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Pseudo monads pseudoalcaligenes gene promoter

A Pseudomonas, promoter technology, applied in genetic engineering, plant genetic improvement, biochemical equipment and methods, etc., can solve problems such as low transcription efficiency

Inactive Publication Date: 2005-05-18
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0011] Because most of the current genetic engineering operating systems in prokaryotes use Escherichia coli or Bacillus subtilis as the operating platform of the model strains, the promoter sequences of these two strains generally have very low transcription efficiency in Pseudomonas. Therefore, it is necessary to Novel promoter that promotes high-level transcription in a broad range of host cells

Method used

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  • Pseudo monads pseudoalcaligenes gene promoter
  • Pseudo monads pseudoalcaligenes gene promoter
  • Pseudo monads pseudoalcaligenes gene promoter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Pseudomonas pseudoalcaligenes total DNA extraction

[0048] Pseudomonas pseudoalcaligenes was activated overnight in 2ml LB, inoculated in 50ml broth medium at a ratio of 1:100, and cultured for 12hr. Put the bacteria into a 50ml centrifuge tube, centrifuge at 3500rmp for 10min, remove the supernatant, turn the centrifuge tube upside down on a paper towel and let the supernatant flow out at night; add a small amount of lysate to suspend the bacteria, and then add lysate to a total volume of 4ml , add 1ml SDS, mix well, 60°C for 10min; add equal volumes of phenol, phenol / chloroform extract, and chloroform to extract once; take the supernatant, add 1 / 10 volume of 3M potassium acetate (pH4.8), mix Mix well, then add 2 times the volume of 100% ice-cold ethanol along the tube wall, shake gently until a filamentous precipitate appears; centrifuge at 2000rpm for 5 minutes, remove the supernatant; add 1ml of 70% ethanol, wash the precipitate once, and let it dry naturally at ro...

Embodiment 2

[0050] produce DNA fragments

[0051] Use Pseudomonas pseudoalcaligenes total DNA extract (1 μ g) to pass through in (100 μ l volume) 100 mM NaCl, 50 mM Tris-HCl (pH7.5), 10 mM MgCl 2 , Dithiothreitol in 1mM was digested with Sau3A I [Bao Biological Engineering (Dalian) Co., Ltd. (TAKARA)] to generate a DNA fragment of Pseudomonas-like alcaligenes. The mixture was incubated at 37°C for 120 minutes and quenched with 10 mM EDTA. The Sau3A1 fragment was precipitated from the digestion mixture with ethanol and washed with ethanol.

Embodiment 3

[0053] Transformation of E. coli and selection of transformants

[0054] The Sau3AI viral fragment produced as described in Example 1 was cloned into plasmid pSUPV4 by the shotgun cloning method described in Sambrook et al., 1989, in Cloning. Plasmid promoter probe vector pSUPV4 (AP r ) was constructed by the Laboratory of Molecular Biology, School of Life, Sichuan University (Zhang Yizheng et al. Journal of Sichuan University (Natural Science Edition), 1998, 35(2): 263-267.). The map of pSUPV4 is shown in Figure 1 . DNA preparation, fill-in reactions and ligation were performed as described by Sambrook et al. (supra) or according to the manufacturer's instructions.

[0055] The DNA fragment of Pseudomonas pseudoalcaligenes was ligated with 1 μg of pSUPV4 pre-treated with BamHI and calf intestinal alkaline phosphatase (CIP) to generate recombinant plasmids PA1; PA2; PA3; PA4: PA5; PA6; PA7; PA8; PA9 . These 9 are related to the heterologous kanamycin resistance marker gene...

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Abstract

The present invention provides new promoter sequence of Pseudomonas pseudoalcaligenes. The present invention includes gene constituent containing the promoter sequence of the present invention, and the promoter sequence is connected to neomycin phosphotransferase gene sequence encoding the structure gene optionally. The present invention also provides the carrier expressing structure gene encoded product of gene constituent in the present invention and the host cell as well as the cell transformed by the heterologous gene connected to the promoter optionally.

Description

technical field [0001] The present invention relates to novel promoters of Pseudomonas pseudoalcaligenes which can be used to express heterologous genes in host cells. Background technique [0002] Locusts belong to the family Locustidae in the order Orthoptera and are worldwide harmful insects. In our country is the national distribution and harm. Especially in the north, it is the most serious. In the northern grasslands, it has become the first pest of grasslands due to its characteristics of "many types, large numbers, wide distribution, and heavy damage" (Liu Shigui, Journal of Sichuan University, 1992, 29: 2-8). Only the northern grasslands and Qinghai-Tibet Plateau grassland, the area that occurs and harms nearly 10 million hectares every year, seriously endangers the growth of forage grass, and makes the amount of grass produced by grassland drop by 30-70% (Li Kefu, Ma Yao, Du Wenliang. Chinese Grassland, 1992, (1): 50-52), causing...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/63C12P21/02
Inventor 杨志荣张杰赵建岳碧松
Owner SICHUAN UNIV
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