DNA fragment of bacillus subtilis with promoter function and application thereof

Abstract

The invention discloses a DNA fragment of bacillus subtilis with a promoter function and application thereof. The DNA fragment is any one of a, the nucleotide sequence as shown in SEQ ID NO.1 or a complementary sequence thereof; b, a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides of the nucleotide sequence as shown in SEQ ID NO.1 and having the promoter function the same as that of the nucleotide sequence as shown in SEQ ID NO.1, or a complementary sequence thereof; and c, a sequence obtained by adding one or more ribosome bind sites to the nucleotide sequence as shown in SEQ ID NO.1. The DNA fragment has the promoter function, and also has high expression activity. High expression of exogenous genes can be achieved without adding an inductor. The DNA fragment can be applied to expression of thermostable beta-galactosidase and transglutaminase, and particularly, an effective element is provided for a bacillus subtilis expression-secretion system.

Description

technical field

[0001] The invention relates to a DNA segment, in particular to a DNA segment with promoter function of Bacillus subtilis and its application. Background technique

[0002] The production of valuable exogenous proteins by gene recombination technology is one of the hot spots in the research and development of biotechnology today. Bacteria have the characteristics of fast growth, easy culture, high expression and simple molecular manipulation, which makes them widely developed and applied to the expression of heterologous proteins. Among them, Bacillus subtilis has become an ideal host cell for industrial use due to its many advantages such as safety and non-toxicity, clear genetic background, and high secretion expression level.

[0003] Various elements are required for expression of heterologous proteins in Bacillus subtilis, among which the promoter is crucial for the effect of expression level. There are usually two ways to genetically modify the promot...

Examples

Embodiment 1

[0032] Culture of bacteria: Bacillus subtilis 168 was taken out from the -80°C glycerol tube and streaked on an LB solid plate at 37°C for 16-24h, and a single colony was picked and placed in 10mL of LB liquid with 1% final concentration of cornstarch culture medium, 37°C, 200rpm to OD 600 20-25 (spectrophotometer, Hitachi, Japan).

Embodiment 2

[0034] Extraction of bacterial total RNA, establishment of transcriptome library and RNA-Seq sequencing: collect 1 mL of the cell culture solution obtained in Example 1 and centrifuge rapidly at 8000g for 1 min to extract total bacterial RNA (see figure 1 ). For the specific extraction method, refer to the Bacterial Total RNA Extraction Kit from OmegaBio-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 into fragments of appropriate size, using the fragmented mRNA as a template, reverse transcriptase and random primers were added to synthesize double-stranded cDNA, and then the synthesized cDNA was purified with the kit QIAquick PCR Purification Kit (Qiagen). Fill in the cohesive ends of the...

Embodiment 3

[0036] Screening and cloning of promoter fragments: Analyze the transcript structure of Bacillus subtilis through RNA-Seq sequencing data and the genome-wide analysis of genes containing transcription start positions, and quantify by RPKM to screen a highly expressed gene and its nucleotide sequence As follows:

[0037]CGTTCTGTTACAGATGGAGGCGACAGCTTAATTTTTCTGCCTAATTCCCTCATCGACAAACGGCTGTCCTTCTTCAGCTCCTCAATGATATTCAGATCAATCTGGTCAAGTTTCATTTCAACATCCTTCTTTTTTGATTTTGTACACATTATCTCGGGTATTTTTGTAAATGACAAGTACAGTTCCCTAGAAAAGGCATGTAAAAATGAATGTTTTCCGAACATTTTTTGAAAGCTGTCATATGCCCCCCCGGATTGTTTATAGTATAAAATGAAAACGTGTCCACAAGGAGGGCGATTT。

[0038] Using the genomic DNA of the strain Bacillus subtilis (Bacillus subtilis 168) as a template, primers F-P ydzA (5'-CGGAATTCCGTTCTGTTACAGATGGAGG-3') and R-P ydzA (5'-GGACTAGTAAATCGCCCTCCTTGTGGAC-3') to amplify a 300bp DNA fragment, namely P ydzA Promoter fragment (see figure 2 ), consistent with the size of the target product. Introduced restriction site...

technical field

[0001] The invention relates to a DNA segment, in particular to a DNA segment with promoter function of Bacillus subtilis and its application. Background technique

[0002] The production of valuable exogenous proteins by gene recombination technology is one of the hot spots in the research and development of biotechnology today. Bacteria have the characteristics of fast growth, easy culture, high expression and simple molecular manipulation, which makes them widely developed and applied to the expression of heterologous proteins. Among them, Bacillus subtilis has become an ideal host cell for industrial use due to its many advantages such as safety and non-toxicity, clear genetic background, and high secretion expression level.

[0003] Various elements are required for expression of heterologous proteins in Bacillus subtilis, among which the promoter is crucial for the effect of expression level. There are usually two ways to genetically modify the promot...

Examples

Embodiment 1

[0032] Culture of bacteria: Bacillus subtilis 168 was taken out from the -80°C glycerol tube and streaked on an LB solid plate at 37°C for 16-24h, and a single colony was picked and placed in 10mL of LB liquid with 1% final concentration of cornstarch culture medium, 37°C, 200rpm to OD 600 20-25 (spectrophotometer, Hitachi, Japan).

Embodiment 2

[0034] Extraction of bacterial total RNA, establishment of transcriptome library and RNA-Seq sequencing: collect 1 mL of the cell culture solution obtained in Example 1 and centrifuge rapidly at 8000g for 1 min to extract total bacterial RNA (see figure 1 ). For the specific extraction method, refer to the Bacterial Total RNA Extraction Kit from OmegaBio-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 into fragments of appropriate size, using the fragmented mRNA as a template, reverse transcriptase and random primers were added to synthesize double-stranded cDNA, and then the synthesized cDNA was purified with the kit QIAquick PCR Purification Kit (Qiagen). Fill in the cohesive ends of the...

Embodiment 3

[0036] Screening and cloning of promoter fragments: Analyze the transcript structure of Bacillus subtilis through RNA-Seq sequencing data and the genome-wide analysis of genes containing transcription start positions, and quantify by RPKM to screen a highly expressed gene and its nucleotide sequence As follows:

[0037]CGTTCTGTTACAGATGGAGGCGACAGCTTAATTTTTCTGCCTAATTCCCTCATCGACAAACGGCTGTCCTTCTTCAGCTCCTCAATGATATTCAGATCAATCTGGTCAAGTTTCATTTCAACATCCTTCTTTTTTGATTTTGTACACATTATCTCGGGTATTTTTGTAAATGACAAGTACAGTTCCCTAGAAAAGGCATGTAAAAATGAATGTTTTCCGAACATTTTTTGAAAGCTGTCATATGCCCCCCCGGATTGTTTATAGTATAAAATGAAAACGTGTCCACAAGGAGGGCGATTT。

[0038] Using the genomic DNA of the strain Bacillus subtilis (Bacillus subtilis 168) as a template, primers F-P ydzA (5'-CGGAATTCCGTTCTGTTACAGATGGAGG-3') and R-P ydzA (5'-GGACTAGTAAATCGCCCTCCTTGTGGAC-3') to amplify a 300bp DNA fragment, namely P ydzA Promoter fragment (see figure 2 ), consistent with the size of the target product. Introduced restriction site...

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