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CN-121975764-A - T7 RNA polymerase mutant with orthogonality and application thereof

CN121975764ACN 121975764 ACN121975764 ACN 121975764ACN-121975764-A

Abstract

The invention relates to a T7 RNA polymerase mutant with K1F promoter recognition activity and application thereof in constructing a gene circuit logic gate design. The T7 RNA polymerase mutant provided by the invention has RNA polymerase activity based on a K1F promoter, can recognize the corresponding K1F promoter, and maintains orthogonality with the T7 RNAP promoter on the whole length and the split polymerase variant with proximity dependence. Compared with the existing K1F RNA polymerase, the RNA polymerase has the advantages of strong specificity, enhanced RNA transcription activity and the like, the performance is obviously improved, the requirement of multi-path orthogonal regulation of the existing gene circuit is more met, and an effective candidate enzyme tool is provided for research and application of RNA.

Inventors

  • PU JINYUE
  • YU SITIE
  • Sun Aiai
  • TANG GONGLI
  • PAN HAIXUE

Assignees

  • 国科大杭州高等研究院

Dates

Publication Date
20260505
Application Date
20251231

Claims (10)

  1. 1. A truncated mutant of T7 RNA polymerase having K1F promoter recognition activity, comprising the 168-883 amino acid sequence of T7 RNA polymerase as shown in SEQ ID NO. 7, and comprising at least one amino acid mutation or any combination thereof of S209Y, H211Y/H211N, R292C, V725M, M750V, G753S, N823N and V833I. .
  2. 2. The truncated mutant of T7 RNA polymerase according to claim 1, which comprises the 168-883 amino acid sequence of T7 RNA polymerase as shown in SEQ ID NO. 7, and comprises the following mutation combination: (1):H211N、G753S; (2):S209Y、H211Y、M750V、V833I; (3) S209Y, H211Y, R292C, N823N, V833I, or (4):S209Y、H211Y、V725M、M750V、V833I。
  3. 3. The truncated mutant of T7 RNA polymerase according to claim 2, wherein the mutant comprises an amino acid sequence as shown in any one of SEQ ID NO. 17-SEQ ID NO. 20.
  4. 4. A T7 RNA polymerase mutant having K1F promoter recognition activity, further comprising the amino acid sequence of positions 1-167 of a T7 RNA polymerase on the basis of the truncated mutant of any one of claims 1-3, thereby forming a full-length mutant (positions 1-883) of the T7 RNA polymerase having K1F promoter recognition activity.
  5. 5. The T7 RNA polymerase mutant according to claim 4, which comprises an amino acid sequence as shown in any one of SEQ ID NO 9-SEQ ID NO 16.
  6. 6. Fusion protein having a protein structure of the form A-B-C, wherein A is a leucine zipper peptide as shown in any one of SEQ ID NO:22-SEQ ID NO:23, or a blue light-induced protein SspB Nano as shown in SEQ ID NO:26, B is a linking peptide, which may be present or absent, C is a truncated mutant of the position 168-883 of T7 RNA polymerase, which contains the amino acid sequence of position 168-883 as shown in SEQ ID NO:7, and further comprises at least one amino acid mutation or any combination thereof S209Y, H211Y/H211N, R292C, V725M, M750V, G753S, N N or V833I.
  7. 7. Fusion protein having a protein structure in the form of D-B-F, wherein D is a truncated mutant at positions 1-167 of the proximity-dependent T7 RNA polymerase SEQ ID NO. 1, B is a linker peptide, which may be present or absent, F is a leucine zipper peptide of the sequence shown as SEQ ID NO. 21, or a blue light-induced protein iLid as shown as SEQ ID NO. 25, or any protein or polypeptide which may interact with the A structural protein in the A-B-C structure of claim 6.
  8. 8. A polynucleotide encoding a T7 RNA polymerase truncated mutant according to any one of claims 1 to 3, a full length mutant according to any one of claims 4 to 5 or a fusion protein according to any one of claims 6 to 7.
  9. 9. A design method for constructing a gene circuit logic gate AND gate (AND) comprises the following steps: (1) Constructing an expression plasmid carrying a gene sequence encoding a T7 RNA polymerase mutant fusion protein with an A-B-C structure, wherein A is a leucine zipper peptide shown in any one of SEQ ID NO. 22-SEQ ID NO. 23 or SspB Nano shown in SEQ ID NO. 26, B is a connecting peptide which can exist or not exist, and C is a C-terminal truncated mutant of the 168-883 position of the T7 RNA polymerase, which comprises an amino acid sequence shown in any one of SEQ ID NO. 17-SEQ ID NO. 20; (2) Constructing an expression plasmid carrying a truncated mutant sequence of the N-terminal of T7 RNA polymerase with a protein structure of D-B-F form, wherein D is 1-167 amino acids of the N-terminal of the adjacent dependent T7 RNA polymerase shown as SEQ ID NO. 24, B is a connecting peptide which can exist or not, F is a leucine zipper peptide with a sequence shown as SEQ ID NO. 21, or blue light induced protein iLid shown as SEQ ID NO. 25, or any protein or polypeptide which can interact with A structural proteins in the A-B-C structure; (3) Constructing an expression plasmid carrying a K1F promoter and a coding luciferase gene sequence, wherein the amino acid sequence corresponding to luciferase LuxAB is SEQ ID NO. 5; (4) The expression plasmids in the steps (1), (2) AND (3) are transfected or transformed into host cells, AND are detected according to luciferase analysis reaction, AND logic gate operation of AND gate (AND) in a genetic circuit is constructed, AND the judgment method comprises the following steps: input 1 is L-arabinose, input 2 is light, output is the expression of luciferase started by the K1F promoter, input is regarded as 1 when the input exists, input is regarded as 0 when the input does not exist, and corresponding high output signals can be detected only when input 1 and input 2 are both 1.
  10. 10. A design method for constructing a genetic circuit logic gate OR gate (OR) comprises the following steps: (1) Constructing an expression plasmid carrying a gene sequence encoding a full-length T7 RNA polymerase having T7 promoter recognition activity; (2) Constructing an expression plasmid carrying a gene sequence encoding a full-length T7 RNA polymerase mutant with K1F promoter recognition activity, wherein the expression plasmid encodes an amino acid sequence shown in any one of SEQ ID NO. 9-SEQ ID NO. 16; (3) Constructing an expression plasmid carrying a T7 or K1F promoter and a gene sequence for encoding luciferase; (4) The expression plasmids described in the steps (1), (2) and (3) are transfected OR transformed into host cells, and the detection is carried out according to luciferase analysis reaction, so that logical gate operation of OR gate (OR) in a genetic circuit is constructed, and the judgment method is as follows: Input 1 is the expression of a full-length T7 RNA polymerase protein with T7 promoter recognition activity, input 2 is the expression of a full-length T7 RNA polymerase mutant with K1F promoter recognition activity, output is the expression of luciferase started by the T7 or K1F promoter, input is regarded as1 when present, input is regarded as 0 when not present, and a corresponding high output signal can be detected when only input 1 or input 2 or both input 1 and input 2 are present.

