Method for regulating gene expression intensity on microbial chromosome by using artificial regulatory element and its library

An artificial regulation element and gene regulation technology, which can be used in the introduction of foreign genetic material using vectors, DNA preparation, recombinant DNA technology, etc. It can solve the problems of large metabolic load of host cells, economic infeasibility, and difficulty in carrying long DNA fragments.

Active Publication Date: 2011-12-21
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this approach has three major drawbacks
First, the use of these inducible promoters in the production of bulk chemicals is not economically feasible due to the need to add expensive inducers such as lactose, IPTG, arabinose, etc. to induce the expression of these promoters
Second, plasmid-based expression has many disadvantages: the maintenance of plasmids will cause a large metabolic load on host cells, especially high-copy plasmids; the genetic stability of many plasmids is not good; only the replication of low-copy number plasmids and cell The reproduction of the cells is synchronized, so only they maintain the same copy number in all cells; the synthesis of many compounds requires the construction of a complex metabolic pathway in the cell, so it is necessary to introduce long DNA fragments containing multiple genes, Most plasmids are more difficult to carry long DNA fragments
At present, there are relatively few reports on the construction of promoter upstream region, messenger RNA stability region and ribosome binding site regulatory elements

Method used

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  • Method for regulating gene expression intensity on microbial chromosome by using artificial regulatory element and its library
  • Method for regulating gene expression intensity on microbial chromosome by using artificial regulatory element and its library
  • Method for regulating gene expression intensity on microbial chromosome by using artificial regulatory element and its library

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Experimental program
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Effect test

Embodiment 1

[0067] Embodiment 1, the construction of Escherichia coli promoter library 1 (P-Lib1)

[0068] The strategy of Escherichia coli promoter library 1 (P-Lib1) construction is to combine Escherichia coli ATCC 8739 (the public can obtain from Tianjin Institute of Industrial Biotechnology, and it has been recorded that Escherichia coli The non-patent literature of bacteria ATCC 8739 is Zhang, X., K.T.Shanmugam, L.O.Ingram.2010.Fermentation of glycerol tosuccinate by metabolically engineered strains of Escherichia coli.Appl Environ Microbiol, 76: 2397-2401) on chromosome The promoter of the glycosidase (lacZ) gene was replaced with an artificial promoter library 1 (P-Lib1) ( figure 1 ), and then determine the strength of each promoter by measuring the enzyme activity of β-galactosidase.

[0069] The promoter library 1 (P-Lib1) was designed based on the PL promoter sequence of bacteriophage lambda (Love et al., 1996, Gene, 176:49-53), and the sequence is sequence 1 in the sequence li...

Embodiment 2

[0080] Embodiment 2, the construction of Escherichia coli promoter library 2 (P-Lib2)

[0081] In order to improve the efficiency of homologous recombination and expand the storage capacity of the promoter library, the Escherichia coli promoter library 2 (P-Lib2) was constructed. When constructing P-Lib2, the length of the left homology arm was extended from 50 bases to 500 bases (500 bases upstream of lacI, relative to the region of -500-0 of the lacI start codon) ( image 3 ).

[0082] The sequence of P-Lib2 is sequence 2 in the sequence listing. Its characteristics are: the promoter-35 core region and the 15 bases upstream of the -35 region are the same as P-Lib1; the sequence of the -10 core region is changed to TATAAT; there are 14 random bases between the promoter-35 and the -10 core region bases and TGR. The downstream sequence of -10 core region was changed to 6 random bases.

[0083] The DNA fragment IV ( image 3 ). First, using the genomic DNA of the recombina...

Embodiment 3

[0094] Embodiment 3, the construction of Escherichia coli messenger RNA stable region library 1 (M-Lib1)

[0095] In order to improve the stability of messenger RNA after gene transcription, a messenger RNA stable region library (M-Lib1) was constructed between the promoter and ribosome binding site.

[0096] The sequence of M-Lib1 is sequence 9 in the sequence table, and it is characterized in that: the promoter sequence is the same as the P2-15 sequence of the strongest promoter in P-Lib2; the sequence of the messenger RNA stable region is 18 random bases and PmeI enzyme cutting site sequence.

[0097] The DNA fragment VI ( Figure 5 ). First, DNA fragment V was amplified using primers lacI-up500 and pL-down-3 using the genomic DNA of the recombinant strain P2-15 as a template. The primer sequences are:

[0098] pL-down-3: GGCTCAATTATATCAACG.

[0099] Then, using DNA fragment V as a template, DNA fragment VI was amplified using primers lacI-up500 and lacZ-pL-R3C. The p...

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Abstract

The invention discloses a method for regulating the expression intensity of genes on microorganism chromosomes by using artificial regulatory elements and libraries thereof. The method for regulating the gene expression intensity on the microbial chromosome by using the artificial regulatory element and its library provided by the present invention includes the following steps: amplify a DNA fragment by PCR, which includes the upstream sequence of the original regulatory region of the gene to be regulated on the microbial chromosome A homologous fragment containing 40-3000 bases, resistance marker gene, artificial regulatory element library or an artificial regulatory element with specific expression strength, homologous to the sequence downstream of the start codon of the gene to be regulated on the microbial chromosome A fragment containing 40-3000 bases; the DNA fragment is electrotransformed into the microorganism, and the original regulatory region of the gene to be regulated on the microbial chromosome is replaced by an artificial regulatory element library or an artificial regulatory element with a specific expression intensity .

Description

technical field [0001] The invention relates to a method for regulating the expression intensity of genes on microorganism chromosomes by using artificial regulatory elements and libraries thereof. Background technique [0002] Gene expression regulation technology is one of the core technologies of metabolic engineering. At present, when transforming microorganisms to improve the synthesis ability of target products, a commonly used strategy is to regulate the expression of genes through the method of plasmid overexpression, and increase the activity of one or several key enzymes, thereby enhancing the metabolism of microorganisms to synthesize target products flow. When the plasmid is overexpressed, inducible promoters (such as lac, trc, ara and other promoters) are usually used to regulate the expression of the gene. However, this approach has three major drawbacks. First, the use of these inducible promoters in the production of bulk chemicals is not economically feas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/67C12N15/10C12N15/09
Inventor 张学礼朱欣娜李清艳卢焦赵婧
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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