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A method and application of excision screening label

A technology for screening tags and recombinant bacteria, applied in the field of genetic engineering, can solve the problems of inability to meet the needs of multi-gene, multi-site genetic modification, and few screening tags, and achieve the effect of simple and easy removal method, high applicability, and low requirements.

Active Publication Date: 2018-04-20
浙江博崤生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are very few screening tags available in Aspergillus terreus, which cannot meet the needs of genetic modification of multiple genes and multiple loci.

Method used

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  • A method and application of excision screening label
  • A method and application of excision screening label
  • A method and application of excision screening label

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Construction of NHEJ Pathway Deletion Genetic Engineering Strain At-Δku80 with Knockout of ku80 Gene

[0053] 1.1 Construct ku80 targeting components

[0054] Primers Uku80-F (5'-gtcgtagctcttcttgccatc-3'), Uku80-R (5'-aatgggatcccgtaatcaattgccctcaatcaccatctcccttatc-3'), Dku80-F (5'-caagagcggctcatcgtcaccccattccggcctcgatgtgttttc-3')ggatg, c Dku80-R (5'-tccacgcggccatcaccgagc-3'), using the genome of Aspergillus terreus strain CICC40205 as a template, using pfu DNA polymerase (Fermentas, Catalog No.: EP0501) for PCR amplification, using primers Uku80-F / Uku80- R can amplify the upstream sequence U-ku80 of ku80 with a size of about 1.5kb, and use primers Dku80-F / Dku80-R to amplify the downstream sequence D-ku80 of ku80 with a size of 1.5kb. Using the plasmid pPTR II (TAKARA, Catalog No.: 3621) as a template, using ptrA-F (5'-gggcaattgattacgggatc-3') and ptrA-R (5'-atggggtgacgatgagccgc-3') as primers to amplify with a size of about The 2.0kb ptrA fragment was detect...

Embodiment 2

[0061] Example 2 Construction of uracil auxotrophic Aspergillus terreus genetically engineered strains

[0062] 2.1 Construction of pyrG targeting elements

[0063] Primers UpyrG-F (5'-tccatccgatggccattcgtggc-3'), UpyrG-R (5'-ctttacgcttgcgatcccgaaaaggtcaattgagacttggacgac-3'), DpyrG-F1 (5'-tttcgggatcgcaagcgtaaagatgat-3')atgcatga, were designed according to the information published in the Aspergillus terreus NIH2624 genome database DpyrG-R (5'-cgaggtcctaccgatgatgttgc-3'), using the genome of Aspergillus terreus strain CICC40205 as a template, using pfu DNA polymerase (Fermentas, Catalog No.: EP0501) for PCR amplification, using primers UpyrG-F / UpyrG- R can amplify the upstream sequence U-pyrG of pyrG with a size of about 2.5kb, and use primers DpyrG-F1 / DpyrG-R to amplify the downstream sequence D-pyrG of pyrG with a size of 2.5kb. Gel electrophoresis detection and gel tapping recovery and purification. The U-pyrG fragment was fused with the D-pyrG fragment by fusion PCR, and ...

Embodiment 3

[0067] Example 3 Establishment of a genetic transformation system based on uracil auxotrophy

[0068] 3.1 Construction of targeting elements using pyrG1 as a screening marker

[0069] Synthesize SEQ ID NO.1 by gene synthesis, the sequence includes two loxP sites in the same direction, PalcA promoter and restriction endonuclease site, the synthetic sequence is finally cloned on the pUC57simple vector to obtain the vector PalcA- syn( Figure 5). Using pyrG1-F(5'-ctaat gctagc cagcagggaatacgagctcca-3') / pyrG-R(5'-ctaat gctagc gcatcaaatcgtcgtaccgc-3') as primers, the Aspergillus niger Co827 genome was used as template for PCR amplification, and the obtained size was about 1.5 The expression element of the pyrG gene of kb Aspergillus niger was used as the selection marker pyrG1, and the pyrG1 fragment was cloned into the PalcA-syn vector by NheI restriction endonuclease to obtain the plasmid pXH103. Using primers Sgfp-F1 (5'-gat ccatggtgagcaagggcgaggagc-3') / TtrpC-R1 (5'-gag ctcgag...

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Abstract

The invention discloses a method and application for excising a screening tag, and belongs to the technical field of genetic engineering. The present invention is based on the recombinant bacteria that integrates pyrG screening tags with loxP sequences in the same direction on the chromosome as the starting bacteria, and uses a site-specific recombination system to excise the pyrG screening tags located between the two loxP sequences, so that the strain can be reused Genetic transformation was performed using the pyrG selection tag as a starting strain. The method of the invention can carry out the excision of the screening tag, so that the screening tag can be recycled, the genetic modification is no longer limited by the shortage of the screening tag, and provides support for the genetic modification of multiple genes and multiple sites.

Description

technical field [0001] The invention relates to a method and application for cutting out screening tags, belonging to the technical field of genetic engineering. Background technique [0002] Aspergillus terreus is an important filamentous fungus that produces a variety of valuable compounds, including organic acids, enzymes, lipids, and biologically active secondary metabolites. Among them, itaconic acid and lovastatin have been industrialized and have a large market. With the continuous development of molecular biology, it is of great significance to transform the production strains through metabolic engineering and improve the production level of the target product. [0003] With the continuous advancement of molecular biology techniques, functional genomics and genetic engineering of strains are becoming more and more important. In recent years, with the continuous release of a large amount of omics data, functional genomics research and genetic engineering often invol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/87C12N1/14C12R1/66
Inventor 吕雪峰黄雪年李建军陈梅
Owner 浙江博崤生物制药有限公司
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