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A specific DNA molecule that regulates promoter strength and expression patterns

A technology of DNA molecules and DNA sequences, which is applied in the field of specific DNA molecules, can solve problems such as limiting the induced expression system, cell physiological burden, affecting production capacity level and production efficiency, etc.

Active Publication Date: 2019-03-19
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the metabolic engineering of yeast, the constitutively high expression of genes or the premature accumulation of metabolites will cause a physiological burden on the cells and affect the final production level and production efficiency. Therefore, time-sharing regulation of gene expression is of great significance very important
[0004] The inducible promoter GAL1p is commonly used in yeast to achieve time-sharing regulation of gene expression, but the inducible expression intensity and sensitivity of the inducible promoter GAL1p are relatively low, and the expression of the target gene cannot be quickly initiated; at the same time, it is strictly repressed by glucose
Glucose is the most commonly used fermentation raw material in the actual fermentation industry. The presence of glucose in the fermentation system will strictly repress the gene expression under the regulation of the inducible promoter GAL1p, which brings difficulties to the control of the fermentation process, thus limiting the inducible expression system. practical application of

Method used

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  • A specific DNA molecule that regulates promoter strength and expression patterns
  • A specific DNA molecule that regulates promoter strength and expression patterns
  • A specific DNA molecule that regulates promoter strength and expression patterns

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1. GAL1p promoter regulates the expression of green fluorescent protein encoding gene

[0070] 1. Construction of expression vector

[0071] 1. Construction of recombinant plasmid YCp-ADH1t

[0072] (1) According to the terminator sequence of Alcohol dehydrogenase 1 (ADH1) of Saccharomyces cerevisiae alcohol dehydrogenase 1 (ADH1) in GenBank (GenBankID: Z74828.1), design and artificially synthesize primer 1: 5′-CCC GAGCTC ATTCGCTTAAAGAATAC-3' (the underlined part is the recognition site of restriction enzyme SacI) and primer 2: 5'-ACGC GTCGAC GGCCATCTCCACACCAG-3' (the underlined part is the recognition site of restriction enzyme SalI).

[0073] (2) Extract the total DNA of Saccharomyces cerevisiae YS58 as a template, use primer 1 and primer 2 synthesized in step (1) as primers, use Q5 ultra-fidelity DNA polymerase for PCR amplification, and recover PCR amplification products.

[0074] Reaction conditions: denaturation at 98°C for 30s; denaturation at 98°C for 10s, annea...

Embodiment 2

[0108] Example 2. GAL1p promoter modification to improve induction intensity and sensitivity

[0109] 1. Construction of recombinant plasmid YCp-UGGA

[0110] (1) According to the Saccharomyces cerevisiae GAL1 gene sequence in GenBank (GenBankID: X76078.1), design and artificially synthesize primer 7: 5′-CCG GAATTC GCATGCCCGCGGTGCTCATTGCTATAT-3′ (the underlined part is the recognition site of restriction enzyme EcoRI) and primer 8: 5′-CCC AAGCTT GCTAGTATTGTAGAATC-3' (the underlined part is the recognition site of restriction endonuclease HindIII).

[0111] (2) Using the PCR amplification product obtained in step 1 of Example 3 (2) as a template, using primer 7 and primer 8 synthesized in step (1) as primers, using Q5 ultra-fidelity DNA polymerase for PCR amplification, The PCR amplification product is recovered.

[0112] (3) Double digestion with restriction enzymes EcoRI and HindⅢ. Step (2) Obtain PCR amplification product, and recover the digestion product.

[0113] (4) Double dig...

Embodiment 3

[0124] Example 3. The transformation of GAL1p promoter breaks through the glucose repressive effect of GAL1p promoter

[0125] 1. Knock out the repressor binding site in the GAL1p promoter

[0126] 1. According to the GAL1 gene sequence of Saccharomyces cerevisiae in GenBank (GenBankID: X76078.1), design and artificially synthesize primer 9, primer 10, primer 11 and primer 12.

[0127] Primer 9: 5'-TTGATTCGTTCATTTGAAGGCCAGGTTACTGCCAATTTTT-3';

[0128] Primer 10: 5'-AAAAATTGGCAGTAACCTGGCCTTCAAATGAACGAATCAA-3';

[0129] Primer 11: 5'-ATCGCTTCGCTGATTAATTATAAGGCTAAAAAACTAATCG-3';

[0130] Primer 12: 5'-CGATTAGTTTTTTAGCCTTATAATTAATCAGCGAAGCGAT-3'.

[0131] 2. Using the recombinant plasmid YCp-UGGA constructed in Example 2 as a template, using primer 7 synthesized in Example 2 and primer 9 synthesized in step 1 as primers, PCR amplification was performed using Q5 ultra-fidelity DNA polymerase, The PCR amplification product 1 of about 418 bp was recovered; the recombinant plasmid YCp-UGGA const...

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Abstract

The invention discloses a specific DNA molecule capable of adjusting promoter strength and an expression pattern. The specific DNA molecule comprises an RREP zone and / or a PCA zone and / or a UASGal4pBS zone and / or a GAL1pdeltam2 zone and / or an ADH (alcohol dehydrogenase)1t zone and / or a GAL1p zone, wherein nucleotide sequences of the RREP zone, the PCA zone, the UASGal4pBS zone and the GAL1pdeltam2 zone are shown from the 4th site to the 12th site, from the 19th site to the 27th site, from the 31th site to the 208th site, from the 215th site to the 660th site starting from the 5' end in a sequence 6, the nucleotide sequence of the GAL1p zone is shown from the 185th site to the 648th site starting from the 5' end in a sequence 4, and the nucleotide sequence of the ADH1t zone is shown in a sequence 1. The specific DNA molecule can adjust the promoter strength and / or the expression pattern and realizes promoter diversity.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a specific DNA molecule that regulates the strength and / or expression pattern of a promoter. Background technique [0002] Metabolic Engineering and Synthetic Biology, which optimize or reconstruct cellular metabolic networks through genetic manipulation, are very important frontiers in the field of modern biotechnology. In the transformation of metabolic pathways, to maximize the synthesis of target metabolites without affecting the physiological functions of the host cell, it is often necessary to coordinate the expression levels of multiple genes. Therefore, synthetic biology methods are used to design and construct different strengths of metabolites. Expression of regulatory elements, timely and appropriate regulation of gene expression levels, can improve the predictability of metabolic pathway modification, thereby simplifying the modification process, improving the effic...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/63
CPCC12N15/113C12N15/63C12N2310/10C12N2800/10
Inventor 何秀萍刘莎郭雪娜王肇悦
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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