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Construction method and application of oxygen response type biosensor

A biosensor and promoter technology, applied in the field of synthetic biology, can solve the problems of not being well adapted to the distribution between the growth and production of metabolic flow, affecting the growth of cells or the production of target compounds, etc.

Pending Publication Date: 2021-06-25
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] The synthesis of compounds using microbial cell factories usually involves the use of metabolic engineering methods to increase the flux of target metabolic fluxes. Traditional metabolic engineering is mostly static irreversible changes, such as gene overexpression and knockout, etc. This method cannot be very fast. Adapt well to changes in intracellular and extracellular conditions to balance the distribution of metabolic flux between growth and production, which can potentially affect the growth of the cell or the production of the target compound

Method used

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  • Construction method and application of oxygen response type biosensor
  • Construction method and application of oxygen response type biosensor
  • Construction method and application of oxygen response type biosensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Construction of an oxygen-responsive biosensor

[0049] Such as figure 1 As shown in B, the sensor plasmid is mainly composed of 3 parts: 1) FNR gene and its upstream promoter P ffs ; 2) anaerobic-inducible promoter P containing FNR transcriptional activator targeting binding site fnrF8 3) GmR resistance gene and its upstream promoter P c .

[0050] Anaerobic inducible promoter P fnrF8 Located between the restriction site Avr II and Xba I of the plasmid pACM4G, and integrated with the FNR transcriptional activator targeting binding site; the promoter P fnrF8 Induces the expression of the target gene; promoter P ffs Located upstream of the restriction site Avr II of the plasmid pACM4G, it induces the expression of the downstream FNR gene; the GmR resistance gene is located downstream of the FNR gene, and is controlled by the upstream P c Promoter induces expression.

[0051] Using the Escherichia coli K12 MG1655 genome as a template, the FNR fragment was...

Embodiment 2

[0056] Example 2 Response Performance Evaluation of Oxygen Responsive Biosensor

[0057] Fumarate and nitrate reductase (FNR) transcriptional activator-dependent biosensors are currently the most studied oxygen-responsive biosensors. As a transcriptional activator, FNR mediates the transition from aerobic to anaerobic metabolism by regulating gene transcription. , whose activity switches between 4Fe-4S and 2Fe-2S clusters with oxygen content ( figure 1 A), under anaerobic conditions, FNR binds to 4Fe-4S clusters to change the conformation of the protein, then dimerizes and binds to specific sites in the promoter region, and finally activates the protein to activate related anaerobic metabolism In the expression of related genes in the pathway, in the presence of oxygen, 4Fe-4S clusters are oxidized to 2Fe-2S clusters, while the dimers decompose and the FNR protein loses its activity.

[0058] The response performance of the biosensor under aerobic and anaerobic conditions was...

Embodiment 3

[0059] Example 3 Screening of different strength anaerobic inducible promoters

[0060] FNR transcriptional activators are known to achieve normal transcription of downstream genes by targeting specific domains of promoters. fnrF8 In , the FNR targeting binding site is located upstream of the promoter-35 region ( figure 2 A), changing the distance between the two may affect the response performance of the biosensor, resulting in differences in the expression levels of genes downstream of the promoter.

[0061] Performance testing was carried out under aerobic and anaerobic conditions respectively, and the blank plasmid pACM4G-F8*-GFP without the FNR targeting binding site was used as a control, and the sensor plasmids pACM4G-F8-GFP, pACM4G- U10F8-GFP, pACM4G-U15F8-GFP, pACM4G-U20F8-GFP, pACM4G-D3F8-GFP, pACM4G-D4F8-GFP and pACM4G-D7F8-GFP were introduced into Escherichia coli JM109 for biosensor performance evaluation.

[0062] The result is as figure 2 As shown in B, inc...

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Abstract

The invention discloses a construction method and application of an oxygen response type biosensor, and belongs to the field of synthetic biology. Based on fumarate nitrate reductase FNR and a promoter PfnrF8 containing an FNR targeted binding site, a biosensor capable of activating anaerobic metabolic pathway related genes is established, and meanwhile, the intensity of the promoter is adjusted through promoter engineering, so that biosensors with different response intensities are obtained. Therefore, a remarkable positive effect is achieved when the strain is applied to optimized fermentation for regulating and controlling escherichia coli to biologically synthesize adipic acid. The dynamic regulation and control on the gene expression level along with the change of the fermentation environment are realized, the method is different from the traditional static fermentation with human intervention, and the method provides a beneficial reference for the production of other high-added-value compounds.

Description

technical field [0001] The invention relates to the construction and application of an oxygen-responsive biosensor, belonging to the field of synthetic biology. Background technique [0002] The use of microbial cell factories to synthesize compounds usually involves the use of metabolic engineering methods to increase the flux of target metabolic fluxes. Traditional metabolic engineering is mostly static irreversible changes, such as gene overexpression and knockout, etc. This method cannot be very fast. Good adaptation to changes in intracellular and extracellular conditions balances the distribution of metabolic flux between growth and production, which can potentially affect the growth of the cell or the production of the target compound. The development and application of intracellular biosensors have alleviated the difficulties faced by traditional methods. They can monitor changes in the internal and external environments of cells in real time and convert environmenta...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/53C12N15/65C12N1/21C12P7/44C12R1/19
CPCC12N15/70C12N9/0044C12N15/65C12P7/44Y02A50/30
Inventor 邓禹周胜虎郝婷婷毛银赵运英李国辉
Owner JIANGNAN UNIV
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