Description

T7 RNA polymerase mutant with orthogonality and application thereof Technical Field The invention relates to the technical field of biology, in particular to a T7 RNA polymerase mutant and application thereof. Background In recent years, biosensors have been increasingly used in industry, medicine, environmental detection, and the like. For many biosensors available today, they are often limited in their practical application by insufficient functionality, one of which is the limited number of bio-elements available today. Heretofore, a split proximity-dependent T7 RNA polymerase (T7 RNAP) biosensor has been constructed by phage-assisted continuous evolution system (PACE). The method has the advantages of high sensitivity, sequence specificity and derived orthogonality of RNA output signals, and diversity of detection means. But the signal response dynamic interval and signal detection dimension of the current split T7 RNAP biosensor itself still have room for further optimization. Theoretically, when an RNA sequence is used as the output signal, the number of signals that can be detected simultaneously can reach 4n (n=nucleic acid length). However, in the practical application of T7 RNAP, the detection dimension is limited by the number of orthogonal mutants. Therefore, in order to expand the application scenario of T7 RNAP, more choices are provided for synthetic biology, and more orthogonalization of T7 RNAP mutants is urgently needed. In particular, in the application direction of the split proximity-dependent T7 RNAP biosensor, more orthogonal T7 RNAP mutants mean more specific recognition response in more dimensions, and can improve the detection diversity and the working predictability of the sensor in practical application. Optimizing and developing a more-dimensional split proximity-dependent T7 RNAP biosensor can further expand the application space of the sensor and apply it to more practical scenes, providing more tools for synthetic biology. . Disclosure of Invention The invention aims to provide T7 RNA polymerase orthogonalization variants and application thereof in constructing a gene circuit logic gate design. The T7 RNA polymerase mutants of the present invention have K1F promoter-based RNA polymerase activity that recognizes the corresponding K1F promoter and maintains orthogonality with the T7 RNAP promoter over both full length and proximity-dependent split polymerase variants. Compared with the existing K1F RNA polymerase, the RNA polymerase has the advantages of strong specificity, enhanced RNA transcription activity and the like, the performance is obviously improved, the requirement of multi-path orthogonal regulation of the existing gene circuit is more met, and an effective candidate enzyme tool is provided for research and application of RNA. Mutant and fusion protein thereof The first object of the present invention is to provide a T7 RNA polymerase mutant having K1F promoter recognition activity. The wild-type T7 RNA polymerase has 883 amino acids, and studies have shown that the wild-type T7 RNA polymerase can split at multiple sites, and that both parts of the split can restore good polymerase activity after binding, alternative cleavage sites include cleavage between amino acids 163/164,167/168,179/180,196/197 and 564/565. The cleavage sites of the proximity-dependent T7 RNA polymerase sensor are screened according to the principle that the C-terminal and the N-terminal with proximity dependence, which do not destroy the specificity of the DNA promoter by the T7 RNA polymerase, and the sites are exposed to solvents in the structural data and are not on the protein-DNA binding surface, so that the improvement of the sensor sensitivity is achieved. The T7 RNA polymerase mutant capable of recognizing the K1F promoter is obtained through screening of the proximity-dependent T7 RNA polymerase based on PANCE orthorhombic evolution scheme. The cleavage site of the proximity-dependent T7 RNA polymerase is based on a T7 RNA polymerase sequence SEQ ID NO.1, and the cleavage site is split at 167/168 amino acid sites to obtain a C-terminal truncated protein of 168-883 aa, which is used as a C-terminal evolution object of the T7 RNA polymerase for orthogonalizing and recognizing the K1F promoter. In the implementation process, the K1F promoter is used as positive selection, negative selection is carried out on the T7 promoter which is the target promoter with orthogonality, and the target functional mutant is obtained through the PANCE evolution strategy. The T7 RNA polymerase truncated mutant with the K1F promoter recognition activity comprises 168-883 amino acid sequences of T7 RNA polymerase shown in SEQ ID NO. 7, and at least one amino acid mutation or any combination of S209Y, H211Y/H211N, R292C, V725M, M750V, G753S, N823N and V833I. In another embodiment, the invention provides a T7 RNA polymerase truncated mutant with K1F promoter recognition activity, which contains the 168-883 am