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Induced t7 RNA polymerase

A polymerase and induced expression technology, which is applied in the field of T7 RNA polymerase and ribonucleic acid polymerase to induce and control gene expression, can solve the problems of no tissue specificity, poor resolution of chemical inducers, and inability to achieve uniform induction. The method is simple and achieves the effect of gene expression level

Active Publication Date: 2019-10-25
UNIV OF SCI & TECH OF CHINA
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Although the method of chemically inducing the production of target proteins is relatively simple and easy to operate, chemical inducers have relatively poor resolution in time and space dimensions (unable to achieve uniform induction of every cell), have no tissue specificity, and are rarely used in biological systems. It is difficult to remove cleanly (there is a risk of contaminating the yeast liquid), which is not conducive to the production of proteins with medical functions, and the cost of chemical inducers is expensive, which limits the wide application of chemical inducers in experimental research and industrial production; photochemically induced genes Although the expression can obtain higher resolution in time and space dimensions, the inducing factor --- ultraviolet rays have potential side effects on cells
In addition, not only the translation system of the host cell needs to be specially modified, but also the unnatural amino acid introduced during the synthesis of T7 RNAP needs to be chemically synthesized, which is a time-consuming, labor-intensive and costly process, which limits its application in metabolic engineering. wide application of

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0054] Example 1 Light-induced protein-protein interaction regulates T7 RNAP activity ( Figure 3a )

[0055] PaT7P-1, an induction system designed in the present invention to recover T7 RNA polymerase (coding sequence is SEQ ID NO.5, corresponding amino acid sequence is SEQ ID NO.30) through light-induced protein-protein interaction. The induction system was obtained by the following method: using the plasmid J61002 (BioBrick code BBa_J61002) as a template, replacing the ampicillin resistance gene with the kana resistance gene, obtaining the p5C plasmid as a template for the light induction system, and introducing multiple Cloning site (MCS) to obtain p5C-MCS ( Figure 1a ). A constitutive promoter Pcat (BioBrick codeBBa_I14033) was inserted between NheI and NcoI, and RBS (BioBrick code BBa_B0034) was inserted between NcoI / BglII and KpnI / NdeI restriction sites respectively. Gene synthesis of VVD-36 9 Sequence (the N-terminus of light-sensitive protein VVD removes 36 amino...

Embodiment 2

[0057] Example 2 Light-induced allosteric regulation of the self-assembly of the T7 RNAP resolution system ( Figure 3b )

[0058] PaT7P-2 is a light-induced allosteric regulation system designed in the present invention. Light activation promotes the self-assembly of protein fragments to restore the activity of T7 RNA polymerase. The induction system was obtained by the following method: plasmid paT7P-1 was digested with NdeI and SacII, using PrimeSTAR HS DNA polymerase and primer pair C565-F2 (SEQ ID NO.21), C565-R1 (SEQ ID NO.20 ) to perform PCR amplification to obtain the C565-I fragment, and use the recombinase Exnase II (Vazyme) to recombine the C565-I fragment to the restriction position, then use PrimeSTAR MAX DNA polymerase and primer pair pMag-F (SEQ IDNO. 22) and pMag-R (SEQ ID NO.23) for site-directed mutation PCR, and then converted and sequenced after DpnI digestion to obtain paT7P-2 ( Figure 1f )(Note: pMag is a mutant of VVD, its nucleic acid sequence and am...

Embodiment 3

[0059] Example 3 Light-induced allosteric regulation of Full-length T7 RNAP activity ( Figure 3c )

[0060] PaT7P-3, light-induced allosteric regulation of T7 RNA polymerase, is a simplified version of paT7P-2 described in Example 2. The induction system is obtained by the following method: paT7P-1 is double digested with EcoRI and BamHI, and the VVD coding sequence is removed. Using pT7P-2 as a template, use PrimeSTAR HS DNA polymerase and primers to perform PCR amplification on pMag1-F (SEQ ID NO.24) and pMag1-R (SEQ ID NO.25) to obtain pMag1 fragment (containing linker1 sequence) , using the recombinase ExnaseMultis (Vazyme) to recombine pMag1 and linker2 into the above-mentioned EcoRI / BamHI restriction sites to obtain paT7P-3 ( Figure 1g ). To detect the light-induced properties of paT7P-3, the pJ61-R reporter system mentioned in Example 1 was still selected, and paT7P-3 and pJ61-R were co-transformed into E.coli, single clones were selected, liquid cultured overnight...

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Abstract

The invention relates to an induced T7 RNA (ribonucleic acid) polymerase, particularly a light or small-molecule-compound induced-regulation T7 RNA polymerase. Specific amino acid sequence positions in the T7 RNA polymerase are used as splitting sites to perform splitting, and the split segments are fused with light-sensitive or small-molecule-compound-sensitive structure fields, proteins or peptides to implement the following induced regulations: a. regulating T7 RNA polymerase activity under the light or small-molecule-compound mediated protein-protein interactions; b. restoring the T7 RNA polymerase activity by split segment self-assembly through light-induced structure regulation; and c. embedding the light-sensitive structure field into the T7 RNA polymerase, and regulating the polymerase activity through the light-induced structure. The induction system provided by the invention implements accurate regulation on the gene expression in time and space dimensions, and the diversification of the T7 RNA polymerase splitting system provides powerful reference values for modular regulatory gene expression.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a light-induced T7 RNA polymerase and a method for inducing and controlling gene expression with an engineered ribonucleic acid polymerase (RNAP). The invention constructs a T7 RNA polymerase that can specifically respond to blue light and shows significant differences in transcriptional activity under light and non-light conditions, thereby realizing transcription initiation activated by light signals, and is suitable for those that need to use external signals to control gene expression Applications, such as recombinant protein production, metabolic pathway regulation, etc. Background technique [0002] Gene switch is the use of artificially manipulated external signals to regulate gene expression. Gene switches have a wide range of applications in biotechnology. For example, using biological cells to produce target proteins with medical use and commercial value is an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63
CPCC07K14/37C12N9/1247C12N15/63C12Y207/07006
Inventor 刘海燕陈泉韩悌云
Owner UNIV OF SCI & TECH OF CHINA
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