3-phosphoglycerate kinase promoter and terminator and their application

A phosphoglycerate kinase and promoter technology, applied in the field of genetic engineering, can solve the problem of unseen reports on the promoter sequence of Trichospora dermatoides, restricting strain transformation, and not applicable to Lipospora stariae and Trichospora dermatoides, etc. problem, to achieve the effect of promoting strain improvement and metabolic engineering research

Active Publication Date: 2018-09-14
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, no promoter sequence suitable for Trichosporium dermatitis has been reported, nor is there any endogenous promoter suitable for Lipoyces stariae, which restricts the targeted strain transformation
[0006] Although the phosphoglycerate kinase promoter of rhizopus oryzae (Rhizopus oryzae) and arbuscular mycorrhizal fungus (Glomus mosseae) was once isolated and used for genetic engineering of filamentous fungi (mold) (US 6528636; US 6465635; Harrier, LA1 ., Paterson, LJ.Curr Genet, 2002, 42(3): 169-178.), but these promoters are not applicable to Lipospora starfield and Trichosporium dermatitis

Method used

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  • 3-phosphoglycerate kinase promoter and terminator and their application
  • 3-phosphoglycerate kinase promoter and terminator and their application
  • 3-phosphoglycerate kinase promoter and terminator and their application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1: Extraction of total RNA from Lipomyces starkeyi NRRL Y-11557

[0088] L. starkeyi NRRL Y-11557 (purchased from the National Agricultural Research Culture Collection (NRRL), Bacterial Foodborne Pathogens & Mycology Research, 1815N. University Street IL 61604. Peoria, Illinois) was inoculated into 50 ml YEPD from the slope. In liquid culture medium (glucose 20.0g / l, yeast extract 10.0g / l, peptone 20.0g / l, pH 6.0), culture at 30°C on a shaker for 24 hours, and then transfer the bacterial solution at a volume ratio of 1:50. Received in 100ml YEPD liquid medium, cultured on a shaker at 30°C for 12h to reach logarithmic growth phase. Centrifuge at 5000 rpm for 4 min at 4°C, collect the bacteria, quickly freeze the bacteria with liquid nitrogen, and grind to break the walls. Use TakaRa's RNAiso kit and follow its standard procedures to extract total RNA.

[0089] RNA was subjected to 1.5% agarose gel electrophoresis, observed and identified with a fluorescence-ultravio...

Embodiment 2

[0090] Example 2: First-strand synthesis of NRRL Y-11557 cDNA and PGK degenerate PCR

[0091] Using the total RNA of Star's lipid yeast NRRL Y-11557 as a template, the first strand of cDNA was synthesized by reverse transcription. Firstly, 1.0μl total RNA (about 2μg), 1.0μl primer SMART IV: 5'-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3' and 1.0μl oligo dT-linker primer CDSIII / 3': 5'-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T) 30N-1 N-3', 2.0μl DEPC treated water (diethyl pyrocarbonate treated water, purchased from Dalian TakaRa Company), add it to the PCR tube and mix well, keep it at 72°C for 2 min, immediately place it on ice and cool for 2 min. μl 5× first chain buffer (Clontech company), 1.0 μl DTT (20mM), 1.0 μl dNTP (10mM), 1.0 μl powerscript reverse transcriptase (Clontech company) were added to the system and mixed. The reaction was extended at 42°C for 60 minutes, and the reaction was finished at 4°C. Store at -20°C for later use.

[0092] Design and synthesize two degenerate primers...

Embodiment 3

[0093] Example 3: Amplification of the genomic DNA of NRRLY-11557 of Lipaseomyces stasii

[0094] The genomic DNA of Starella oleaginous yeast NRRLY-11557 was extracted by glass bead breaking method (Chapter 13 of the Third Edition of the Compiled Molecular Biology Experiment Guide, Osber et al., translated by Yan Ziying et al., published by Science Press). The prepared genomic DNA is measured by Nanodrop 1000, and the OD is measured 260 / OD 280 = 1.85, indicating that the quality of genomic DNA is very good. The concentration is 120ng / μl, 500μl in total, and the genomic DNA samples are frozen at -20°C for later use.

[0095] According to the 3-phosphoglycerate kinase cDNA sequence obtained in Example 2, a pair of gene-specific primers were designed, PGK-p1: 5'-ATGCTTAACCTCAAAGTATCTGTT-3' and PGK-p2: 5'-TTAGAACTTCTGCTCGACATCT-3', The genomic DNA of NRRLY-11557 was used as a template, and PCR amplification was performed according to conventional methods to obtain a PCR product of a...

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PUM

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Abstract

In the present invention, by amplifying the upstream and downstream sequences of the 3-phosphoglycerate kinase genomic DNA of Lipomyces starii, and performing biological information analysis and functional verification, the invention can be widely used in the genus Lipomyces (Lipomyces) and Trichosporon in oleaginous yeasts. The promoter and terminator for gene expression, genetic engineering operation and strain improvement in the genus (Trichosporon), the nucleotide sequences of which are SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The present invention also relates to a DNA expression cassette or a recombinant vector containing these elements, and a method for constructing a genetically engineered bacterial strain of the genus Liposaccharomyces or Trichosporon by using the relevant elements and the corresponding bacterial strain.

Description

Technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to the promoter terminator of Lipomyces starkeyi and its use, including transformation methods necessary for the construction of genetic engineering strains. Background technique [0002] Microorganisms are one of the most widely distributed species in nature. They have excellent biosynthesis capabilities and can synthesize almost all organic chemicals on the earth. Compared with multicellular organisms, although the metabolic pathway of microorganisms is relatively simple, the production of its compounds is efficient and fast, with mild reaction conditions, strong controllability, and easy mass production. It can be used as an excellent cell factory. [0003] Some microorganisms in nature can store more than 20% of their dry cell weight in their cells under certain conditions (such as lack of nitrogen). Among them, triglycerides are the main ones. Microorganisms wit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82C12N15/70C12N15/81C12N1/21C12N1/19
Inventor 赵宗保林心萍张素芳王雅南
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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