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Application of an n-terminal sequence element in regulating protein expression in Bacillus subtilis

A Bacillus subtilis, N-terminal technology, applied in the field of genetic engineering, can solve the problems of no discovery effect, imperfect standard elements, long promoter and terminator sequences, etc.

Active Publication Date: 2021-03-30
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the standard elements that have been developed to regulate the expression of target proteins in Bacillus subtilis are still incomplete
For example, promoter and terminator sequences are relatively long, increasing the difficulty of genetic manipulation and the cost of DNA sequence synthesis
In addition, most standard elements focus on regulation at the transcriptional level, with fewer tools and elements for translational regulation
At the same time, the current regulatory elements at the translational level only focus on regulating the expression of the target gene, but have not found its role in dynamic regulation

Method used

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  • Application of an n-terminal sequence element in regulating protein expression in Bacillus subtilis
  • Application of an n-terminal sequence element in regulating protein expression in Bacillus subtilis
  • Application of an n-terminal sequence element in regulating protein expression in Bacillus subtilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Construction of recombinant plasmid

[0023] The composition of the recombinant plasmid is to sequentially insert a sequence of any of the N end shown in SEQ IDNO.1 to 97 after the P43 promoter of the PP43NMK plasmid (SEQ ID N-terminal sequence element, SEQ ID No. 24, as an example) The green fluorescent protein gene (sequence is shown in SEQ ID NO. 98). In order to introduce the sequence encoding N-terminal into the P43 promoter, the primer RH_GLNA-0.75K_P43NMK-GFP_F (shown in Seq ID No. 99), RH_GLNA-0.75K_P43NMK-GFP_R (shown in SEQ ID NO.100).

[0024] Escherichia coli containing a green fluorescent protein (GFP, Genbank: AF324408.1) is a template, and a green fluorescent protein fragment is obtained by colony PCR;

[0025] The primer fx_glna-6.7k_p43nmk-gfp_f (shown in SEQ ID No. 101); FX_GLNA-6.7K_P43NMK-GFP_R (shown in SEQ ID No. 102), with plasmid PP43NMK as a template, expanded by PCR The plasmid fragment was obtained; finally recombinant plasmids were cons...

Embodiment 2

[0026] Example 2 Construction of Bacillus PP43NMK-GLNA-GFP Plasminate Plasmite

[0027] The PP43NMK-GLNA-GFP plasmid was constructed to transform the strain of Bacillus strain. The transformant was selected by YZ_ZONG-P43NMK_F (sequence such as SEQ ID No. 103) and YZ_ZONG-P43NMK_R (sequence such as SEQ ID NO.104) primers selected a transformator for colonial PCR, and 1.8 kb strips were detected, and the recombinant subtilis was constructed. success.

Embodiment 3

[0028] Example 3 Influence of N-Terminal Sequence on Expression of Green Fluorescent Protein in Engineering Bacteria

[0029] The seeds were cultured from 37 ° C, 750 rpm, 700 μl Lb medium, and 96 wells in a deep hole plate to 190 μl of LB medium at 37 ° C, 750 rpm, and cultured under 37 ° C, 750 rpm. Under the same conditions, if only PP43NMK is expressed, the final fermentation liquid is measured in the final fermentation liquid is about 5159, 20 hours to approximately 48181; after adding the N-ternation sequence of the GLNA protein, it is measured. The fluorescence intensity is 4 hours average of 20912.3, and the 20-hour average is 105441.4. The control is 4 times, twice, respectively.

[0030] The effects of N-terminal sequence shown in SEQ ID NO.1 to 97 on the expression of green fluorescent protein in engineering bacteria are summarized as follows:

[0031](1) Effect of regulatory protein expression: Taking fluorescence intensity characterization of protein expression, accor...

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Abstract

The invention discloses an N-terminal sequence element capable of statically and dynamically regulating and controlling expression amount of bacillus subtilis target protin and belongs to the field ofgene engineering. According to the invenetion, bacillus subtilis is taken as an expression host; 15 nucleotide sequences of amino acids before N-end encoding of endogenous protein are added before the target protein encoding genes, and free expression in plasmids is realized, so that the expression of the bacillus subtilis target protein realizes regulation and control over expression differenceand expression quantity at different growth stages, wherein regulation and control types include growth coupling type (NHbs and the like), a hysteresis expression type (NPdhD and the like), a sustained expression type (NGlnA and the like), and an intense inhibitory type (NSat and the like). Meanwhile, by virtue of regulation and control, the relative expression quantity of the target protein spansover four orders of magnitude, and a basis is laid for efficient expression and gene engineering reconstruction of the bacillus subtilis target protein.

Description

Technical field [0001] The present invention relates to an N-terminal sequence element and application of a static and dynamically regulatory protein expression of Bacillus, which belongs to the field of genetic engineering. Background technique [0002] Bacillus Subtilis is a representative of Gram-positive bacteria that does not produce endotoxin, and is a non-pathogenic microorganism, and is certified by the US Food and Drug Administration (FDA) Certification is a safe (GRAS) food. Level microorganisms. As the most common strain in the laboratory, it also serves as a strain that is widely used in the production of industrial enzyme preparations, high additional products and scientific research, and Bacillus Bacillus have a lot of advantages, including: fast growth, short culture time, and culture The system is low; the system of secretion signal peptides is very efficient, which has strong secretion protein capability, which provides a great convenience of subsequent protein e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/75C07K19/00C12P21/00C12R1/125
CPCC07K2/00C07K2319/00C12N15/75C12P21/00
Inventor 刘延峰堵国成田荣臻陈坚
Owner JIANGNAN UNIV
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