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Method for rapidly constructing filamentous fungus expression vector

A filamentous fungus and expression vector technology, applied in the field of filamentous fungal vector construction, can solve the problems of high cost, many non-specific fusion fragments and high error rate, and achieve the effect of high accuracy

Inactive Publication Date: 2019-10-08
NANJING NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the research of our laboratory, we have found that the existing kits are not suitable for the construction of vectors with multiple fragments or long pathways. There are many non-specific fusion fragments, the error rate is high, the cost is high, and the correct rate of vector construction is low.

Method used

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  • Method for rapidly constructing filamentous fungus expression vector
  • Method for rapidly constructing filamentous fungus expression vector
  • Method for rapidly constructing filamentous fungus expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Acquisition of Escherichia coli Replication Module, Yeast Replication Module and Filamentous Fungi Replication Module

[0023] The medium used is: LB medium (1L): 10g peptone, 5g yeast extract, 10g sodium chloride, and 20g agar should be added to the solid medium.

[0024] The plasmid pCT74 was hydrolyzed with restriction endonuclease SalI to obtain the skeleton structure of the expression vector of filamentous fungi, which contains the replication module of E. Mycin resistance gene and replication initiation element), and the part of HygR excised by SalI restriction endonuclease was cloned using pCT74 as template. The clones of yeast self-replicating sequence 2μOri and auxotrophic selection marker URA3 in the yeast plasmid replication module were amplified using the pCRCT plasmid as a template, and the promoter, target gene, and terminator sequences were amplified by PCR using pCT74 as a template. The sequence is shown in Table 3, and the assembled fragment...

Embodiment 2

[0031] Embodiment 2: Each fragment co-transforms yeast

[0032] The medium used is: SC-URA medium (600mL): 1g YNB, 3g ammonium sulfate, 0.414g CMS-URA, 550mL single distilled water, autoclaved (115°C, 30min); add 50mL 24% glucose solution (filter sterilized).

[0033] YPD medium (1L): 10g yeast extract, 20g peptone, 20g glucose, 20g agar should be added to the solid medium.

[0034] Production of competent yeast cells: (1) Pick a single yeast clone and culture it in 2mL YPD medium at 30°C and 250rpm overnight. (2) Determination of bacterial liquid OD 600nm , draw part of the bacterial solution into fresh YPD medium (make the solution OD 600nm =0.2) Cell OD after continuing to culture for 4-5h 600nm reach 0.8, centrifuge at 4000 rpm for 5 min at 4°C, and remove the supernatant. (3) The cells were resuspended and washed with 5 mL of cold sterile water, centrifuged at 4000 rpm for 10 min at 4°C, and the supernatant was discarded. (4) Add 1 mL of cold sterile water to resusp...

Embodiment 3

[0036] Example 3: Extraction and enrichment of yeast assembly plasmids

[0037] The reagents used are: yeast wall breaking buffer: 2% Triton X-100, 1% SDS, 100mmol / L NaCl, 10mmol / L Tris-Cl (pH 8.0), 1mmol / L Na2EDTA.

[0038] TE buffer: 10mmol / L Tris-HCl, 1mmol / L EDTA, pH 8.0.

[0039] Extraction of yeast transformant plasmids: (1) Randomly select 10 single clones on the SC-URA plate, insert each single clone into 10 mL of SC-URA liquid medium, and culture overnight at 30°C (500 μL of bacterial solution + 500 μL 50% glycerol, frozen at -80° refrigerator for preservation). (2) Centrifuge the remaining yeast liquid at 13,000 rpm for 1 min, discard the supernatant, and add 1.2 mL of yeast wall breaking buffer to suspend the yeast. (3) Dispense the suspension into 1.5mL EP tubes filled with glass beads (about 0.3g per tube), 200 μL per tube, vortex for 5 minutes, and then freeze the bacteria repeatedly in liquid nitrogen and in boiling water at 95°C Each time for 1 min (about 3 ...

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Abstract

The invention discloses a method for rapidly constructing a filamentous fungus expression vector. The method comprises the following main processes: respectively cloning a yeast plasmid replication module, a bacterial plasmid replication module, a filamentous fungal replication module, and the corresponding promoter sequences, wherein each fragment has a homologous arm of about 20 to 50 bp connected end to end, and employing an electrotransformation technology to obtain a plasmid containing three modules, which can be used for rapid identification of the strength of a non-mode filamentous fungi self-promoter, key enzyme gene expression, secondary metabolite biosynthesis, and interaction between endophytes and plants. The method provided by the invention has the advantages of rapidity and high precision, and no need of restriction endonuclease, and can be used for the construction or replacement of multi-fragment and long-fragment vectors, which makes up the defects of difficult selection of restriction endonuclease during the construction of the existing filamentous fungal vector and non adaption in multiple segments or long segments.

Description

technical field [0001] The invention belongs to a method for constructing a filamentous fungal vector, in particular to a method for rapidly constructing a filamentous fungal expression vector. Background technique [0002] As a cell factory, filamentous fungi are widely used in the production of a variety of products, and have also become indispensable expression hosts for the production of fungal-derived enzymes and non-fungal-derived enzymes. In addition, filamentous fungi themselves are capable of producing abundant secondary metabolites and serve as a treasure trove for clinically meaningful drug development. In particular, endophytic filamentous fungi have attracted more and more attention due to their mutualistic symbiosis with plants, which can promote plant growth, increase yield, and disease resistance. The advantages of filamentous fungi in practical applications are mainly reflected in: 1) Filamentous fungi have strong growth ability, can grow in simple and chea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/66C12N15/65C12N15/80
CPCC12N15/66C12N15/65C12N15/80
Inventor 梅艳珍戴传超杨倩
Owner NANJING NORMAL UNIVERSITY
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