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Method for non-trace knocking out cyanobacteria gene by one-step transformation

A traceless knockout, gene technology, applied in the field of traceless knockout of a gene in cyanobacteria, gene knockout

Active Publication Date: 2020-02-28
PEKING UNIV
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  • Claims
  • Application Information

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Problems solved by technology

[0003] However, according to the existing literature reports, the method of gene seamless knockout in cyanobacteria can only be realized after 2 or more steps of genetic transformation, so that the cycle of constructing a traceless knockout is about 10 times that of a resistant knockout. 3 to 4 times the division period

Method used

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  • Method for non-trace knocking out cyanobacteria gene by one-step transformation
  • Method for non-trace knocking out cyanobacteria gene by one-step transformation
  • Method for non-trace knocking out cyanobacteria gene by one-step transformation

Examples

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Embodiment 1

[0029] Example 1. Construction and homozygosity verification of cyanobacteria hetR traceless knockout mutants

[0030] (1) Construction of hetR seamless knockout vector PRL277Cre

[0031] The schematic diagram of the construction of the hetR seamless knockout vector PRL277Cre is as follows figure 1 As shown in middle B, where FlankA and FlankB represent homologous fragments of 3 KB upstream and downstream of hetR, respectively (see figure 1 Middle A). Using the total genomic DNA of wild-type Anabaenasp. Cre fragment, the erythromycin Em gene fragment was amplified with the primer pair P9 / P10; then the whole gold one-step seamless cloning kit ( Seamless Cloning and Assembly Kit) simultaneously ligated the five fragments of FlanKA, PpetE, Cre, Em, and FlanKB into the pRL277 plasmid to obtain the scarless knockout vector PRL277Cre. Since the 5' ends of primers P2, P5, P10, and P3 are all designed with a 34bp loxP sequence in the same direction, the core element on the scarle...

Embodiment 2

[0041] Example 2. Phenotype verification of cyanobacteria hetR traceless knockout mutant

[0042] (1) Phenotype verification

[0043] After the successful construction of the traceless knockout mutant △hetR was identified by PCR, the phenotype of the △hetR mutant was verified. HetR protein is the main regulatory protein of heterocyst differentiation in filamentous cyanobacteria, and filamentous cyanobacteria lacking hetR gene will not be able to differentiate heterocysts in the absence of N. The WT wild-type Anabaena sp.PCC 7120 and △hetR were respectively cultured in the non-antibiotic medium containing N and N-free, and it was found that in the N-containing medium, there was no difference in phenotype between WT and △hetR ( image 3 A and B of A and B); while in the anti-free medium without N, WT can differentiate heterocysts ( image 3 Middle C, indicated by the arrow), while △hetR cannot differentiate heterocysts ( image 3 Middle D). These results indicated that the p...

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Abstract

The invention discloses a method for non-trace knocking out cyanobacteria gene by one-step transformation. The method includes replacing purpose gene in two loxP sequences in the same direction and asequence including induction type promoter-driven Cre gene and selection marker gene between the sequences; then inducing the expression of Cre recombinant enzymes by using an induction type promoter;and finally, acquiring a non-trace knockout mutant. The method only needs one-step genetic transformation, by controlling the induction conditions in a culture medium to achieve non-trace knocking out of cyanobacteria gene, so that the time of non-trace knocking out cyanobacteria is greatly shortened, and the training cost can be reduced, the use of antibiotic and the pollution of gene to the environment are reduced, and the method provides a way for more rapidly developing all kinds of high-quality nonresistant cyanobacteria engineering bacteria chassis organisms in the future.

Description

technical field [0001] The invention belongs to the gene knockout technology in the field of microbial genetic engineering, and specifically relates to a method and application that can finally realize the traceless knockout (no antibiotic marker) of a certain gene in cyanobacteria by only one step of genetic transformation. Background technique [0002] Cyanobacteria have been regarded as an ideal chassis organism in synthetic biology due to their advantages of photosynthesis, simple genetic background, and fast growth, and this involves many genetic manipulations and gene editing of cyanobacteria. Most of the current gene knockout methods in cyanobacteria use homologous recombination to replace the target gene with a resistance gene, and then obtain homozygotes under antibiotic culture conditions. However, the disadvantages of this method mainly include: 1. The introduction of an exogenous resistance gene may make the functional analysis of the target gene inaccurate, and ...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/65C12N15/66
CPCC12N15/65C12N15/66C12N15/74
Inventor 郑正高董春霞王宏蕊赵进东
Owner PEKING UNIV
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