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Chimeric phenylalanine translation system introduced with non-natural amino acid and construction method of chimeric phenylalanine translation system

An unnatural amino acid, phenylalanine technology, applied in the field of molecular biology, can solve the problems of signal-to-noise ratio improvement, unreachable insertion efficiency of unnatural amino acids, and obstacles to the widespread application of chimeric phenylalanine translation systems, reaching The effect of high signal-to-noise ratio

Pending Publication Date: 2022-03-04
杭州嵌化合生医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its unnatural amino acid insertion efficiency has not yet reached the level of endogenous aminoacyl-tRNA synthetase to recognize natural amino acids, and the signal-to-noise ratio needs to be further improved
The mutual orthogonality between this system and the commonly used non-natural amino acid introduction system is unknown, and the insertion efficiency of the non-natural amino acid integrated into the genome is also unknown, which hinders the widespread application of the chimeric phenylalanine translation system

Method used

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  • Chimeric phenylalanine translation system introduced with non-natural amino acid and construction method of chimeric phenylalanine translation system
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  • Chimeric phenylalanine translation system introduced with non-natural amino acid and construction method of chimeric phenylalanine translation system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1. Library construction of chimeric phenylalanine-tRNA

[0089] (1) The nucleotide sequence of the wild-type phenylalanine-tRNA is shown in SEQ ID NO: 1; the 2nd to 7th base pairs of the selected chimera phenylalanine-tRNA acceptor arm region, with pNEG-chPheT-GFP190TAG (see for details figure 1 , nucleotide sequence referring to SEQ ID NO: 45) as a template, with the primer PheT-Lib-AU-F / R (ie SEQ ID NO: 17 and SEQ ID NO: 18 shown in Table 1) saturation mutagenesis to produce approximately 1.7x10 7 The mutant library was cloned into pNEG-CAT-112TAG-GFP-190TAG-tRNA vector by Gibson assembly.

[0090] (2) Transform pBK-chPheRS-1 into Escherichia coli DH10B to prepare electroporation competent cells.

[0091] (3) Electrotransform the mutant library prepared in (1) into the electroporation-competent cells in (2), add 1mMAzF and incubate at 37°C for 3h, and then spread the bacterial solution on the cells containing 50μg / ml Kanamyces 100 μg / ml ampicillin, 10 μg / ...

Embodiment 2

[0094] Example 2. Screening chimeric phenylalanine-tRNA mutants by GFP fluorescence reporter assay

[0095] (1) Pick the single clone with fluorescent signal in Example 1 and culture overnight.

[0096] (2) Inoculate the bacterial solution in (1) according to the ratio of 1:100, and cultivate to OD at 37°C 600 When =0.6-0.8, add 0.2% arabinose to induce expression, and at the same time take 1 ml of bacterial liquid and add 1 mM AzF, and express at 30° C. for 20 h.

[0097] (3) After centrifuging 750 μl of the bacterial solution in (2), add 150 μl of 1×Bugbuster (Millipore, Lot: 3492682) and place it at 25°C for 30 min, then centrifuge, take 100 μl of the supernatant to a 96-well plate, and at the same time Take 100 μl (2) of the bacterial solution, and measure the GFP fluorescence signal intensity and OD by a microplate reader 600 , to calculate the mutant recognition efficiency of unnatural amino acids.

[0098] (4) Sequence the chimera phenylalanine-tRNA mutant that signi...

Embodiment 3

[0102] Example 3. Directed evolution of the catalytic domain of chimeric phenylalanyl-tRNA synthetases

[0103] (1) Using the C-terminal catalytic domain sequence (SEQ ID NO: 8) of the chimeric phenylalanyl-tRNA synthetase (chPheRS) as a template, perform error-prone PCR amplification to obtain the C-terminal catalytic domain mutation of chPheRS Libraries, mutant libraries were cloned into the pBK vector by Gibson assembly.

[0104] (2) Combine pNEG-chPheT-CAT112TAG-GFP190TAG (see figure 1 , the nucleotide sequence is SEQ ID NO:47) The plasmid was transformed into Escherichia coli DH10B to prepare electroporation competent cells.

[0105] (3) Electrotransforming the mutant library of the chimeric phenylalanyl-tRNA synthetase in (1) into the electroporation competent cells in (2), and culturing to obtain a single clone.

[0106] (4) Identify mutants that significantly improve the efficiency of the chimera phenylalanine translation system through GFP fluorescent signal report...

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Abstract

The invention discloses a chimera phenylalanine translation system introduced with non-natural amino acid and a construction method of the chimera phenylalanine translation system. The invention provides a construction method of a chimeric phenylalanine translation system introduced with non-natural amino acid, and an AzF-dependent escherichia coli strain and the chimeric phenylalanine translation system constructed by the construction method. The directed evolution strategy established by the translation system provided by the invention has broad-spectrum applicability and can be applied to directed evolution of other genetic password extension systems; in addition, the signal-to-noise ratio of a chimera phenylalanine translation system for identifying 4-azido-phenylalanine (AzF), which is established by the translation system provided by the invention, is up to 65 times.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a chimera phenylalanine translation system introducing an unnatural amino acid and a construction method thereof. Background technique [0002] Genetic code expansion technology uses orthogonal aminoacyl-tRNA synthetases / tRNA pairs (Aminoacyl-tRNAsynthetases / tRNA pairs) to insert unnatural amino acids into proteins at specific sites, and has been widely used in protein function research and protein function modification , protein function remodeling and so on. Genetic code expansion technology can also control protein translation through the addition of unnatural amino acids, and then play the role of a molecular switch, which has been applied to the construction of attenuated E. coli. However, due to the lack of broad-spectrum orthogonal, high-efficiency, and high-signal-to-noise ratio aminoacyl-tRNA synthetase / tRNA pairs in eukaryotes and prokaryotes, not...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N9/00C12N15/70C12N15/65C12N1/21C12R1/19
CPCC12N15/11C12N9/93C12N15/70C12N15/65C12Y601/0102C12N2840/55
Inventor 林世贤赵红霞丁文龙柳光龙
Owner 杭州嵌化合生医药科技有限公司
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