A Bacillus subtilis promoter with improved activity and its construction and application

A Bacillus subtilis, promoter technology, applied in the field of promoter engineering, can solve problems such as the inability to meet artificial metabolic pathways

Active Publication Date: 2019-08-20
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, natural promoters are the most widely used, but due to the species specificity of natural promoters, they cannot meet the requirements of research such as constructing more complex artificial metabolic pathways.

Method used

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  • A Bacillus subtilis promoter with improved activity and its construction and application
  • A Bacillus subtilis promoter with improved activity and its construction and application
  • A Bacillus subtilis promoter with improved activity and its construction and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: P srfA Promoter mutant library construction strategy

[0038] (1) with P srfA-7bp -1 and P srfA-7bp -2 (Table 1) is primer, with pBSG03 as template, to P srfA Promoter [C.Guan, W.Cui, J.Cheng, L.Zhou, J.Guo, X.Hu, G.Xiao, Z.Zhou, Construction and development of an auto-regulatory gene expression system in Bacillus subtilis, Microb. Cell Fact.14(2015)]σ A Identify the upstream 7bp base of the core region "-10 region" for degenerate mutation ( figure 1 ).

[0039] The primers used in the present embodiment of table 1

[0040]

Embodiment 2

[0041] Example 2: P srfA Promoter mutant library screening

[0042] P srfA The promoter mutant library was transformed into B. subtilis 168, and the plates were spread to obtain transformants. The obtained Bacillus subtilis transformants were picked and cultured in a 96-well cell culture plate, and the OD of the bacterial solution was measured with a microplate reader 600 and GFP fluorescence intensity to screen mutant promoters with different GFP expression levels.

[0043] It can be seen from the screening results of the 96-well plate ( figure 2 ), all transformants have successfully expressed the GFP whose sequence is shown in SEQ ID NO.5, the GFP fluorescence intensity of most transformants has decreased, and the GFP fluorescence intensity of 3 transformants has increased, indicating that these 3 mutant promoters It is a forward mutation, and the promoters of the three mutants obtained are respectively PV1, PV2 and PV3, and the base sequences are respectively shown in...

Embodiment 3

[0044] Example 3: P srfA Shake flask experiment verification of mutant promoters

[0045] The 3 Ps obtained by screening in Example 2 srfA The promoter was used for shake flask experiment verification. In the 250ml shaking flask system, verify that these transformants express GFP activity, the results show that the growth curve of the bacterial strain with the mutant promoter plasmid is consistent with the wild-type bacterial strain ( image 3 -a), and both the wild-type and mutant promoters increased rapidly in the middle and late stages of cell logarithmic growth ( image 3 -b), which conforms to P srfA Promoters are activated by quorum sensing signals. The activities of the three mutant promoters screened were significantly improved compared with the wild-type promoter, and the highest PV1 activity was about 1.76 times that of the wild-type promoter ( image 3 -c), PV2, PV3, the expression fluorescence intensity is the wild-type promoter P srfA 1.5, 1.67 times. SDS-P...

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Abstract

The invention discloses a bacillus subtilis promoter with improved activity, and construction and applications thereof, and belongs to the field of promoter engineering. A PsrfA promoter is taken as the template to carry out directed evolution. The mutant promoters with greatly improved activity are screened out. Green fluorescent protein (GFP) is taken as the reporter gene, and the highest expression amount of promoters is increased by 70% after mutation, compared with that of original promoters. Then the expression of aspartase AspA is carried out, and the mutant expression amount is increased by 90%, compared with the original promoter. Compared with a method of using a flow cytometry to establish database, the workload is largely reduced, at the same time, the method is applied to other promoter modification, and the performance of modified promoters on expressing target genes is largely improved.

Description

technical field [0001] The invention relates to a bacillus subtilis promoter with improved activity and its construction and application, belonging to the field of promoter engineering. Background technique [0002] Bacillus subtilis (Bacillus subtilis) is a Gram-positive bacterium that has been identified as biosafety GRAS (Generally regarded as safe) by the US FDA. It has the advantages of good protein secretion ability and simple fermentation conditions, so it is widely used in the production of industrial and food enzyme preparations. Although many industrial and food enzyme preparations have achieved recombinant expression in B. subtilis, the heterologous protein production capacity of the existing protein expression system is far from meeting the needs of large-scale production. Among them, the promoter element plays a central role in the gene expression system, and most of the promoters being used in B. subtilis have the problem of low activity. Promoter is the most...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/75C12N15/67C12N1/21C12N9/88C12R1/125
Inventor 索菲娅周哲敏崔文璟韩来闯程锦涛刘中美周丽
Owner JIANGNAN UNIV
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