1mL (inWater), supplier: Suo Laibao.

[0085] Plasmids were synthesized from General Biology (Anhui), the classifier plasmid backbone used SWB-Blasticidin lentiviral backbone, and the synthetic promoter vector backbone used SWP-Puromycin.

[0086] Lipofectamine 3000, specification: 1mL, batch number: L3000015, supplier: ThermalFisher.

[0087] Lentivirus packaging kit, specification: 100mL, batch number: GM-040801-100, supplier: Jimon Biotech.

[0088] Opti-MEM medium, specification: 100mL, batch number: 31985-062, supplier: Beijing Zhongsheng Ruitai Technology Co., Ltd.

[0089] instrument:

[0090]CX41 ...

experiment example 1

[0092] gene synthesis image 3 The gene structure in , and the gene fragment was integrated into the DNA3.1 plasmid backbone by the Gibson assembly method, and the integrated plasmid was transfected into HEK293T cells by Invitrogen Lipofecta mine 3000, and after 3 days, the Leica fluorescent confocal Observe under a microscope whether there is mVenus fluorescence expression (mVenus fluorescence confocal parameters, Exλ:515, Emλ:527).

[0093] Gibson assembly operation steps: 10 μL of Gibson Assembly reaction system: 3 μL of pcDNA 3.1 vector backbone, 1 μL of CasRx protein split protein clone fragment, 6 μL of mixed enzyme, and react at 50°C for 20 minutes.

[0094] Invitrogen Lipofectamine 3000 transfection steps:

[0095] 1. Transfect when the cell confluency reaches 70%;

[0096] 2. Dilute Lipofectamine 3000 with Opti-MEM medium and mix well;

[0097] 3. Dilute plasmid DNA with Opti-MEM medium to prepare plasmid DNA master mix, then add P3000 reagent and mix thoroughly; ...

Embodiment 2

[0114] Using synthetic promoters: the first promoter, the second promoter and the CasRx split of miRNA 1, miRNA 2 merged gene circuits to significantly knock down the gene expression of MYC and SOX3 in A549 cells:

[0115] 1. Transfect when the cell confluency reaches 70%;

[0116] 2. Use Opti-MEM TM Dilute Lipofectamine 3000 in the culture medium and mix thoroughly;

[0117] 3. Use Opti-MEM TM Dilute the plasmid DNA in the medium, prepare the plasmid DNA master mix, and then add P3000 TM Reagents, mixed thoroughly;

[0118] 4. Dilute Lipofectamine 3000 in each tube TM Diluted DNA (1:1 volume ratio) was added to the reagent.

[0119] 5. Incubate at room temperature for 15 minutes;

[0120] 6. Add plasmid DNA-Lipofectamine 3000 TM complexes into cells;

[0121] 7. Incubate the cells at 37 degrees for 2-4 days. Observe mVenus fluorescence expression.

[0122] The mRNA of HEK293T cells positively expressed by mVenus was extracted by BD flow cytometry, using TakaraPrimeS...

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Abstract

The invention provides an AND gate gene circuit and a method for obtaining the AND gate gene circuit, and belongs to the technical field of gene engineering. The AND gate gene circuit comprises a first end sequence, a guide sequence and a second end sequence which are connected in sequence; the first terminal sequence comprises a CasRx-C terminal sequence and a first promoter which are connected in sequence; the guide sequence at least comprises a guide promoter and a gRNA sequence which are connected in sequence; andthe second terminal sequence at least comprises a second promoter and a CasRx-N terminal sequence, and resolution sites of the CasRx-C terminal sequence and the CasRx-N terminal sequence are located in a secondary structure hinge region of the three-dimensional prediction structure of the amino acid sequence of CasRx. The invention further discloses a method for obtaining the AND gate gene circuit. When the first promoter and the second promoter are different promoters, the CasRx fused again can exert a gene editing effect only under the precondition that the two promoters can normally exert functions by utilizing the characteristics similar to those of an AND gate circuit, so that whether the two promoters interfere with each other or not can be quickly judged.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an AND gate gene circuit and a method for obtaining the AND gate gene circuit. Background technique [0002] With the continuous development of genetic engineering technology and cell biology, human beings can fully understand the life process and predict and even finely control it, which further makes the rapid development of synthetic biology. As one of the fields of synthetic biology research, gene circuits help to solve problems such as cancer and genetic defects by designing and constructing biological components and systems that do not exist in living organisms. In genetic engineering, it often involves the use of multiple promoters to respectively activate different functional regions. [0003] However, with the increasing complexity of promoters, multiple promoters often interfere with each other and fail to work properly when used in combination. SpCas9 or S...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/55C12N15/113C12N15/65C12N15/64
CPCC12N15/85C12N9/22C12N15/113C12N15/65C12N2800/107C12N2310/20
Inventor 余裕姜长安
Owner 珠海中科先进技术研究院有限公司
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