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A kind of blue light-mediated regulation plasmid and its construction method and application

A construction method and blue light technology, applied in the field of microorganisms, can solve the problems of strong toxicity of the inducer methanol, unfavorable application of Pichia pastoris, unstable transport delay, etc., and achieve the effects of good reversibility, simple operation and fast response.

Active Publication Date: 2022-05-03
HUAZHONG UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the induction and regulation of Pichia pastoris is still in the stage of exogenous chemical inducers. The most commonly used inducer, methanol, is highly toxic and flammable, which is not conducive to the application of Pichia pastoris in the fields of food, medicine, and agricultural product processing.
Therefore, it is an urgent need in this field to develop a new light regulation method of Pichia pastoris, which can effectively solve the defects of toxicity, instability and transport delay of traditional chemical inducers, and to expand the methods of expression regulation of Pichia pastoris. A technical problem solved

Method used

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  • A kind of blue light-mediated regulation plasmid and its construction method and application
  • A kind of blue light-mediated regulation plasmid and its construction method and application
  • A kind of blue light-mediated regulation plasmid and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] In this embodiment, the gene sequence is optimized.

[0043] The VP16 and EL222 gene sequences and protein sequences retrieved from NCBI were optimized according to the codon preference of Pichia pastoris, and the DNA sequences were resynthesized.

[0044] The specific optimization method is: according to the gene synthesis company GenScript codon optimization software OptimumGene TM To optimize the gene, mainly refer to the codon preference of Pichia pastoris, then combine the filter element and balance the GC content of the gene to finally complete the optimization of the gene codon, and obtain the SV40-VP-EL nucleic acid sequence, as shown in SEQ ID NO.3 shown; PGAP-SV40-VP-EL nucleic acid sequence, as shown in SEQ ID NO.4; PC120-GFP-TADH1 nucleic acid sequence, as shown in SEQ ID NO.5.

Embodiment 2

[0046] In this example, the blue-light regulatory plasmid pGSVEACA was designed and constructed using the commonly used plasmid pAO815 of Pichia pastoris as the backbone.

[0047] The specific construction method is:

[0048] The PGAP-SV40-VP-EL gene sequence optimized in Example 1 was fully synthesized, and then the pAO815 plasmid AOX1 promoter and the sequence between the AOX1 promoter and AOX1 terminator were replaced by Gibsonassembly technology to obtain the plasmid pGSVEA;

[0049] The PC120-GFP-TADH1 gene sequence optimized in Example 1 was fully synthesized, and then the CGTTCGTTTGTGC sequence of the plasmid pGSVEA was replaced by Gibsonassembly technology to obtain the blue light-regulated plasmid pGSVEACA.

[0050] In this example, the PGAP-SV40-VP-EL gene sequence does not contain a terminator sequence, so it is necessary to borrow the AOX1 terminator on the pAO815 plasmid backbone to construct a complete SV40-VP-EL open reading frame and realize the SV40-VP - Cons...

Embodiment 3

[0053] In this example, the blue light-regulated plasmid pGSVEACA was transformed, induced, expressed and analyzed.

[0054] (1) Plasmid extraction and quantitative detection

[0055] 1) Take 10 mL of the overnight bacterial culture, centrifuge at 13400 g for 1 min, discard the supernatant and collect the precipitate.

[0056] 2) Add 500 μL of solution Ⅰ, solution Ⅱ and solution Ⅲ in sequence, immediately flip up and down gently for 6 to 8 times, let stand for 5 minutes, and centrifuge at 13400 g for 10 minutes.

[0057] Wherein the composition of solution I is 25mM Tris-HCl (pH=8.0), 10mM EDTA, 50mM glucose;

[0058] The composition of solution II is 250mM NaOH, 1% (W / V) SDS;

[0059] The composition of solution III is 3M potassium acetate, 5M acetic acid.

[0060] 3) Add the supernatant collected in the previous step into the filter column, centrifuge at 13400 g for 1 min, add 450 μL of isopropanol and mix well. Then add to the adsorption column, centrifuge at 13400g for...

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Abstract

The invention discloses a blue light-regulated promoter PC120 that can be recognized and bound by the blue light protein SV40‑VP‑EL, and belongs to the technical field of microorganisms. The present invention also discloses a blue-light-mediated regulation plasmid comprising the blue-light-regulated promoter PC120, and the plasmid also includes a fusion gene PGAP-SV40-VP-EL for transcribing and translating blue-light protein. The invention further discloses the preparation method of the plasmid and the application of the plasmid and / or construction method in the regulation of Pichia pastoris. The blue light-mediated regulatory plasmid of the present invention provides a non-invasive, reversible and high-spatial-resolution blue light regulation method for the induced expression of Pichia pastoris, which is beneficial to broaden the regulation methods of Pichia pastoris in production and life.

Description

technical field [0001] The invention relates to the technical field of microbes, in particular to a blue light-mediated regulatory plasmid and its construction method and application. Background technique [0002] The traditional regulation methods of gene expression process are mostly exogenous chemical inducers combined with soluble transcription factors to achieve artificial control of gene expression. This method is the main driving force for the rapid development of biotechnology, synthetic biology and medical research in the last century one. Although these traditional regulatory methods can initially control the time and amount of gene expression, their limited reversibility and inducer-based transport regulation mechanism make them have various limitations and defects, such as: its potential off-target Effects, delay in transport process and toxicity. Ideally, biological regulatory elements that can be quickly and precisely turned on or off at will will effectively...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/81C12N15/65C07K14/195
CPCC07K14/195C12N15/815C12N15/65C12N2800/102
Inventor 张后今闫云君王志乾龚梦瑶韩娟张婷
Owner HUAZHONG UNIV OF SCI & TECH
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