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Expression regulatory sequence

An expression regulation and sequence technology, applied in biochemical equipment and methods, applications, and botanical equipment and methods, etc., can solve the problems of complex translation of leader peptides, and achieve the effect of increasing yield

Inactive Publication Date: 2002-10-23
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Translation of the leader peptide in vivo can be further complicated when the sense amino acid is in excess (and more precisely, the corresponding cargo tRNA) or is deficient in bacterial cells

Method used

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  • Expression regulatory sequence
  • Expression regulatory sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Example 1. Construction of recombinant plasmids with natural attenuators and artificial anti-attenuators and fragments thereof

[0105] 1. Vector plasmid pML-P tac - Construction of ter_thrL-cat

[0106] Ap with a ColE1-like replicon as a selectable marker was constructed in the following manner R -gene, tac promoter (P tac ) (Russel D.R., Bennett G., Gene 20 (1982) 231), the rho-independent transcription terminator of the synthetic E. coli threonine operon (ter_thrL) leader peptide and the P tac Plasmid vector pML-P of downstream cat-gene structural part tac -ter_thrL-cat. Will be from P tac The gene cat, which initiates transcription, was used as a reporter gene for further experiments.

[0107] 1.1. Construction of pML-pp-vector

[0108] Two plasmids were used as starting plasmids for the construction of the pML-pp-vector. The first plasmid was the previously described plasmid pML24 (Trukhan et al. Biotechnologiya (in Russian) 4, No. 3 (1988) 325-334). The s...

Embodiment 2

[0148] Example 2. Detection of Cat protein accumulation level in bacterial strains containing recombinant plasmids with natural trpL, artificial anti-attenuator and fragments thereof.

[0149] According to conventional implementation, the aforementioned plasmid (see Example 1): pML-P tac -an4: an5-cat, pML-P tac -an3:an4(an4:an5)-cat, pML-P tac -trpL-cat, pML-P tac -anti_att-I-cat, pML-P tac -anti_att-II-cat introduced strain Escherichia coli TG1 (supE, hsd, thi, Δ(lac-proAB), F'(traD36, proAB + , lacl Q , lacZΔM15]) and Escherichia coli B7248 (trpB - : Tnl0, Str R ) and select plasmid vector cells in medium supplemented with ampicillin (100 μg / ml) (Sambrook et al., "Molecular Cloning Laboratory Manual". (1989) 2nd edition, Cold Spring Harbor Laboratory Press). The resulting cell cultures were grown in test tubes containing liquid medium at 37°C with good ventilation. Regarding the medium, L-broth with added ampicillin is used for TG1-driven plasmid-vector strains, whi...

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Abstract

An expression control sequence that depends on the concentration of amino acids in the cell to regulate the expression of a target gene that is connected downstream of the expression control sequence, wherein the expression control sequence includes a promoter connected upstream of the expression control sequence and a promoter connected downstream of the expression control sequence In the bacteria of the DNA construct of the target gene, the expression of the target gene can be increased by increasing the concentration of amino acids in the cell to reduce the frequency of termination in the expression control sequence and the frequency of transcription starting from the promoter.

Description

technical field [0001] The present invention relates to the microbiology industry, and in particular to the development of novel methods of regulated gene expression in bacterial cells. Background technique [0002] Inducing the expression of cloned genes by adding relatively simple and inexpensive compounds is a very attractive idea for many bioengineering approaches based on the development of recombinant bacteria, especially E. coli. Obviously, natural L-amino acids are very good candidates for use as such inducers. However, to the inventors' knowledge, the regulatory region of the tryptophanase gene is a unique natural system induced by the addition of tryptophan to the culture medium [Landick R., Tumbough C.L., Yanofsky C. "Transcription Decay" / " Escherichia coli and Salmonella (Salmonella.) Cellular and Molecular Biology" (Second Edition, F.C. Neidhardt-Ed.), (1996), pp. 1263-1286]. In contrast, there are several systems that reduce the expression of regulated genes ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N1/21C12N9/10C12N15/00C12N15/11C12N15/63C12N15/70C12N15/71C12P13/00C12R1/125C12R1/19
CPCC12N15/71
Inventor S·V·马什科D·V·兹门科夫
Owner AJINOMOTO CO INC
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