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Method of high-effectively detecting cell cycle of cyanobacteria

A technology for cyanobacteria and Anabaena, which is applied in biochemical equipment and methods, determination/inspection of microorganisms, and introduction of foreign genetic material using vectors, and can solve problems such as the inability of cyanobacteria to detect cell cycle well

Active Publication Date: 2017-10-24
PEKING UNIV
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Problems solved by technology

[0004] The purpose of the present invention is to provide a method for efficiently detecting the division cycle corresponding to each cell of cyanobacteria in vivo, which solves the technical problem that the current cyanobacteria cannot perform cell cycle detection well, and provides a method for studying the relationship between cyanobacteria cell division and differentiation. It also provides a convenient and intuitive method for assessing the growth status of cyanobacteria by detecting the cell cycle

Method used

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  • Method of high-effectively detecting cell cycle of cyanobacteria
  • Method of high-effectively detecting cell cycle of cyanobacteria
  • Method of high-effectively detecting cell cycle of cyanobacteria

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Embodiment 1, the construction of integration vector and the screening of positive bacterial strain

[0027] (1) Construction of integration vector 1730D

[0028] The structure of the integration vector 1730D is as follows figure 1 Shown in middle B, wherein FlankA and FlankB represent homologous fragments on the Anabaena PCC7120 genome respectively (seefigure 1 Middle A). Using the total genomic DNA of wild-type Anabaena sp.PCC 7120 as a template, use primer pairs P1 / P2, P3 / P4 (see Table 1 for primer sequences) to amplify FlanKA and FlanKB fragments, and use primer pairs P5 / P6, P7 / P8 amplifies P amiC3 and amiC3 fragment, use primer pair P9 / P10 to amplify the green fluorescent protein gfp gene fragment, and primer pair P11 / P12 to amplify the erythromycin Em gene fragment; The connection was recovered by enzyme digestion, and the final recovery product was used as a template to amplify the about 7kb fragment of FlanKA-Em-FlanKB with P1 / P4, and then digested with SphI...

Embodiment 2

[0040] Embodiment 2, utilize fluorescence microscope to detect the cell cycle of positive bacteria

[0041] After the positive homozygous strain 1730D-amiC3 was cultured in liquid BG11, samples were taken and then observed under a fluorescent microscope. Using the excitation fluorescence at 488nm to observe the green fluorescence of 1730D-amiC3, by image 3 Triangular arrows indicate the result of the cell division ring can be seen: if there is no green fluorescence in the center of the cell, this indicates that the cell is not in the cell division phase, the cell is in interphase ( image 3 Middle A). If there is faint green fluorescence in the center of the cell, this indicates that the cell division ring is assembling and the cell is in prophase ( image 3 Middle B). If there is strong green fluorescence in the center of the cell, this indicates that the cell division ring has been assembled and the cell is in metaphase ( image 3 Middle C). If the green fluorescence i...

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Abstract

The invention discloses a method of high-effectively detecting cell cycle of cyanobacteria. In the method, cyanobacteria cell division rings are labeled by means of a fusion protein of amidase AmiC3 or a homologous protein thereof and a fluorescent protein, and the cell cycle of the cyanobacteria is determined by observing the existence status of the cyanobacteria cell division rings through fluorescent display. The method solves a technical problem that cell cycle of the cyanobacteria cannot be detected well and is a way for researching the relationship between cell division and cell differentiation of the cyanobacteria. The method also is convenient and intuitive for evaluating the growth status of the cyanobacteria by detecting the cell cycle.

Description

technical field [0001] The invention relates to a cell cycle detection method, in particular to a method for efficiently detecting cyanobacteria cell cycle. Background technique [0002] It is of great significance to efficiently detect the cell cycle of cyanobacteria. First of all, cyanobacteria, as the simplest model organism to study cell differentiation, detects the division cycle of each cell in vivo and studies the corresponding differentiation potential, which provides a basis for studying the relationship between cell division and differentiation. a path. Secondly, due to the characteristics of photosynthesis, easy genetic manipulation, and fast growth rate of cyanobacteria, there are many examples of using it as a bioreactor to express foreign genes in the literature and industrial fermentation, such as the production of fatty acids and cellulose. Efficient detection of cell cycle can evaluate the growth state of cyanobacteria and determine which period of growth ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/74
CPCC12N15/74C12Q1/6897
Inventor 郑正高董春霞赵进东
Owner PEKING UNIV
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