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Temperature-sensitive gene loop system as well as construction method and application thereof

A temperature-sensitive, genetic technology, applied in the fields of synthetic biology and metabolic engineering, can solve problems such as cell growth damage and achieve the effect of relieving metabolic stress

Pending Publication Date: 2022-07-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the pentose phosphate and glycolysis pathways are both necessary for the normal growth of microorganisms, so directly knocking out these two competing pathways using a static strategy will cause damage to cell growth

Method used

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  • Temperature-sensitive gene loop system as well as construction method and application thereof
  • Temperature-sensitive gene loop system as well as construction method and application thereof
  • Temperature-sensitive gene loop system as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1. Design and construction of thermosensitive promoter

[0036] (a) Artificially synthesized gene fragment P 43 -CI 857 (see SEQ ID NO.1 for the sequence), using the Bacillus subtilis 168 genome as a template, using primers amye-U-F and amye-U-R to amplify the gene amye-U, and using primers amye-D-F and amye-D-R to amplify the gene amye -D. Using plasmid p7s6 as a template, the gene amye-S was amplified by primers S-amye-F and S-amye-R. with P 43 -CI 857 As a template, using P43-CI-F, P43-CI-R amplification to obtain fragment P 43 -CI. (Plasmids p7s6 and p7z6 plasmids were both derived from Cre / lox System and PCR-Based Genome Engineering in Bacillus subtilis, DOI: 10.1128 / AEM.01156-08).

[0037] (b) The four fragments amye-U, amye-D, amye-S and P43-CI gene fragments obtained in step (a) are configured in an equimolar ratio for one-step fusion PCR processing. The ligated fragments were transferred into the competent Bacillus subtilis 168, and then plated ...

Embodiment 2

[0063] Example 2. Construction of SIMO gene circuit

[0064] (a) Synthesized artificially with promoter P veg32 Gene fragment P that controls CFP expression veg32 -CFP (SEQ ID NO. 7), artificially synthesized with promoter P veg34 Gene fragment P that controls GFP expression veg34 -GFP (SEQ ID NO. 8), artificially synthesized with promoter P veg37 Gene fragment P that controls mcherry expression veg37 -mcherry (SEQ ID NO. 9). Using the genome of Bacillus subtilis 168 as a template, the gene npre-U was amplified with primers npre-U-F and npre-U-R, and the gene npre-D was amplified with primers npre-D-F and npre-D-R. The primers gana-U-F, gana - The gene gana-U was obtained by U-R amplification, and the gene gana-D was obtained by amplification with primers gana-D-F and gana-D-R. Using plasmid p7z6 as a template, the gene npre-Z was amplified by primers npre-Z-F and npre-Z-R. Using the plasmid p7s6 as a template, the fragment gana-S was obtained by amplifying the primers ...

Embodiment 3

[0081] Example 3. Construction of bidirectional gene circuits

[0082] (a) Synthesized artificially with promoter P veg5 Gene fragment P that controls dcpf1 expression veg5 -dcpf1 (SEQ ID NO. 10), artificially synthesized with promoter P veg Controls the gene fragment P that suppresses mcherry expression of crRNA veg -crrnam (SEQ ID NO. 11). Using the genome of Bacillus subtilis 168 as a template, the gene npre-U2 was amplified with the primers npre-U-F and npre-U-R2, and the gene npre-D was amplified with the primers npre-D-F and npre-D-R, and the primer gana-U-F was used , gana-U-R was amplified to obtain the gene gana-U, and the primers gana-D-F2 and gana-D-R were used to amplify the gene gana-D2. Using the plasmid p7z6 as the template, the gene npre-Z2 was amplified with the primers npre-Z-F2 and npre-Z-R, and the fragment gana-S2 was amplified with the primers gana-C-F and gana-C-R2 with the plasmid p7S6 as the template.

[0083] (b) The npre-U2, npre-D, npre-Z2 and ...

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Abstract

The invention relates to a temperature-sensitive gene loop system as well as a construction method and application thereof. According to the invention, a series of temperature-sensitive biosensors suitable for B. subtilis are designed and reconstructed; a bidirectional gene loop is constructed based on a CRISPRi system, the bidirectional gene loop is combined with a single-input multi-output gene loop to construct a high-version single-input multi-output gene loop, and activation expression or / and down-regulation expression of a plurality of metabolism modules at different fermentation time points is / are realized. And then dynamically regulating and controlling three modules participating in metabolic synthesis of 2 '-FL by using the obtained gene loop: a 2'-FL synthesis module, a cofactor supply module and a competitive pathway module. The cell growth stage and the 2 '-FL synthesis stage can be decoupled by dynamic regulation and control through a gene loop, the metabolic pressure of host cells is relieved, metabolic flux is reasonably distributed, and efficient synthesis of 2'-FL is achieved.

Description

technical field [0001] The invention belongs to the technical field of synthetic biology and metabolic engineering, in particular to a temperature-sensitive gene circuit system and a construction method and application thereof. Background technique [0002] 2'-fucosyllactose (2'-fucosyllactose, 2'-FL), molecular formula C 18 H 32 O 15 , is an oligosaccharide composed of three monosaccharide molecules, and its content accounts for about 31% of the total breast milk oligosaccharides. 2'-FL can inhibit the growth of intestinal pathogenic bacteria, thereby enhancing the intestinal protective barrier function and regulating the human immune system. At present, both the EU and the FDA have approved that 2'-FL can be added to infant formula milk powder, and the market prospect is broad. [0003] As a Gram-positive host of GRAS (Generally Regard as Safe), B. subtilis has been widely used in the biosynthesis of high value-added compounds such as L-asparaginase, hyaluronic acid an...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/75C12N1/21C12P19/00C12R1/125
CPCC12N9/2491C12N9/1051C12N9/0006C12N9/1205C12N9/1229C12N9/22C12N15/75C12P19/00C12Y302/01025C12Y204/01069C12Y101/01049C12Y207/01011C12Y207/04006C12Y207/04008Y02A50/30
Inventor 刘龙陈坚吕雪芹堵国成李江华刘延峰于文文
Owner JIANGNAN UNIV
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