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A strong broad-spectrum promoter and its application

A promoter and promoter sequence technology, applied in the field of genetic engineering, can solve the problems of lack of prokaryotic bacteria and fungal shuttle promoters, reducing the importance of research, limiting the scope of application, etc.

Active Publication Date: 2019-12-24
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the limitation of the host range of the promoter, the currently developed transformation tools and optimized synthetic pathways can only be used in a specific range of hosts, which greatly limits the scope of its application and seriously reduces the importance of some research. significance
At present, there are only a few reports on multi-host promoters, and they mainly focus on the construction of broad-spectrum promoters in bacteria, such as the shuttle promoter Pveg in Escherichia coli and Bacillus subtilis, and there is no shuttle promoter between prokaryotic bacteria and fungi sub report

Method used

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  • A strong broad-spectrum promoter and its application
  • A strong broad-spectrum promoter and its application
  • A strong broad-spectrum promoter and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0036] The construction of embodiment 1 broad-spectrum promoter

[0037] Artificially designed promoter P sh (as shown in SEQ ID NO.1) and P min (as shown in SEQ ID NO.2), with #1 and #2, #3 and #2 as primers, by PCR amplification, subsequently with #4 and #5 as primers, the green fluorescent protein amplified by PCR GFP (as shown in SEQ ID NO.13) DNA fragments are fused to construct P sh -gfp expression cassette and P min -GFP expression box. Amplify P with #6 and #7, #8 and #9 as primers sh -gfp expression cassette, and then inserted into BamHI / SacII digested Escherichia coli and Saccharomyces cerevisiae shuttle vector pY26 and BamHI / PstI digested Bacillus subtilis plasmid pUCB18 to produce recombinant plasmid pY26-P sh -gfp, pUCB18-P sh -gfp, pY26-P min -gfp and pUCB18-P min -gfp, followed by pY26-P sh -gfp and pUCB18-P sh-gfp as a template, for #10 and #11, #10 and #12 primers, PCR to obtain the plasmid backbone, and then through the method of self-ligation after...

Embodiment 2

[0042] The intensity analysis of embodiment 2 broad-spectrum promoter

[0043] Using #13 and #14 as primers, PCR amplifies the strong promoter P of Escherichia coli J23119 (As shown in SEQ ID NO.7) DNA fragment, with #15 and #16 as primers, PCR amplifies green fluorescent protein GFP gene (as shown in SEQ ID NO.13) DNA fragment, produces P by fusion PCR J23119 -gfp expression cassette, inserted into BamHI / SacII digested pY26-P sh -gfp plasmid, while replacing the original P sh Promoter, resulting in pY26-P J23119 -GFP plasmid.

[0044] Plasmid pY26-P J23119 -gfp into E. coli, with E.coli / pY26-P sh -gfp was cultured in the LB medium containing 100mg / L geneticin at the same time, and the fluorescence intensity was measured by a fluorescent microplate reader after 12h, and the results showed that: E.coli / pY26-P sh -gfp with E.coli / pY26-P J23119 The relative fluorescence intensities of -GFP are 15370 and 7318, respectively, so E.coli / pY26-P sh -GFP fluorescent protein cont...

Embodiment 3

[0050] Example 3 Construction of broad-spectrum geneticin resistance gene expression cassette

[0051] Using #25 and #26 as primers, PCR amplifies the strong broad-spectrum promoter P sh (as shown in SEQ ID NO.1) DNA fragment, with #27 and #28 as primers, PCR amplification geneticin resistance gene kanR (as shown in SEQ ID NO.10) DNA fragment, with #29 and # 30 is a primer, and PCR amplifies the artificially synthesized prokaryotic terminator T 0 (as shown in SEQ ID NO.11) and eukaryotic terminator T E (as shown in SEQID NO.12) DNA fragment, produces P by fusion PCR sh -kanR-T 0 -T E (as shown in SEQ ID NO.5) an expression cassette, which is a broad-spectrum geneticin resistance gene expression cassette.

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Abstract

The invention belongs to the field of gene engineering and discloses a strong broad-spectrum promoter and application thereof. By molecular means, a prokaryotic bacterium conserved promoter sequence is inserted into a non-core area with a high-activity saccharomyces cerevisiae promoter to obtain the strong broad-spectrum promoter which has multiple hosts. The strong broad-spectrum promoter has strong promoter activity in escherichia coli, bacillus subtilis and saccharomyces cerevisiae and meets current demands on broad-spectrum molecular modification tools and gene expression cassettes.

Description

technical field [0001] The invention relates to a strong broad-spectrum promoter and its application, belonging to the field of genetic engineering. Background technique [0002] The promoter is a DNA sequence located upstream of the 5' end of the structural gene, which can activate RNA polymerase to accurately combine with the template DNA and have the specificity of transcription initiation. Due to the differences in RNA polymerases between different hosts, there are also differences in the conserved sequences of the promoters they recognize, so the currently identified promoters can only be used in a specific host or a few hosts active in. [0003] With the continuous development of molecular biology, the development of molecular modification tools and the optimization of metabolic pathways are becoming mainstream research directions. However, due to the limitation of the host range of the promoter, the currently developed transformation tools and optimized synthetic pa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/70C12N15/75C12N15/81
CPCC12N15/113C12N15/70C12N15/75C12N15/81C12N2830/34
Inventor 康振陈坚杨森堵国成
Owner JIANGNAN UNIV
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