阅读说明：本技术 Trc启动子突变文库及其应用 (Trc promoter mutation library and application thereof ) 是由 邓禹 赵梅 张佳南 娜荷芽 周宇辰 汪敏 叶乘锋 于 2020-06-28 设计创作，主要内容包括：本发明涉及Trc启动子突变文库及其应用,属于生物技术领域。本发明通过大量筛选获得了SEQ ID NO.1～41所示的启动子,他们调控目的基因表强度的范围较广,以此建立了Trc启动子突变文库。本发明通过构建启动子文库,将“开量”式微调基因表达成为可能,在代谢分析和改造中,突破了原来只能通过基因敲除和过量表达两种非“开”即“关”的手段来实现对细胞基因型的扰动,即可通过对目标基因表达进行梯度式的精确微调来分析和控制其对表型和代谢流分布的影响。(The invention relates to a Trc promoter mutation library and application thereof, belonging to the technical field of biology. The invention obtains the promoters shown in SEQ ID NO. 1-41 through mass screening, and the promoters have wide range of regulating and controlling the table intensity of target genes, so that a Trc promoter mutation library is established. The invention makes it possible to express the 'on-the-go' type fine-tuning gene by constructing the promoter library, breaks through the disturbance of the cell genotype only by two non 'on' or 'off' means of gene knockout and over-expression in the metabolic analysis and modification, and analyzes and controls the influence of the gradient type fine-tuning on the target gene expression on the phenotype and the metabolic flow distribution.)

1. A promoter library capable of regulating and controlling the expression quantity of a target protein is characterized in that the promoter library comprises promoters shown by SEQID NO. 1-41; wherein the expression intensity of the promoters is sequentially increased according to the sequence of SEQ ID NO. 1-41.

2. A promoter is characterized in that a nucleotide sequence is shown as any one of SEQ ID NO. 7-41.

3. An expression vector carrying the promoter of claim 2.

4. The expression vector of claim 3, wherein the plasmid is linked to the promoter; the downstream of the promoter comprises a multiple cloning site or at least one enzyme cutting site.

5. A recombinant microbial cell comprising the promoter of claim 2.

6. A method for controlling the expression level of a target protein, which comprises expressing a gene encoding the target protein using a promoter in the promoter library of claim 1, in accordance with the expression level of the target protein in a desired up-regulation manner.

7. The method according to claim 6, wherein the gene encoding the target protein is ligated to the expression vector of claim 3 or 4 and then transformed into a host cell for expression.

8. The method according to claim 7, wherein the target gene encoding the target protein is ligated to a multiple cloning site downstream of the promoter, thereby ligating the target gene to the promoter.

9. The method according to any one of claims 6 to 8, wherein the target gene is a foreign gene relative to the promoter.

10. The method according to any one of claims 6 to 8, wherein the protein of interest is galactosidase.

Technical Field

The invention relates to a Trc promoter mutation library and application thereof, belonging to the technical field of biology.

Background

Promoters are important regulatory elements for gene expression, and determine the strength and timing of gene expression. With the development of synthetic biology and metabolic engineering, people have higher and higher requirements on the regulation range and strength of a promoter in the regulation and control of cell metabolism. Promoters with a wider regulatory range can provide finer regulation of expression of different genes in metabolic pathways, while higher strength promoters provide sufficient motive force for achieving efficient expression of target proteins. The functional promoters can be divided into an inducible promoter and a constitutive promoter, the inducible promoter regulates and controls the transcription level of a target gene under the action of corresponding regulatory protein and an inducer, and the greatest advantage of the inducible promoter is high expression strength and accurate regulation and control. However, since the expression strength of the inducible promoter is closely related to the inducer, a plurality of different inducers are required to be added in the precise regulation of the multi-gene metabolic pathway, so as to regulate the expression levels of different genes, which limits the use thereof in the metabolic regulation. In addition, most inducible promoters have leaky expression, and the addition of an inducer greatly increases the fermentation cost. Constitutive promoters have the advantage of stable expression levels under different growth conditions, but the existing constitutive promoters in nature are generally weak in strength and cannot meet the actual production requirements.

Disclosure of Invention

The first purpose of the invention is to provide a promoter library capable of regulating and controlling the expression quantity of a target protein, wherein the promoter library comprises promoters shown in SEQ ID NO. 1-41; wherein the expression intensity of the promoters is sequentially increased according to the sequence of SEQ ID NO. 1-41.

