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Orotate phosphoribosyltransferase promoter, application, construct and vector

A technology of orotate phosphoribosyl transferase, applied in the field of genetic engineering, can solve problems such as difficult-to-target bacterial strain transformation

Inactive Publication Date: 2013-03-13
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

With Rhodosporidium toruloides as the host bacteria, whether it is genetic engineering operation or metabolic engineering strain improvement, it is restricted by the genetic operating system, and it is difficult to carry out targeted strain transformation

Method used

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  • Orotate phosphoribosyltransferase promoter, application, construct and vector
  • Orotate phosphoribosyltransferase promoter, application, construct and vector
  • Orotate phosphoribosyltransferase promoter, application, construct and vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: Extraction of Rhodosporidium toruloides ATCC 10788 total RNA

[0044] Inoculate fresh Rhodosporidium toruloides ATCC 10788 (purchased from the American Standard Biological Collection Center, ATCC) into 50ml YEPD liquid medium from a slant, culture it on a shaker at 30°C for 24 hours, and then inoculate it at a volume of 1:50 Ratio Transfer the bacterial solution to 100ml YEPD liquid medium respectively, and culture it on a shaker at 30°C for 12h to reach the logarithmic growth phase. Centrifuge at 5000 rpm for 4 minutes at 4°C to collect the cells, freeze the cells quickly with liquid nitrogen, and grind to break the wall. Total RNA was extracted using the RNAiso kit from TaKaRa Company and following its standard procedures.

[0045] The RNA was subjected to 1.5% agarose gel electrophoresis, observed and identified using a fluorescence-ultraviolet analyzer, and two clear bands could be seen. Analyze the total RNA sample with a UV / Vis spectrometer and mea...

Embodiment 2

[0046] Example 2: Rhodosporidium toruloides ATCC 10788 cDNA first-strand synthesis and ura5 degenerate PCR

[0047] Using the total RNA of Rhodosporidium toruloides ATCC 10788 as a template, the first strand of cDNA was synthesized by reverse transcription. First, mix 1.0 μl total RNA (about 2 μg), 1.0 μl primer SMART IV: 5′-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3′ and 1.0 μl ligo dT-linker primer CDS III / 3′: 5′-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T) 30N-1 N-3′, 2.0 μl of DEPC-treated water (diethyl pyrocarbonate-treated water, purchased from Dalian TaKaRa Company), was added to the PCR tube and mixed evenly, kept at 72°C for 2 minutes, immediately placed on ice for 2 minutes, and 2.0 μl 5×first strandbuffer, 1.0 μl DTT (20 mM), 1.0 μl dNTP (10 mM), 1.0 μl powerscript reversetranscriptase (Clontech Company) were added to the system, and mixed well. Extend the reaction at 42°C for 60 minutes, and end the reaction at 4°C, and store it at -20°C for later use.

[0048] According to ...

Embodiment 3

[0049] Embodiment 3: the amplification of Rtura5 gene genomic DNA sequence

[0050]1. The genomic DNA of R. toruloides ATCC 10788 (purchased from the American Standard Biological Collection Center, ATCC) was extracted using the glass bead wall breaking method (Chapter 13 of the third edition of the refined Molecular Biology Experiment Guide, Osper et al., Translated by Yan Ziying and others, published by Science Press). The prepared genomic DNA was measured by Nanodrop 1000, and the OD was measured 260 / OD 280 =1.85, indicating that the quality of genomic DNA is very good. The concentration was 120ng / μl, 500μl in total, and the genomic DNA samples were frozen at -20°C for future use.

[0051] 2. According to the orotate phosphoribosyltransferase cDNA sequence obtained in Example 2, design a pair of gene-specific primers, ura5-ORF-p1: 5'-ATGAGCGCCACGTCCTACGC-3' and ura5-ORF-p2: 5'- For CTAGTTAACGCCCCACTTTGCG-3', using the genomic DNA of Rhodosporidium toruloides ATCC 10788 ...

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Abstract

With the present invention, upstream sequence and downstream sequence of orotate phosphoribosyltransferase genomic DNA of rhodosporidium toruloides are amplified, biological information analysis and functional verification are performed for the amplified sequences to obtain the promoter and the terminator, wherein a target gene can be effectively expressed in the rhodosporidium toruloides throughthe promoter and terminator, such that the promoter and terminator can be applicable for the rhodosporidium toruloides genetic engineering operation and the strain improvement. The invention further relates to the DNA construct containing the components and the vector containing the components.

Description

technical field [0001] This patent belongs to the technical field of genetic engineering, and specifically relates to Rhodosporidium toruloides promoters, terminators and their uses, 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 biosynthetic ability and can synthesize almost all organic chemicals on earth. The species diversity and genetic diversity of microorganisms determine their metabolic diversity. Compared with multicellular organisms, although the metabolic pathways of microorganisms are relatively simple, the production of their compounds is efficient and fast, and is closely related to human daily production and life. [0003] As a natural production strain of a certain chemical or an environmental treatment application strain, its specific production performance is often not optimal. How to op...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12R1/645
Inventor 张素芳赵宗保林心萍
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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