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Method for constructing highly expressed trehalose synthase engineering bacteria by using Pcry3Aa promoter

A trehalose synthase and promoter technology, applied in the field of genetic engineering, can solve the problems of difficult manufacturing, complicated process and high extraction cost

Active Publication Date: 2016-07-20
山东开盾生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Due to the complicated process of microbial extraction, fermentation, and genetic recombination, the extraction cost is high, manufacturing is difficult, and it is difficult to achieve large-scale production

Method used

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  • Method for constructing highly expressed trehalose synthase engineering bacteria by using Pcry3Aa promoter
  • Method for constructing highly expressed trehalose synthase engineering bacteria by using Pcry3Aa promoter
  • Method for constructing highly expressed trehalose synthase engineering bacteria by using Pcry3Aa promoter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0138] The Pcry3Aa promoter derived from Bacillus thuringiensis and the PhoD signal peptide of Bacillus subtilis were connected into a fragment Pcry3Aa-PhoD by overlapping PCR, which was constructed into the shuttle plasmid PHT01 to obtain the recombinant plasmid Pcry3Aa-PhoD-PHT01.

[0139] (i) extracting the genome of Bacillus thuringiensis LM79 thallus, using the genome as a template, performing PCR amplification to obtain the Pcry3Aa promoter fragment;

[0140] Described PCR primer sequence is as follows:

[0141] Upstream primer (Pcry3Aa-F): 5'- GGTACC GTGCTTTTTTTGTTGCAATT-3';

[0142] Downstream primer (Pcry3Aa-R): 5'-GACTGTCGTATGCCATTTTTCTTCCTCCCTTTCTTAT-3';

[0143] The underline is the enzyme cutting site KpnI;

[0144] Described PCR amplification system is as follows:

[0145] 2×TaqPCR MasterMix 25 μl, 2.5 μl upstream primer with a concentration of 10 μmol / L, 2.5 μl downstream primer with a concentration of 10 μmol / L, 2.5 μl template, ddH 2 O make up 50 μl;

[...

Embodiment 2

[0204] The gene fragment SpoIIID-1 derived from Bacillus subtilis 168 was combined with the Km from plasmid pBRNeo by overlapping PCR technique r Ligated into fragments SpoIIID-1-Km r , and then the fragment was electrotransformed into Bacillus subtilis WB800n after being digested with BamHI restriction enzyme to obtain a strain Bacillus subtilis WB800n[ΔSpoIIID] that does not express the SpoIIID gene.

[0205] (1) Using the DNA of the Bacillus subtilis 168 genome as a template, PCR amplification obtains the fragment SpoIIID-1; the nucleotide sequence of the SpoIIID-1 gene fragment is shown in SEQ ID NO.4;

[0206] The PCR amplification primer sequence of described fragment SpoIIID-1 is as follows:

[0207] Upstream primer (SpoIIID-1-F): 5'-CGC GGATCC GCGGGCGCTTTGGTTGATTTGAT-3';

[0208] Downstream primer (SpoIIID-1-R):

[0209]5'-TCTTGTTCAATCATTGGAAACACCAAATTCCTTCGCAATGAC-3';

[0210] The single underline is the BamHI restriction site; the fragment SpoIIID-1 is the enti...

Embodiment 3

[0251] The production of trehalose synthase, the operation steps are as follows:

[0252] 1) Preparation of the first-class species: transfer the high-expression trehalose synthase engineered bacteria WB800n[ΔSpoIIID]+[Pcry3Aa-PhoD-PHT01-TreS] prepared in Example 2 from the LB plate containing chloramphenicol 20ug / mL A single colony of the strain was cultured overnight in 3mLLB liquid medium at 37°C and 200rpm, and the obtained strain was a first-class species;

[0253] 2) Preparation of the secondary species: the primary species was inoculated in 800mL LB liquid medium containing chloramphenicol 20ug / mL, cultivated to OD at 37°C and 200rpm 600 is 0.9 (4.5 hours);

[0254] 3) Preparation of the tertiary species: Inoculate the secondary species into an 80LLB liquid fermenter at 37°C, control the pH to about 7.0 with citric acid and NaOH, ventilate and stir, control the dissolved oxygen at 20-30%, and cultivate to OD 600 is 0.9 (4.5 hours);

[0255] 4) Fermentation in the pro...

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Abstract

The invention relates to a method for constructing highly expressed trehalose synthase engineering bacteria by using a Pcry3Aa promoter. For a recombinant carrier, a Pcry3Aa-PhoD fragment by which a Pcry3Aa promoter fragment and a PhoD signal peptide fragment are connected by an overlap PCR (Polymerase Chain Reaction) is inserted at the upstream of a restriction enzyme cutting site BamHI of a shuttle plasmid PHT01, and a target protein trehalose synthase TreS fragment is inserted between two restriction enzyme cutting site, i.e., BamHI and AatII. The invention further relates to a method for constructing the highly expressed trehalose synthase engineering bacteria by using the recombinant carrier. According to the method disclosed by the invention, the Pcry3Aa promoter is adopted to naturally induce the synthesis of trehalose synthase; because the Pcry3Aa promoter contains a special STAB-SD structure, the stability of the Pcry3Aa promoter to transcribe mRNA is improved, the half-life period of mRNA is prolonged, the mRNA translation level of a downstream target gene is improved, and therefore the trehalose synthase is highly expressed.

Description

technical field [0001] The invention relates to a method for constructing high-expression trehalose synthase engineering bacteria by using a Pcry3Aa promoter, which belongs to the technical field of genetic engineering. Background technique [0002] Trehalose, also known as cocoon honey, mushroom sugar or mushroom sugar, is not reducing, and its molecular formula is C 12 h 22 o 11 2H 2 O. Trehalose can not only store energy, but also protect the body under special circumstances. For example, under harsh conditions such as high osmotic pressure, high temperature, and dryness, trehalose can protect organisms against external adverse factors and maintain the stability of organisms. Trehalose can only be synthesized in harsh environments, and will be quickly degraded in suitable environments, which brings difficulties to its production, which also leads to its high price. Therefore, finding a way to synthesize trehalose in large quantities is a technical problem that needs...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N15/66C12N15/61C12N1/21C12N9/90C12R1/125
CPCC12N9/90C12N15/66C12N15/75C12N2800/101C12N2810/55C12N2830/34C12Y504/99016
Inventor 王腾飞刘强王瑞明
Owner 山东开盾生物科技有限公司
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