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TALER protein for displaying transcription inhibiting action through steric hindrance and application thereof

A protein and protein expression technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, the use of vectors to introduce foreign genetic material, etc., can solve the problem of low inhibition efficiency

Active Publication Date: 2014-12-03
TSINGHUA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that in the context of mammalian gene regulation, placing the LacI binding site downstream of the cytomegalovirus promoter (CMV) or CAG promoter in the synthetic gene circuit can inhibit gene expression, although the inhibition efficiency is lower than that of mammals. Animal expression system is lower than prokaryotic expression system

Method used

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  • TALER protein for displaying transcription inhibiting action through steric hindrance and application thereof
  • TALER protein for displaying transcription inhibiting action through steric hindrance and application thereof
  • TALER protein for displaying transcription inhibiting action through steric hindrance and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1, Functional verification and specificity analysis of TALER protein

[0051] The schematic diagram of the action mechanism of pCMV-TALERx plasmid, pTx+Tx+ plasmid and pEF1a-TagBFP-2A plasmid is shown in figure 2 . Under the action of the pEF1a promoter, TagBFP and Gal4 / vp16 are expressed (the 2A connecting peptide between TagBFP and Gal4 / vp16 is a self-splicing peptide, so TagBFP can represent the expression level of Gal4 / vp16). Gal4 / vp16 activated the 5×UAS sequence, thereby activating the transcriptional initiation of the CMVmini promoter, and mKate2 was expressed. Under the action of the CMV promoter, EYFP and TALER1 proteins are expressed (the 2A connecting peptide between EYFP and TALER1 proteins is a self-splicing peptide, so EYFP can represent the expression level of TALER1 proteins). The TALER1 protein binds to the T1 sequence and exerts a transcriptional repression effect through steric hindrance, and the CMVmini promoter between the two T1 sequence...

Embodiment 2

[0117] Embodiment 2, further ductility research

[0118] The pEF1a-TagBFP-2A plasmid is the pEF1a-TagBFP-2A plasmid in Example 1.

[0119] The pCMV-TALER1 plasmid is the pCMV-TALER1 plasmid in Example 1.

[0120] The pCMV-TALER2 plasmid is the pCMV-TALER2 plasmid in Example 1.

[0121] The pCMV-TALER4 plasmid is the pCMV-TALER4 plasmid in Example 1.

[0122] The pCMV-TALER5 plasmid is the pCMV-TALER5 plasmid in Example 1.

[0123] The pCMV-TALER32 plasmid is the pCMV-TALER32 plasmid in Example 1.

[0124] The pT1+T1+72-DsRed plasmid is shown in SEQ ID NO:54. In sequence 54, nucleotides 2441-2533 from the 5' end are 5×UAS sequences, nucleotides 2549-2562 are T1 sequences (target sequence of TALER1 protein), nucleotides 2569-2628 It is a CMVmini promoter, the 2635-2648th nucleotide is the T1 sequence, and the 2668-3345th nucleotide is the coding gene of DsRed (red fluorescent protein).

[0125] The pT1+T2+72-DsRed plasmid is shown in SEQ ID NO:55. In sequence 55, nucleoti...

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Abstract

The invention discloses a TALER protein for displaying transcription inhibiting action through steric hindrance and application thereof. The invention protects application of the TALER protein in inactivating a target promoter. The upper stream and lower stream of the target promoter are respectively provided with at least one target spot of the TALER protein; or the upper stream of the target promoter is not provided with the target spot of the TALER protein, but the lower stream is provided with at least one target spot of the TALER protein. The invention also protects application of the TALER protein in inactivating the coding gene of the target protein. The expression of the coding gene of the target protein is started by the target promoter. The upper stream and lower stream of the target promoter are respectively provided with at least one target spot of the TALER protein; or the upper stream of the target promoter is not provided with the target spot of the TALER protein, but the lower stream is provided with at least one target spot of the TALER protein. The TALER protein has important value for regulation expression modes and function studies of proteins.

Description

technical field [0001] The present invention relates to TALER protein exerting transcriptional inhibition effect through steric hindrance and application thereof. Background technique [0002] The synthetic gene circuit is carefully designed, and the gene regulation device is assembled according to the function, and the molecular information in the cell is sensed, integrated, and processed to perform certain functions. Various synthetic gene circuits have been developed to achieve customizable, programmable functions within cells, including dynamic behavior, switching and memory, intercellular communication, fitness, cell polarization, digital and analog computation, and complex biosynthetic pathways. Most of these genetic circuits were constructed using limited genetic elements and costly, inefficient "trial-and-error" methods. Therefore, to simplify design and optimize complex manipulations in living cells, it is necessary to develop a large-scale library of functionally ...

Claims

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Application Information

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IPC IPC(8): C12N15/63
CPCC07K14/195C12N15/113C12Q1/68
Inventor 谢震威斯李寅青蒋云廖微曦陈赫
Owner TSINGHUA UNIV
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