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Method for one-step transformation and marking of blue-green algae chromosomes and application

A marking method and chromosome technology, which is applied in the field of fluorescent labeling of cyanobacterial chromosomes, can solve the problems of limited research on cyanobacterial chromosome replication and separation, lack of effective labeling of cyanobacterial chromosomes, and the number of chromosomes is not fixed.

Inactive Publication Date: 2021-06-29
PEKING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, as a prokaryotic bacterium, cyanobacteria have very different chromosomes from other bacteria: 1. A cyanobacteria cell contains multiple copies of the same chromosome, usually 2-8
2. The number of chromosomes in each cyanobacteria cell is not fixed. The current literature tends to believe that the number of chromosomes in a cyanobacteria cell is related to the cell volume
3. The chromosome replication and separation of cyanobacteria may not have a particularly strict mechanism. Through staining and real-time quantitative PCR, it is found that the amount of chromosomes contained in the newly divided daughter cells of cyanobacteria will be different, and the replication of each chromosome of cyanobacteria is in the cell. Chromosomes are independent between each other and even within a cell, there is no obvious time period in which multiple chromosomes are replicated at the same time
[0004] Unlike other bacteria, studies on the replication and segregation of cyanobacterial chromosomes are currently very limited, mainly due to the lack of effective methods for labeling cyanobacterial chromosomes

Method used

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  • Method for one-step transformation and marking of blue-green algae chromosomes and application
  • Method for one-step transformation and marking of blue-green algae chromosomes and application
  • Method for one-step transformation and marking of blue-green algae chromosomes and application

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Embodiment 1

[0029] Example 1, Construction of Chromosomal Marker Vector 277ARY

[0030] (1) Construction of TetO repeat sequence TetO×120

[0031] The schematic diagram of constructing the TetO sequence array TetO×120 formed by repeating the TetO sequence 120 times is as follows figure 1As shown in A, wherein the base sequence of TetO is tctctatcactgataggga. Because a continuous repeated sequence is very unstable in the genetic replication of bacteria, a random base sequence R of 10-30 nt is inserted between every two TetO. In a case we implemented, the entire nucleotide sequence of the designed TetO×120 is shown in SEQ ID No:2 in the sequence listing. Then TetO×120 was obtained by Beijing Qingke Biotechnology Co., Ltd. through gene synthesis technology.

[0032] (2) Construction of chromosome marker vector 277ARY

[0033] The schematic diagram of the construction of the chromosome marker vector 277ARY is shown in figure 1 As shown in B, this carrier has the core element Upstream—P ...

Embodiment 2

[0034] Example 2, Screening of Chromosomal Marker Positive Strain ARY and Chromosomal Fluorescent Detection

[0035] (1) Screening and homozygosity verification of chromosomal marker-positive strain ARY

[0036] The constructed chromosomal marker carrier 277ARY was transferred into the genome of wild-type Anabaena sp. figure 1 In C, the chromosomal marker vector 277ARY in P 1 The —TetR-GFP—TetO×120—Em fragment was exchanged between the all1730 and all1729 genes of the genome chromosome of Anabaena sp.PCC 7120. The method of three-parent mating was as follows: 1. Combining the plasmid pRL443 and the helper plasmid pRL528 into Escherichia coli HB101, Recombinant strain A containing resistance genes Amp (ampicillin), Sm (streptomycin), Cm (chloramphenicol) and Tc (tetracycline) was obtained. 2. Transfer the chromosomal marker carrier 277ARY into the recombinant bacteria A to obtain the recombinant bacteria B, and wash with LB medium for 3 times. 3. Wash wild-type Anabaena sp. ...

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Abstract

The invention discloses a method for one-step transformation and marking of blue-green algae chromosomes and application. The method comprises the following steps: combining specificity of an inhibiting protein TetR in a tetracycline operon with a manipulated DNA sequence TetO; transferring a TetR-GFP fusion protein and a TetOxn sequence array onto blue-green algae genome chromosomes through one-step genetic transformation; then inducing expression of the TetR-GFP fusion protein by utilizing a promoter; combining the expressed TetR-GFP fusion protein with a TetOxn sequence on the chromosomes; and finally, obtaining a blue-green algae positive strain with the chromosomes emitting green fluorescence. The number and dynamic information of the chromosomes can be observed under a fluorescence microscope, so that the chromosome fluorescence-marked algae strain can be used as an ideal material for researching a copying and separating mechanism of the blue-green algae chromosomes.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering, and specifically relates to a method and application for finally realizing fluorescent labeling of cyanobacteria chromosomes by only one-step genetic transformation. Background technique [0002] Bacterial chromosome replication and segregation have been intensively and extensively studied in species such as Escherichia coli, Bacillus crescentus, Vibrio cholerae, and Bacillus subtilis. Bacteria do not have a nucleus, but only a nucleoid surrounded by a membrane. In order to distinguish it from plasmid DNA, scholars generally call the macrocircular DNA molecule in the nucleoid a chromosome. Bacteria generally have 1 or 2 different chromosomes, and the second chromosome usually plays an auxiliary role. [0003] However, as a prokaryotic bacterium, the chromosomes of cyanobacteria are very different from other bacteria: 1. A cyanobacteria cell contains multiple copies of the same chromo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/65C12N15/90
CPCC12N15/65C12N15/74C12N15/902
Inventor 郑正高刘轶群董春霞赵进东
Owner PEKING UNIV
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