Co-regulatory sequence based on tetracycline and Cumate

A tetracycline and sequence technology, applied in the field of nucleic acid sequences transcribed by nucleic acid fragments, can solve the problems of low transcription activity and the like

Active Publication Date: 2021-11-26
EUREKA BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In the current development and improvement of various inducible expression systems, the main optimization is focused on reducing the control of the basal leakage transcriptional activity of the regulated target nucleic acid fragment under non-inducing conditions, while the transcriptional activity under inducing conditions is not the focus of optimization. The post-induced transcriptional activity of the target nucleic acid fragment regulated by the inducible expression system is lower than that under the control of a common constitutively active promoter (constitutively active promoter)

Method used

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  • Co-regulatory sequence based on tetracycline and Cumate
  • Co-regulatory sequence based on tetracycline and Cumate
  • Co-regulatory sequence based on tetracycline and Cumate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: plasmid construction method

[0042]Molecular cloning techniques used in the following examples, for example, PCR amplification of DNA fragments, restriction endonuclease digestion of DNA fragments, gel recovery of DNA fragments, T4 DNA ligase connection of two or more DNA fragments, Methods such as transformation of competent cells of ligated products, small-scale preparation and identification of plasmids are all well-known techniques in the art. The following reagents are involved in the following examples: PCR enzyme (Thermo, F-530S); restriction endonuclease (NEB); T4 DNA ligase (Invitrogen, 15224041); DNA fragment gel recovery kit (Omega, D2500-02 ); Plasmid small extraction kit (TIANGEN, DP105-03); Competent cells (XL-10Gold, Hunan Fenghui Biotechnology Co., Ltd., JZ011); The nucleotide sequence shown in SEQ ID NO:1 to SEQ IDNO:22 is composed of GenScript synthesized and used for the construction of the plasmid described in the present invention, a...

Embodiment 2

[0063] Embodiment 2: The influence of CuO operator position and quantity on the induced expression level and the expression level without induction leakage

[0064] The experiment described in this example is to study and verify the influence of the position and copy number of the CuO operon in the Tet-On and Cumate composite response elements on the induced expression of the composite response element and the expression without induced leakage, and to optimize and confirm the optimal CuO operation Sublocation and copy number. This embodiment is based on TRE 3G The 7xTetO sequence and the minimal promoter sequence #1 (SEQ ID NO:25) in the response element are connected to the CuO operator sequence design at intervals of 14bp, 30bp, 50bp and 100bp in the range of 10bp to 100bp downstream of the TATA box, respectively TRE 3G CuO 14 (10-317bp in SEQ ID NO:8), TRE 3G CuO 30(SEQ ID NO:28), TRE 3G CuO 50 (SEQ ID NO:23) and TRE 3G CuO 100 (10-403bp in SEQ ID NO: 10) response...

Embodiment 3

[0071] Embodiment 3: the impact of single regulation / composite regulation and intron on induced expression level and non-induced leakage expression level

[0072] In the Tet-On inducible expression system, the TetO operator junction sequence and minimal promoter sequence of the TRE response element and the different mutants of the transactivator rtTA comprehensively affect the induced transcriptional activity and leaky transcriptional activity. In addition, downstream of the 3' end of the response element, the cleavable intron sequence connected upstream of the 5' end of the regulated target nucleic acid fragment may enhance the transport and stability of the messenger ribonucleic acid, but may also affect the inducible transcriptional activity and transcriptional activity of the response element of the induced expression system Leaked transcriptional activity. Based on this, this embodiment compares (1) based on TRE adv and TRE 3G Tet-On and Cumate Composite Response Elemen...

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Abstract

The invention provides a nucleic acid sequence for regulating transcription of a target nucleic acid fragment. The nucleic acid sequence comprises at least two copies of a TetO-operator sequence capable of being combined with a trans activator rtTA regulated by tetracycline or derivatives thereof, one copy of a minimum promoter sequence comprising a TATA box sequence and at least one copy of a CuO-operator sequence combined with a transcription repressor CymR regulated by cumate. The invention also provides a vector containing the nucleic acid sequence, a host cell and a method for inducible expression of a target nucleic acid fragment in the host cell.

Description

[0001] field of invention [0002] The present invention relates to tetracycline- and Cumate-dependent nucleic acid sequences for regulating the transcription of target nucleic acid fragments. Specifically, the present invention relates to a nucleic acid sequence for regulating the transcription of a nucleic acid fragment of interest, said nucleic acid sequence comprising at least 2 copies of TetO-operator sequence (Tet operator, TetO), 1 copy of minimal promoter sequence containing TATA box sequence and at least 1 copy of CuO-operator sequence (Cumate operator, CuO) binding CymR transcription repressor (transcription repressor). Further, there is also a spliceable intron sequence between the downstream of the 3' end of the above nucleic acid sequence and the upstream of the 5' end of the regulated target nucleic acid fragment, which can be used to further increase the transcriptional activity after induction without leakage of induction . Still further, the present invention ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/63C12N15/85C12N5/10
CPCC12N15/113C12N15/63C12N15/85C12N2830/003C12N2830/002C12N5/10C12N2800/10C12N2830/42C12N2800/90C12N2800/22C12N15/635C12N2830/001
Inventor 薛博夫杨银辉刘科马墨
Owner EUREKA BIOTECH LTD
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