Method for constructing and screening mutant library of maltose transcriptional activation factor MalR and application of method

A transcription activator, maltose technology, applied in the biological field, can solve problems such as the inability to balance expression intensity and expression stringency, low promoter expression, increased production costs, etc., and achieve good wide adaptability and good scalability.

Active Publication Date: 2019-12-20
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The commonly used promoter systems in Bacillus subtilis are Pspac, Pxyl and PsacB systems. These three promoter systems are widely used in the research work of Bacillus subtilis, but they also have their own shortcomings: the inducer IPTG of Pspac promoter not only costs High and toxic; the price of xylose, the inducer of the Pxyl promoter, is high, which is easy to increase the production cost; the expression level of the PsacB promoter is relatively low, and these defects make them have certain limitations in industrial production
However, the above-mentioned transformation strategies often cannot take into account the expression intensity and expression stringency, that is, there are serious leaky expressions while improving the expression level of the promoter.

Method used

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  • Method for constructing and screening mutant library of maltose transcriptional activation factor MalR and application of method
  • Method for constructing and screening mutant library of maltose transcriptional activation factor MalR and application of method
  • Method for constructing and screening mutant library of maltose transcriptional activation factor MalR and application of method

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preparation example Construction

[0030] 3. DNA preparation and transformation:

[0031] Use Tiangen or Omega's DNA preparation kit, according to the manufacturer's instructions, isolate DNA from E. coli and Bacillus subtilis or from agarose gel. Standard molecular techniques are used in all examples. The plasmid DNA as described by Chung C.T. et al., Proc. Natl. Acad. Sci. USA 86, 1989, 21722175 was used to transform E. coli. According to the improved "Paris method" (Harwood C.R. Molecular Biological Methods for Bacillus, 1990, John Wiley & Sons Ltd., England), plasmid DNA or DNA fragments are used to transform Bacillus subtilis.

[0032] 4. Determination of green fluorescent protein fluorescence intensity RFU:

[0033] The tested strains cultured to an OD600 of about 0.6-0.8 were centrifuged at 4°C and 4000 rpm for 10 minutes to collect the bacteria, washed twice with pre-cooled PBS solution, and transferred 150 μL to a 96-well black bottom permeable plate (Corning , USA), placed in a microplate SpectraMax M2 mi...

Embodiment 1

[0040] Example 1 Construction of Bacillus subtilis malR gene deletion strain

[0041] In order to screen for MalR transcription activator mutants with low leakage expression and high inducible expression intensity, it is necessary to construct an engineered strain that knocks out the wild-type malR gene on the Bacillus subtilis genome to eliminate the influence of wild-type MalR transcription activator on the screening system. interference. Therefore, the present invention uses the malR-KO-UP.FOR / malR-KO-UP.REV primers to amplify the upstream recombinant fragment UP of the gene malR to be knocked out on the basis of the aforementioned seamless genome knockout method, and uses malR -KO-DN.FOR / malR-KO-DN.REV primers amplify the downstream recombinant fragment DN of the gene to be knocked out malR, and use malR-KO-neo.FOR / malR-KO-neo.REV primers to amplify resistance screening Gene neo, the above-mentioned three DNA fragments of UP, neo, and DN are overlapped and extended by PCR, a...

Embodiment 2

[0042] Example 2 Construction of Bacillus subtilis pDRG vector

[0043] In order to screen for malR gene mutants in the Bacillus subtilis strain △R00 with the malR gene knocked out, a constitutive type containing the maltose operon PmalA promoter derived from Bacillus subtilis and its regulated gfp gene and reverse insertion was constructed. Promoter The malR gene regulated by the PhpaII promoter can be integrated into the amyE gene locus of the Bacillus subtilis genome, named pDRG. The method for constructing the above-mentioned integrated expression vector is: based on the pDL plasmid (BGSC, USA), and the PmalA-insert.for / rev primer to amplify the Maltose operon MalA promoter or its mutant DNA fragment derived from Bacillus subtilis And connected to pDL plasmid through KpnI / BamHI restriction site. Using the CPECPCR cloning method (Nature Protocols, 6,242-251, 2011), the bgaB gene on the pDL plasmid was replaced with the gfp gene regulated by the Bacillus subtilis PmalA promote...

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Abstract

The invention provides a method for quickly constructing and screening a maltose transcriptional activation factor MalR mutation library through fluorescent protein by using bacillus as an original strain, and besides, through screening, a promoter mutant which can reduce leakage expression of the maltose promoter and increase the induction expression intensity is obtained by screening, so that anapplication of the maltose promoter to protein expression is facilitated.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular to a Maltose transcription activator MalR mutant, and a method for obtaining the mutant and application. Background technique [0002] The heterologous expression production of polypeptides and recombinant proteins is of great significance in protein engineering. Among them, the use of prokaryotes for large-scale production of polypeptides and recombinant proteins has the advantages of high protein expression, high fermentation density, and low fermentation costs. [0003] The Bacillus subtilis promoter is one of the key elements to achieve high gene expression. In recent years, a lot of work has been carried out in the study of promoters and considerable progress has been made. A batch of promoters that can be applied to Bacillus subtilis have been cloned. However, the existing promoters of Bacillus subtilis have many problems in terms of quantity, expression level and regulation met...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C40B50/06C07K14/32
CPCC07K14/32C12N15/75C40B50/06
Inventor 张大伟付刚
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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