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A double-plasmid co-transformation genetically engineered bacteria with high expression of exogenous gene

A technology of genetically engineering bacteria and genes, applied in the field of bioengineering, can solve problems such as disadvantages

Active Publication Date: 2021-10-15
天津昕因达生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] By constructing a co-expression vector, the functional gene and the exogenous target gene are connected to an expression vector at the same time, and a transformation screening of this patent can achieve the purpose of expressing two genes in the transformant at the same time; but the disadvantage of this co-expression method is the function The gene and the exogenous target gene can only be transferred into the recipient cells at the same gene dosage, but in fact, the ultimate goal of our construction of engineering bacteria is to express the exogenous target gene as high as possible, the functional gene and the exogenous target gene at the same gene dosage On the contrary, it is not conducive to the realization of this goal

Method used

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  • A double-plasmid co-transformation genetically engineered bacteria with high expression of exogenous gene
  • A double-plasmid co-transformation genetically engineered bacteria with high expression of exogenous gene
  • A double-plasmid co-transformation genetically engineered bacteria with high expression of exogenous gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Construction and screening of dual auxotrophic receptor strains

[0070] 1. Obtain the Hansenula adenine gene (Hp-ADE2△) with structural gene mutation and construct the plasmid vector

[0071] Primers were designed according to the nucleotide sequence of the reported Hansenula adenine (ADE2) gene as follows:

[0072] Primer ADE-1 (SEQ ID NO: 1):

[0073]

[0074] (The underline is the restriction enzyme cutting site EcoRI, and the bold TGGA is the insertion mutation base of the ADE2 structural gene)

[0075] Primer ADE-2 (SEQ ID NO: 2):

[0076] 5'-AC GAGCTC CTATTTATTAAGGTATTCTTCAT-3'

[0077] (Stroke is the restriction enzyme cutting site SacI)

[0078] Using the total DNA of Hansenula polymorpha (ATCC34438) as a template, use primers ADE-1 and primers ADE-2 to amplify the mutant Hp-ADE2△, PCR system: premixed Pfu DNA polymerase 25 μL, template 1 μL, primers 1 μL, supplemented with ultrapure water to 50 μL and mix well; use a PCR instrument (PC707-0...

Embodiment 2

[0112] Embodiment 2 expression vector construction

[0113] 1. Obtain MOX promoter terminator and Hansenula autonomously replicating sequence and construct recombinant vector pBlu-MP(18L1)-HARS

[0114] Digest our own plasmid vector pMPT-HPV18L1 (see invention patent CN201210013236.X, the HPV18L1 gene is inserted between the MOX promoter and terminator) with restriction endonucleases BssHII and KpnI, and agarose gel electrophoresis to recover and purify the large fragment MP(18L1)+HARS (containing MOX promoter terminator and Hansenula autonomously replicating sequence, 4433bp, SEQ ID NO:20), used for ligase connection to construct expression vector.

[0115] Using pBlueScript SK(+) as a template, design primers:

[0116] pB-01 (SEQ ID NO: 11):

[0117] 5'-TA GCGCGC TTGGCGTAATCATG-3'

[0118] (Stroke is the restriction enzyme site BssHI)

[0119] pB-02 (SEQ ID NO: 12):

[0120] 5'-GC GGTACC TG ACGCGT CACTGGCCGTCGTTTTACAACG-3'

[0121] (Strokes are restriction enzy...

Embodiment 3

[0143] Example 3 Construction of functional carrier

[0144] 1. Obtain Saccharomyces cerevisiae mother adenine (ADE2sc) gene and carry out the first step of vector replacement

[0145] Primers were designed according to the nucleotide sequence of the cerevisiae cerevisiae adenine (ADE2sc) gene as follows:

[0146] Primer Ad-01 (SEQ ID NO:20):

[0147] 5'-CA ACGCGT CGCTATCCTCGGTTCTGCATTG-3'

[0148] (Stroke is the restriction enzyme cutting site MLUI)

[0149] Primer Ad-02 (SEQ ID NO:21):

[0150] 5'-TA GGTACC TAACGCCGTATCGTGATTAACG-3'

[0151] (Stroke is the restriction enzyme cutting site KpnI)

[0152]Use the total DNA of brewer's yeast (S288c) as a template, primer Ad-01, primer Ad-02 to amplify the brewer's yeast adenine gene ADE2sc, and the PCR product is alcohol-precipitated and air-dried with restriction endonucleases MluI and KpnI, and agar Glycogel electrophoresis recovered and purified the digested fragment ADE2sc (SEQ ID NO: 22, the first and last 6 base...

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Abstract

The invention provides a genetically engineered bacterium and a preparation method thereof. The double auxotrophic strain is used as an expression host, and the expression host is simultaneously transferred into two vectors through one electric transformation, wherein one of the vectors contains an exogenous target gene and an expression host Compensation gene for auxotrophy deficiency, another vector contains UPR key gene HAC1p, and another compensation gene for auxotrophy in the expression host. The engineering bacteria provided by the invention contain functional genes of different gene doses and exogenous target genes, and can obtain high-yield engineering strains that secrete and express the exogenous target genes through screening.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a genetically engineered bacterium and a method for improving the high expression of exogenous genes in a yeast system. Background technique [0002] In recent years, with the in-depth research on the biological characteristics of Hansenula polymorpha, its advantages as a host for exogenous target gene expression have gradually emerged. Hansenula is also a methanolotrophic yeast, similar to Pichia pastoris. The expression of various key enzymes in the process of methanol metabolism, including MOX, DHAS and CAT, etc. are regulated at the transcriptional level, they are induced by methanol, glycerol and sorbitol, and repressed by glucose and ethanol, but when the concentration of glucose When it is lower than 0.1%, the repression is released. Under methanol fully induced conditions, peroxisomes can account for 80% of the total cell volume, MOX and DHAS can acco...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/81C12R1/78
CPCC07K14/39C07K14/395C12N15/815
Inventor 王昌华杨珺刘刚徐俊孙正祥
Owner 天津昕因达生物技术有限公司
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