In one embodiment, the promoter library consists of the promoters shown in SEQ ID NO. 7-41.

The second purpose of the invention is to provide a promoter, the nucleotide sequence of which is shown in any one of SEQ ID NO. 7-41.

The third purpose of the invention is to provide an expression vector carrying the promoter.

In one embodiment, the expression vector is a plasmid.

In one embodiment, the plasmid has the promoter attached thereto; the downstream of the promoter comprises a multiple cloning site or at least one enzyme cutting site.

In one embodiment, the expression vector may further comprise (in the 5 'to 3' direction): a promoter for directing the transcription of a target gene and the target gene. If desired, the recombinant vector may also include a 3 'transcription terminator, a 3' polyadenylation signal, other untranslated nucleotide sequences, transit and targeting nucleotide sequences, resistance selection markers, enhancers or operators.

In one embodiment, the plasmid includes, but is not limited to, pTrc99 a.

The fourth purpose of the invention is to provide a method for regulating the expression level of target protein by using the promoter library; the method comprises the steps of utilizing a promoter shown in any one of SEQ ID NO. 7-41 to express a gene encoding the target protein, or connecting the gene encoding the target protein to an expression vector containing the promoter, and converting the gene into a host cell for expression.

In one embodiment, the method comprises ligating a gene of interest encoding the protein of interest to a multiple cloning site downstream of the promoter, thereby ligating the gene of interest to the promoter.

In one embodiment, the gene of interest may be exogenous (heterologous) with respect to the promoter.

In one embodiment, the gene of interest is one that encodes a protein having a particular function, e.g., certain proteins having an important property or function.

In one embodiment, the protein of interest is green fluorescent protein or galactosidase.

In one embodiment, the method comprises the steps of:

(1) amplifying a fragment containing a target gene, an Nco I enzyme cutting site and a Hind III enzyme cutting site;

(2) connecting the fragment obtained in the step (1) to a plasmid pTrc99a to obtain a recombinant plasmid pTrc-lacZ;

(3) replacing a wild type Trc promoter by a mutant promoter obtained by error-prone PCR on the plasmid pTrc-lacZ obtained in the step (2) to obtain a recombinant plasmid mpTrc-lacZ;

(4) transforming the recombinant plasmid obtained in the step (3) into a host cell, and screening a positive transformant;

(5) culturing the recombinant cells expressing the correct protein obtained by screening in the step (4) in a suitable culture medium.

In one embodiment, transformation of a host cell with a recombinant DNA may be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase, treated with TB ions and dimethyl sulfoxide (super competent preparative method) or treated with 10% glycerol (electroporation competent).

In one embodiment, the host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Representative examples are: yeast, Escherichia coli, animal tissue cells, plant tissue cells, and the like. It will be clear to one of ordinary skill in the art how to select an appropriate vector and host cell. In a preferred embodiment of the present invention, the host cell is an E.coli cell.

Has the advantages that: according to the invention, a series of promoters are obtained through mass screening, and the scope of the regulation and control of the target gene table intensity is wider, so that a Trc promoter mutation library is established; the invention makes it possible to express the 'on-the-go' type fine-tuning gene by constructing the promoter library, breaks through the disturbance of the cell genotype only by two non 'on' or 'off' means of gene knockout and over-expression in the metabolic analysis and modification, and analyzes and controls the influence of the gradient type fine-tuning on the target gene expression on the phenotype and the metabolic flow distribution.

Drawings

FIG. 1 is a schematic diagram of recombinant plasmid construction; (A) the construction of the recombinant plasmid pTrc-sfGFP is shown schematically, and (B) the construction of the recombinant plasmid pTrc-lacZ is shown schematically.

FIG. 2 shows fluorescence intensity of GFP of 41 promoters in the Trc promoter mutation library.

FIG. 3 shows the enzyme activities of 41 promoters expressing beta-galactosidase in the Trc promoter mutation library.

FIG. 4 is an alignment of the results of the sequencing of mpTrc1-mpTrc41 (the shaded area indicates the site of mutation).

Detailed Description

The terms:

as used herein, the term "promoter" refers to a nucleotide sequence, which is usually present upstream (5' to) the coding sequence of a gene of interest, and which is capable of directing transcription of the nucleotide sequence into mRNA. Generally, promoters provide recognition sites for RNA polymerase and other factors necessary for proper transcription.

