Construction and Screening of Mutant Library of Maltose Transcription Activator Malr and Its Application

A technology of transcription activator and maltose, which is used in chemical libraries, introduction of foreign genetic material using vectors, library creation, etc., can solve the problems of inability to balance expression strength and expression rigor, low promoter expression, and increased production costs. , to achieve the effect of good wide adaptability and good scalability

Active Publication Date: 2021-10-22
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • 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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  • Construction and Screening of Mutant Library of Maltose Transcription Activator Malr and Its Application
  • Construction and Screening of Mutant Library of Maltose Transcription Activator Malr and Its Application
  • Construction and Screening of Mutant Library of Maltose Transcription Activator Malr and Its Application

Examples

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preparation example Construction

[0030] 3. Preparation and transformation of DNA:

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

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

[0033] Centrifuge at 4°C and 4000rpm for 10 minutes to collect the bacterial cells cultured to an OD600 of about 0.6-0.8, wash the bacterial cells twice with pre-cooled PBS solution, and transfer 150 μL to a 96-well black-bottomed microtiter plate (Corning , USA), placed in a microplate Spectra...

Embodiment 1

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

[0041] In order to screen MalR transcriptional activator mutants with low leaky 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, which is used to exclude the wild-type MalR transcriptional activator from affecting the screening system. interference. Therefore, on the basis of the genome traceless knockout method described above, the present invention uses the primers malR-KO-UP.FOR / malR-KO-UP.REV to amplify the upstream recombination fragment UP of the gene malR to be knocked out, and utilizes malR - KO-DN.FOR / malR-KO-DN.REV primers amplify the downstream recombinant fragment DN of the gene malR to be knocked out, and use malR-KO-neo.FOR / malR-KO-neo.REV primers to amplify resistance screening For the gene neo, the above-mentioned three DNA fragments of UP, neo, and DN are ov...

Embodiment 2

[0042] Example 2 Construction of Bacillus subtilis pDRG vector

[0043] In order to screen the malR gene mutants in the Bacillus subtilis strain △R00 with the deletion of the malR gene, a gfp gene containing the maltose operon PmalA promoter and its regulation from Bacillus subtilis was constructed and the reverse insertion was constitutively Promoter The malR gene regulated by the PhpaII promoter can be integrated into the integrated expression vector of the amyE gene site of the Bacillus subtilis genome, named pDRG. The construction method of the above-mentioned integrated expression vector is: based on the pDL plasmid (BGSC, USA), amplify the maltose operon MalA promoter or its mutant DNA fragment derived from Bacillus subtilis with the PmalA-insert.for / rev primer And through the KpnI / BamHI restriction site into the pDL plasmid. 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 b...

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Abstract

The present invention provides a method for rapidly constructing and screening maltose transcription activator MalR mutation library using fluorescent protein as the starting strain, and at the same time screens to obtain a promoter mutation that can reduce the leaky expression of the maltose promoter and increase its induced expression intensity body, which is beneficial to the application of maltose promoter in protein expression.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a maltose transcriptional activator MalR mutant, a method for obtaining the mutant, and an application thereof. Background technique [0002] The heterologous expression and production of polypeptides and recombinant proteins is of great significance in protein engineering. The large-scale production of polypeptides and recombinant proteins using prokaryotes has the advantages of high protein expression, high fermentation density, and low fermentation cost. [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 research of promoters and considerable progress has been made. A number 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 mode...

Claims

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Application Information

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Patent Type & Authority Patents(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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