DNA for high-level expression of N-acyl homoserine lactonase in yeasts and engineering bacteria constructed thereby

A technology of acyl homoserine lactone and DNA molecules, applied in the direction of genetic engineering, recombinant DNA technology, microorganism-based methods, etc., to achieve the effects of reducing production costs, improving safety and effectiveness, and high expression ability

Active Publication Date: 2010-12-15
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the mechanism of action in quorum sensing quenching is mainly studied by constructing transgenic plants and transgen

Method used

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  • DNA for high-level expression of N-acyl homoserine lactonase in yeasts and engineering bacteria constructed thereby
  • DNA for high-level expression of N-acyl homoserine lactonase in yeasts and engineering bacteria constructed thereby
  • DNA for high-level expression of N-acyl homoserine lactonase in yeasts and engineering bacteria constructed thereby

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1, Optimization of N-acyl homoserine lactonase gene codons

[0051] 1. Optimization of codons of homoserine lactonase gene

[0052] The N-acyl homoserine lactonase gene aiiaB546 (shown in sequence 1 of the sequence listing) is codon-optimized, and the optimized gene aiiaB546M is shown in sequence 2 of the sequence listing. For comparison of gene aiiaB546 and optimized gene aiiaB546M see figure 1 .

[0053] 2. Construction of recombinant plasmid aiiaB546 / pUC57

[0054] 1. Artificially synthesize the aiiaB546 gene shown in sequence 1 of the sequence listing.

[0055] 2. Digest the gene synthesized in step 1 with restriction endonucleases EcoR I and Not I, and recover the digested product.

[0056] 3. Digest the pUC57 vector with restriction enzymes EcoR I and Not I to recover the vector backbone.

[0057] 4. Ligate the digestion product of step 2 with the vector backbone of step 3 to obtain a ligation product.

[0058] 5. The ligation product was sequenced, ...

Embodiment 2

[0065] Embodiment 2, construction of recombinant expression vector

[0066] 1. Construction of pPIC9-aiiaB546M

[0067] 1. Extract recombinant plasmid aiiaB546M / pUC57 and plasmid pPIC9 respectively

[0068] Plasmids were extracted using a plasmid mini-extraction kit from Beijing Tiangen Biotechnology Co., Ltd., and detected by 1.2% agarose gel electrophoresis (electrophoresis buffer 1×TAE, voltage 1-5 V / cm, time about 30 min). The electropherogram of the recombinant plasmid aiiaB546M / pUC57 is shown in figure 2 .

[0069] 2. Digest plasmid pPIC9 with restriction endonucleases EcoR I and Not I to recover the vector backbone. 1.0% agarose gel electrophoresis detection, see electrophoresis image 3 .

[0070] 3. Digest the recombinant plasmid aiiaB546M / pUC57 with restriction endonucleases EcoR I and Not I, and recover a DNA fragment (aiiaB546M) of about 750 bp. 1.0% agarose gel electrophoresis detection, see electrophoresis image 3 .

[0071] 4. Ligate the vector backbon...

Embodiment 3

[0083] Embodiment 3, the preparation of recombinant bacteria

[0084] 1. Preparation and screening of recombinant bacteria with aiiaB546M gene

[0085] 1. Mass extraction of plasmid DNA

[0086] ① Take the Escherichia coli Trans1 containing the recombinant plasmid pPIC9-aiiaB546M obtained in Example 2 and inoculate it in 50 mL LB medium containing 100 μg / mL ampicillin, and cultivate overnight at 37°C with shaking;

[0087] ② Take 50 mL of the overnight culture solution, centrifuge at 10,000 rpm, 4°C for 5 minutes, discard the supernatant, invert the centrifuge tube on absorbent paper, and absorb the excess liquid;

[0088] ③Resuspend the precipitate in 2mL of solution I, shake vigorously, and suspend fully; add 4mL of freshly prepared solution II, mix upside down, and place on ice for 4min; add 3mL of solution III, mix upside down, place on ice for 5min, 13,000rpm , Centrifuge at 4°C for 10 minutes;

[0089] ④Remove the supernatant and filter it with lens-cleaning paper; ad...

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Abstract

The invention discloses a DNA for high-level expression of N-acyl homoserine lactonase in yeasts and engineering bacteria constructed thereby. The DNA provided by the invention is the DNA expressed by the sequence 2 in a sequence list. On the basis of the traditional homoserine lactonase gene aiiaB546, the DNA sequence thereof is optimized to obtain the optimized gene aiiaB546 M by the method. The expression ability of the optimized gene in Pichia pastoris is obviously higher than that of the gene before optimization. An aiiaB546 M-containing recombinant expression plasmid is constructed; and recombination strains of the high-efficient homoserine lactonase can be obtained by introducing the recombinant expression plasmid into the Pichia pastoris. The expression ability of one of the recombination strains is particularly high. The DNA and the engineering bacteria have great value to the production of the homoserine lactonase.

Description

technical field [0001] The invention relates to highly expressing DNA of N-acyl homoserine lactonase in yeast and engineering bacteria constructed thereof. Background technique [0002] Due to the degeneracy of the genetic code, one amino acid can have 1-6 synonymous codons with different usage frequencies. For a specific species, highly expressed genes often use some specific synonymous codons, which are considered to be optimal codons for highly expressed genes of that species, a phenomenon known as codon bias. bias). Codon bias makes it difficult for cloned foreign genes to be efficiently expressed in heterogeneous biological cells. The exogenous genes that are highly expressed in yeast are often the genes encoded by the yeast preferred codons. The statistical analysis of the codon usage of yeast genes has confirmed that 25 of the 61 codons are preferred by yeast. Therefore Modification of codon preference can greatly increase the expression of recombinant protein in t...

Claims

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Application Information

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IPC IPC(8): C12N15/57C12N9/50C12N15/81C12N15/63C12N1/19C12N5/10C12R1/84
Inventor 周志刚张宇婷姚斌曹雅男张美超何夙旭刘玉春孟昆
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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