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Method for in-vitro rapid optimization of strain metabolic pathway

A pathway and strain technology, applied in the field of rapid optimization of strain metabolic pathways in vitro, can solve problems such as insufficient gene rearrangement diversity, and achieve the effects of rapid optimization of metabolic pathways, efficient and rapid joint analysis, and wide application fields.

Active Publication Date: 2018-01-12
TIANJIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The design of the Creator system is similar to that of the Gateway system. Both are used in gene cloning and expression, but they can only conduct functional research on a single gene, and the diversity of gene rearrangement is not rich enough.

Method used

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  • Method for in-vitro rapid optimization of strain metabolic pathway
  • Method for in-vitro rapid optimization of strain metabolic pathway
  • Method for in-vitro rapid optimization of strain metabolic pathway

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: the synthesis of each functional gene fragment of β-carotene

[0041] Starting from the commercial vector pUC19, the DNA structure "loxPsym site-BsaI restriction site-RFP (red fluorescent protein gene)-BsaI restriction site-URA gene-loxPsym site" was amplified by overlapping PCR, and then passed the enzyme The method of cut ligation inserts the sequence into the multiple cloning site of pUC19 plasmid and assembles it into a universal vector pYW0120. This universal vector can quickly replace the RFP gene with the BsaI restriction sites at both ends of RFP, so that the exogenous function Gene fragments are quickly added to screening tags 1 and loxPsym sequences at both ends.

[0042] The functional gene fragment refers to the complete transcription unit of the target gene. Use PCR to amplify the functional gene on the genome template or use chemical methods to synthesize it from scratch, so that the end of the functional gene fragment carries a BsaI restrict...

Embodiment 2

[0044] Example 2: Obtaining of rearranged plasmid libraries

[0045] According to the reaction system (50 μL) in Table 1, each functional gene fragment of β-carotene in Example 1 was mixed with the donor plasmid pYW0113, Cre enzyme, Cre enzyme buffer and ddH introduced loxPsym sequence and screening tag HIS. 2 O undergoes in vitro rearrangement.

[0046] Table 1 rearrangement reaction system

[0047] Element

[0048] According to the amount of each substance in the table (the mass of the above 11 fragments is 200ng, and the receiving vector pYW0113 is 400ng), add them into PCR tubes respectively, and mix well. React at 37°C for 60 minutes and inactivate at 70°C for 10 minutes, then the reaction of the rearrangement system is completed, and a rearranged plasmid library is obtained.

Embodiment 3

[0049] Embodiment 3: the acquisition of high-yielding bacterial strain

[0050] 1. Transformation of Saccharomyces cerevisiae with rearranged plasmid library

[0051] Pick a single colony of Saccharomyces cerevisiae yYW0301 in 5mLYPD liquid medium, culture overnight at 30°C; measure the OD of Saccharomyces cerevisiae culture medium cultured overnight 600 , inoculate overnight culture solution into 5mL YPD (0.125OD 600 / ml), cultivated to OD at 30℃, 220rpm 600 Reach 0.5 (about 3.5-4.5h);

[0052] Pipette 1mL of Saccharomyces cerevisiae culture solution into a 1.5mL EP tube, centrifuge at 4000rpm for 2min, and collect the cells; resuspend the cells in 1mL of sterile water, centrifuge as above, and collect the cells; resuspend the cells in 1mL 0.1M LiOAc, centrifuge as above, and collect the cells; Remove 900 μL supernatant with a pipette, and resuspend the cells in the remaining 100 μL LiOAc, place on ice to obtain competent cells.

[0053] Prepare the transformation system:...

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Abstract

The invention relates to the field of biological technology, and in particular discloses a method for in-vitro rapid optimization of a strain metabolic pathway. According to the method, on the basis of the property of specific recombination of Cre-loxP site, random insertion of a gene is achieved by introducing an improved loxPsym sequence to a functional gene segment and a receiving vector, so that DNA of random insertion is obtained, and diverse rearrangement plasmid libraries are formed; the random combination of various metabolic pathway functional genes can be achieved; a process of synthesizing long-segment DNA by various functional genes can be avoided; combined analysis of the various functional genes can be efficiently and rapidly achieved; a metabolic pathway can be rapidly optimized; a key gene can be searched, without the limitation of a host; therefore, the method is broad in application fields.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to a method for rapidly optimizing the metabolic pathway of bacterial strains in vitro. Background technique [0002] The popularity of DNA sequencing technology has revealed the whole genome information of more and more species. In addition, the development of related omics technologies such as transcriptome, proteome, and metabolome has enabled scientists to understand various complex metabolic pathways and fine-grained pathways in biological cells. A deeper understanding of the regulatory mechanism. For example, the necessary gene elements for the production of β-carotene by Saccharomyces cerevisiae are GGPP synthase gene crtE, phytoene synthase gene crtB, phytoene dehydrogenase gene crtI, and lycopene cyclase gene crtY; The essential gene elements for yeast to produce lycopene are GGPP synthase gene crtE, phytoene synthase gene crtB and phytoene dehydrogenase gene c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/65C12N15/90C12R1/865
Inventor 元英进吴毅朱瑞莹刘瑞马璐
Owner TIANJIN UNIV
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