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Family shuffling technology system for modifying multiple genes of dihydroxyl dioxygenase

A technology of dihydroxydioxygenase and hydroxydioxygenase, which is applied in plant gene improvement, recombinant DNA technology, genetic engineering and other directions, can solve the problems of difficult target shape, low mutation rate and small mutation potential.

Inactive Publication Date: 2012-03-21
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to provide a family recombination technology system for the improvement of multiple genes of dihydroxydioxygenase, specifically to provide a technology system for directional molecular evolution in vitro to overcome the existing dihydroxydioxygenase Oxygenase transformation technology has the disadvantages of low mutation rate, low mutation potential, and difficulty in obtaining the target shape caused by shuffling with a single gene

Method used

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  • Family shuffling technology system for modifying multiple genes of dihydroxyl dioxygenase
  • Family shuffling technology system for modifying multiple genes of dihydroxyl dioxygenase
  • Family shuffling technology system for modifying multiple genes of dihydroxyl dioxygenase

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: PTDS method synthesizes dihydroxydioxygenase gene PSBPHCI

[0026] (1) Test method:

[0027] 1. The gene synthesis of the present invention adopts the PTDS method [Xiong'aisheng et al., 2004, Nucl Acids Res 32, e98], and a total of 23 primers are designed for gene synthesis, see Table 1.

[0028] Amplified Fragment 1: In a 50 μl reaction system, the amount of inner primers (PSBPHCI2-PSBPHCI11) was 1.5 pmol, and the amount of outer primers (PSBPHCI1, PSBPHCI12) was 30 pmol. The amplification conditions were: 94°C preheating for 10 minutes; 94°C , 30s; 52°C, 30s; 72°C, 30s; 30 cycles; finally 72°C extension for 10min. The Taq DNA polymerase used was pyrobest taq enzyme (Takara Company Dalian) to amplify the target gene.

[0029] Amplified Fragment 2: In a 50 μl reaction system, the amount of inner primers (PSBPHCI14-PSBPHCI22) added is 1.5 pmol, and the amount of outer primers (PSBPHCI13, PSBPHCI23) added is 30 pmol. The amplification conditions are: 94°C ...

Embodiment 2

[0036] Embodiment 2: PTDS method synthesizes dihydroxydioxygenase gene RRBPHCI

[0037] The gene synthesis of the present invention adopts the PTDS method [Xiong'aisheng et al., 2004, Nucl Acids Res32, e98], and a total of 22 primers are designed for gene synthesis, and the length of each primer is 60 bp. See Table 2. Both ends of the primers were respectively introduced with Bam H I and Sac I enzyme cutting sites.

[0038] Wherein the amplified fragment 1 (Fragment 1): in the 50 μ l reaction system, the addition amount of inner primers (RRBPHCI-2~RRBPHCI-9) is 1.5 pmol, and the addition amount of outer primers (RRBPHCI-1 and RRBPHCI-10) is 30 pmol, The amplification conditions were: preheating at 94°C for 10 min; 94°C for 30 s; 52°C for 30 s; 72°C for 30 s; 30 cycles; and finally extension at 72°C for 10 min. The TaqDNA polymerase used was pyrobest taq enzyme (Takara Company Dalian) to amplify the target gene.

[0039] Amplified Fragment 2 (Fragment 1): In a 50 μl reaction...

Embodiment 3

[0047] Embodiment 3: PTDS method synthesizes dihydroxydioxygenase gene MTBPHCI

[0048] The gene synthesis of the present invention adopts the PTDS method [Xiong et al., 2004, Nucl Acids Res 32, e98], and a total of 23 primers are designed for gene synthesis, wherein the length of the primers MTBPHCI-1 to MTBPHCI-22 is 60bp, and the primer MTBPHCI-23 The length is 35bp. See Table 3. Both ends of the primers were introduced with BamHI and SacI enzyme cutting sites.

[0049] Amplified Fragment 1 (Fragment 1): In a 50 μl reaction system, the amount of inner primers (MTBPHCI-2~MTBPHCI-9) was 1.5 pmol, and the amount of outer primers (MTBPHCI-1 and MTBPHCI-10) was 30 pmol. The amplification conditions were: preheating at 94°C for 10 min; 94°C for 30 s; 52°C for 30 s; 72°C for 30 s; 30 cycles; and finally extension at 72°C for 10 min. The TaqDNA polymerase used was pyrobesttaq enzyme (Takara Company Dalian) to amplify the target gene.

[0050] Amplified Fragment 2 (Fragment 1): ...

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Abstract

The invention relates to a family shuffling technology system for modifying multiple genes of dihydroxyl dioxygenase. Firstly, four different types of dihydroxyl dioxygenase genes are designed and synthesized, and an individual dihydroxyl dioxygenase mutant with changed characters such as high activity and the like is obtained through gene family shuffling and a high-flux screening system. Since the dihydroxyl dioxygenase is a key enzyme in biphenyl / polychlorinated biphenyl biodegradation, the obtained dihydroxyl dioxygenase with increased activity can be used for increasing the efficiency of biphenyl / polychlorinated biodegradation and reducing environmental pollution and has a higher ecological benefit.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering, and specifically relates to a family reorganization technology system for improving multiple genes of dihydroxydioxygenase, in particular to a method for improving enzyme-related genes in the biodegradation process of biphenyl / polychlorinated biphenyl Gene in vitro directed molecular evolution technology system, especially the application of this technology system in the in vitro directed transformation of dihydroxydioxygenase, and then in the biodegradation of biphenyl / PCB. Background technique [0002] Chemical DNA synthesis (Chemical DNA synthesis) is an important transformation method of genetic engineering. In the past 30 years, the maturity of gene synthesis methods and technologies has made the length of gene synthesis from less than 1kb in the 1980s to Up to now, gene families up to 32kb can be synthesized. At present, the chemical synthesis of genes has been successfully app...

Claims

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Application Information

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IPC IPC(8): C40B50/06C40B40/08C12N15/53C12N15/10A62D3/02A62D101/20A62D101/22
Inventor 熊爱生姚泉洪彭日荷帅建军田永生赵伟付晓燕金晓芬朱波许晶韩红娟陈晨
Owner SHANGHAI ACAD OF AGRI SCI
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