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Sic1 gene knock-in saccharomyces cerevisiae genetically engineered bacterium as well as construction method and application thereof

A technology of genetically engineered bacteria and Saccharomyces cerevisiae, applied in the field of genetically engineered bacteria of Saccharomyces cerevisiae and its construction, can solve the problems of prolonged fermentation period, high resistance, hindering mass transfer and conduction, etc.

Active Publication Date: 2021-08-31
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, too much biofilm will cause excessive bacterial aggregation and immature aggregates, high resistance, which hinders mass transfer and conduction, resulting in a longer fermentation cycle

Method used

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  • Sic1 gene knock-in saccharomyces cerevisiae genetically engineered bacterium as well as construction method and application thereof
  • Sic1 gene knock-in saccharomyces cerevisiae genetically engineered bacterium as well as construction method and application thereof
  • Sic1 gene knock-in saccharomyces cerevisiae genetically engineered bacterium as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Construction of recombinant bacteria S288c-pSic1.

[0048] 1. Construction of the Sic1 gene knock-in module

[0049] (1) Using the genomic DNA of the original Saccharomyces cerevisiae S288c as a template, the upstream, Sic1 gene and downstream homology arm amplification fragments were amplified by common PCR (upstream homology arm amplification primer sequences up-F, up -R are respectively shown in SEQ ID NO.2 and SEQ ID NO.3; Sic1 gene amplification primer sequences Sic1-F, Sic1-R are respectively shown in SEQ ID NO.4 and SEQ ID NO.5; downstream homology The sequences of the arm amplification primers down-F and down-R are respectively shown in SEQ ID NO.6 and SEQ ID NO.7); the PCR reaction system is shown in Table 1, and the PCR reaction conditions are as follows: 1) Pre-denaturation at 95°C for 3 minutes; 2 ) Denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 1 min, three steps for 35 cycles, and extension at 72°C for 5 min. ...

Embodiment 2

[0094] (1) Take 10 μL of glycerol bacteria S288c (original bacteria) and S288c-pSic1 (knock-in bacteria constructed in Example 1) and add them to 5 mL of sterilized LYPD liquid medium for overnight culture and activation;

[0095] (2) According to the inoculation ratio of 1% by volume, transfer the bacterial liquid obtained in step (1) to 100 mL of YPD liquid medium, and continue to cultivate at 30 ° C and 200 r / min until the OD600 of the bacterial liquid is about 2.0;

[0096] (3) Take 2 mL of the bacterial solution obtained in step (2) and measure the absorbance value at OD600, and dilute the bacterial solution with sterilized YPD liquid medium so that the OD600 of the diluted bacterial solution is 1;

[0097] (4) Add 190 μL of YPD medium and 10 μL of the diluted bacterial solution in step (3) to a 96-well plate, and incubate at 30°C for 24 hours;

[0098] (5) Pour out the 96-well plate bacterial solution, buffer with 0.01M PBS buffer 3 times, and pat dry;

[0099] (6) Add ...

Embodiment 3

[0102] (1) Take 10 μL of glycerol bacteria S288c (original bacteria) and S288c-pSic1 (knock-in bacteria constructed in Example 1) and add them to 5 mL of sterilized LYPD liquid medium for overnight culture and activation;

[0103] (2) According to the inoculation ratio of 1% by volume, transfer the bacterial liquid obtained in step (1) to 100 mL of YPD liquid medium, and continue to cultivate at 30 ° C and 200 r / min until the OD600 of the bacterial liquid is between 0.8 and 1.2 ;

[0104] (3) Transfer the seed solution in step (2) to 100 mL of fermentation medium at an inoculation volume ratio of 10%, and divide it into free fermentation and immobilized fermentation.

[0105] When carrying out immobilized fermentation: add cotton fiber material to the fermentation medium as the immobilized material, add 4g cotton fiber medium to each shake flask; ferment at 30°C, 200r / min, after the glucose is exhausted, the reaction ends The instrument measures the amount of residual sugar i...

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Abstract

The invention discloses a Sic1 gene knock-in saccharomyces cerevisiae genetically engineered bacterium. The saccharomyces cerevisiae is formed by knocking the gene Sic1 into original saccharomyces cerevisiae by utilizing a CRISPR-Cas9 technology and then modifying the saccharomyces cerevisiae. Original strain S288c aggregates too much, aggregates are immature and resistance is high in immobilized fermentation, mass transfer and conduction are hindered, and the fermentation period of the strain is prolonged, while compared with the original saccharomyces cerevisiae strain, the Sic1 gene knock-in saccharomyces cerevisiae genetically engineered bacterium has the advantages that the flocculation characteristic is obviously weakened in free fermentation, in immobilized fermentation, cell adhesion and aggregation conditions are reduced, adhesiveness is reduced, free cells are increased, mass transfer and conduction are enhanced, and the fermentation period is shortened.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and microorganisms, and in particular relates to a Sac1 gene knock-in Sac1 genetically engineered bacterium and its construction method and application. Background technique [0002] Biofilm, also known as biofilm, is a biological aggregate composed of microbial cells and their secreted extracellular matrix. The microbial clusters and their secreted polysaccharides, proteins, fatty acids, etc. form a film-like structure and attach to the surface of the carrier. The existence of biofilm not only creates a stable internal environment for the life activities of cells as a barrier, mediates the connection between cells and cells, and between cells and substrates, but also undertakes material transport, information transmembrane transmission and energy conversion. And other functions, more importantly, biofilm is also used as an important industrial application - immobilized fermentation. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12P7/06C12R1/865
CPCC07K14/395C12N15/81C12P7/06C12N2310/20Y02E50/10
Inventor 应汉杰蒋颖陈勇赵伟朱家庆张涛梁偲策余斌
Owner NANJING TECH UNIV
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