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DNA fragment with promoter function and use thereof

A technology of promoter and function, applied in the field of DNA fragments, can solve the problems of difficult search, high activation intensity, unsustainable expression, etc., and achieve the effect of high activity

Active Publication Date: 2016-11-09
SOUTH CHINA UNIV OF TECH
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Problems solved by technology

Studies have shown that subtilis is rich in σ factors (12 types), and the specific binding of RNA polymerase to promoters depends on σ factors, so it is difficult to search for efficient promoters in Bacillus
At present, there are three types of promoters used to express recombinant proteins in Bacillus. The first is the inducible promoter, which needs to be added with different inducers, and its weakness is that the efficiency of starting is low; Different growth stages are activated with high promoter strength but not sustained expression; lastly an autoinducible promoter

Method used

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  • DNA fragment with promoter function and use thereof
  • DNA fragment with promoter function and use thereof
  • DNA fragment with promoter function and use thereof

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Embodiment 1

[0035] (1) Bacterial cultivation: Take out the Bacillus licheniformis ATCC14580 (-80°C) glycerol tube and streak it on an LB solid plate for 16 hours at 37°C, pick a single colony in 10 mL of LB liquid medium containing 1% final concentration of starch , 37°C, 200rpm to OD 600 20-25 (spectrophotometer, Hitachi, Japan).

[0036] (2) Extraction of bacterial total RNA and RNA-Seq sequencing: 1 mL of cell culture solution obtained in step (1) was collected and centrifuged at 8000 g for 1 min to extract total bacterial RNA (see figure 1 ). For the specific extraction method, refer to the Cellular Bacterial Total RNA Extraction Kit from Omega Bio-tek Company. The samples used for RNA-Seq sequencing library preparation were qualified by the Agilent Technologies 2100 Bioanalyzer, and the mixed DNA molecules were treated with DNaseI (RNase Free), and Ribo-Zero (Gram-Positive Bacteria) kit (USA) was used to remove most of the total RNA rRNA, purified mRNA. The mRNA was first broken...

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Abstract

The invention discloses a DNA fragment with a promoter function and a use thereof. The DNA fragment has a sequence selected from (a), a nucleotide sequence shown in the formula of SEQ ID NO. 1 or its complementary sequence, (b), a nucleotide sequence which is derived from the nucleotide sequence shown in the formula of SEQ ID NO. 1 through replacement, deletion or addition of one or multiple nucleotides and has a promoter function the same to that of the nucleotide sequence shown in the formula of SEQ ID NO. 1, or its complementary sequence, and (c), a sequence derived from the nucleotide sequence shown in the formula of SEQ ID NO. 1 through addition of one or more ribosome binding sites. The DNA fragment has a promoter function and strong specific expression activity, realizes high exogenous gene expression without an inducer and provides an effective element for bacillus subtilis expression of an exogenous gene.

Description

technical field [0001] The invention relates to a DNA segment, in particular to a DNA segment with promoter function and application thereof. Background technique [0002] Protein expression system is an important research content of modern biotechnology, widely used in food, pharmaceutical, detergent production and other fields. Among them, the prokaryotic expression system has the characteristics of fast growth, easy culture, high expression level, clear genetic background, simple molecular manipulation, and is suitable for engineering strain transformation. It has been widely used in the expression of heterologous proteins. At present, relatively mature prokaryotic expression systems include Escherichia coli and Bacillus. Since Escherichia coli is not suitable as a host for large-scale expression of heterologous proteins, Bacillus is a common system for large-scale production of industrial enzyme preparations. The advantages of the Bacillus host are: recognized safe str...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/75C12N15/56C12N15/54C12N1/21C12R1/07C12R1/125
CPCC12N9/1044C12N9/2471C12N15/113C12Y203/02013C12Y302/01023C12N2830/00
Inventor 潘力刘欣王斌廖瑜玲
Owner SOUTH CHINA UNIV OF TECH
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