As used herein, the "promoter library" refers to a collection of nucleotides from a series of mutant Trc promoters obtained by the screening of the present invention.

As used herein, the "Trc promoter" is abbreviated as pTrc.

As used herein, the term "pTrc-sfGFP" refers to a recombinant plasmid containing the promoter of sfGFP and Trc, which are genes expressing green fluorescent protein.

As used herein, the term "pTrc-lacZ" refers to a recombinant plasmid containing the promoter of the gene lacZ expressing beta-galactosidase and Trc.

As used herein, the term "mpTrc-sfGFP" refers to a recombinant plasmid containing sfGFP, a gene expressing green fluorescent protein, and the promoter of mpTrc1-mpTrc 41.

As used herein, the term "mpTrc-lacZ" refers to a recombinant plasmid containing the gene lacZ expressing β -galactosidase and the promoter of mpTrc1-mpTrc 41.

As used herein, the term "ECTrc-sfgfp" refers to a colony of E.coli transformed with the above plasmid pTrc-sfgfp.

As used herein, the term "ECTrc-lacZ" refers to a colony of E.coli transformed with the above plasmid pTrc-lacZ.

As used herein, the term "ECmTrc-sfgfp" refers to E.coli colonies transformed with the above plasmid mpTrc-sfgfp.

As used herein, the term "ECmpTrc-lacZ" refers to a colony of E.coli transformed with the above-mentioned plasmid mpTrc-lacZ.

As used herein, "isolated" refers to a substance that is separated from its original environment (in the case of a natural substance, the original environment is the natural environment). For example, a polynucleotide or polypeptide in a natural state in a living cell is not isolated and purified, but the same polynucleotide or polypeptide is isolated and purified from other substances which are naturally present in the same state.

As used herein, "gene of interest" refers to a gene whose expression can be directed by the promoter of the present invention. The invention is not particularly limited with respect to suitable genes of interest, including, for example, but not limited to: structural genes, genes encoding proteins with specific functions, enzymes, reporter genes (such as green fluorescent protein sfGFP, galactosidase gene lacZ).

As used herein, "exogenous" or "heterologous" refers to the relationship between two or more nucleotide or protein sequences from different sources, e.g., a promoter is exogenous to a gene of interest if the combination of the promoter and the sequence of the gene of interest is not normally found in nature. A particular sequence is "foreign" to the cell or organism into which it is inserted.

Fluorescence intensity and OD₆₀₀Detection of the value:

utilize multi-functional ELIASA Cytation 3imaging ReaderSystem (BioTek, Berton instruments, USA) for measuring fluorescence intensity and OD₆₀₀The excitation wavelength in green fluorescence detection was 485nm and the emission wavelength was 528nm using wild type e.coli MG1655 as a control strain. Final measured fluorescence intensity and cell OD₆₀₀The ratio of (a) to (b) is defined as the expression intensity of the promoter.

Enzyme activity assay for beta-galactosidase

Centrifuging 3mL strain culture solution containing β -galactosidase, discarding supernatant, adding lysis solution Tris-HCl 25 μ L and PMSF 12.5 μ L, oscillating, breaking cell wall, adding 25 μ L Tris-HCl after breaking cell wall, centrifuging, collecting supernatant as enzyme solution, adding Coomassie brilliant blue 150 μ L enzyme solution 30 μ L, and measuring OD₅₉₅Absorbance. Collecting 20 μ L enzyme solution, adding 90 μ L ZBuffer, incubating at 30 deg.C for 5min, adding 10 μ L ONPG, adding 50 μ L Na immediately when the solution turns light yellow₂CO₃The reaction was stopped and the OD measured₄₂₀Absorbance.

Culture medium:

LB culture medium: 10g/mL peptone, 5g/mL yeast powder and 10g/mL NaCl, and supplementing 1.0 mu g/mL ampicillin and 1.0 mu g/mL streptomycin after sterilization;

m9 medium: 6.78g/L Na₂HPO₄，3g/L KH₂PO₄，1g/L NH₄Cl, 0.5g/L NaCl, 10g/L glucose (sterilized separately), 0.24g/L MgSO₄(sterilized separately), 0.115g/L CaCl₂(sterilization alone). Ampicillin at a final concentration of 100. mu.g/mL and streptomycin at a final concentration of 50. mu.g/mL were added after sterilization.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.