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Fructose-1,6-bisphosphate aldolase promoter and its application and construct, vector

A bisphosphate aldolase and promoter technology, which is applied in the field of genetic engineering and can solve problems such as difficult-to-target strain transformation

Inactive Publication Date: 2011-12-07
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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  • Fructose-1,6-bisphosphate aldolase promoter and its application and construct, vector
  • Fructose-1,6-bisphosphate aldolase promoter and its application and construct, vector
  • Fructose-1,6-bisphosphate aldolase promoter and its application and construct, vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: Extraction of Rhodosporidium toruloides ATCC 10788 total RNA

[0040] 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 TakaRa RNAiso kit and following its standard procedures.

[0041] 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 measure the OD ...

Embodiment 2

[0042] Embodiment 2: Rhodosporidium toruloides ATCC 10788 cDNA first strand synthesis and FBA degenerate PCR

[0043] 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 (Clontech Company), 1.0 μl DTT (20 mM), 1.0 μl dNTP (10 mM), 1.0 μl powerscript reverse transcriptase (Clontech Company) were added to the system, and mixed. 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.

[00...

Embodiment 3

[0045] Example 3: Amplification of RtFBA Genomic DNA

[0046] 1 Genomic DNA of R.toruloides ATCC 10788 (purchased from the American Standard Biological Collection, ATCC) was extracted by glass bead wall breaking method (Chapter 13 of the third edition of the refined edition of Molecular Biology Experiment Guide, Osper et al., Yan Ziying etc., 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.

[0047]2 According to the fructose-1,6-bisphosphate aldolase cDNA sequence obtained in Example 2, design a pair of gene-specific primers, FBA-ORF-p1: 5'-ATGGGTGTCCTCGATGTTGTCCC-3' and FBA-ORF-p2: 5'-TTAGAGGGTTCCCTCGGAGCGGAGG-3', using the genomic DNA of Rhodosporidium toruloides ATCC10788 as a template, PCR amplification was performed accord...

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Abstract

A promoter and a terminator which can be used for effectively expressing a target gene on rhodosporidium toruloides and can be therefore used for genetic engineering operation and strain improvement of the rhodosporidium toruloides are obtained by amplifying an upstream sequence and a downstream sequence of rhodosporidium toruloides fructose-1,6-diphosphate aldolase genome DNA (Deoxyribonucleic Acid) and performing biological information analysis and functional verification. The invention also relates to a DNA construct comprising the elements and a vector.

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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IPC IPC(8): C12N15/113C12N15/81C12N1/19C12N15/60C12N15/63C12N9/88C12R1/645
Inventor 张素芳赵宗保朱志伟
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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