A conjugative transfer method for Rhodobacter sphaeroides

A technology of Rhodobacter sphaeroides, conjugation and transfer, which is applied in the field of conjugation and transfer of Rhodobacter sphaeroides, can solve the problems of generally low efficiency of conjugation and transfer of Rhodobacter sphaeroides, and achieves the effect of improving the efficiency of conjugation and transferring and shortening the time.

Active Publication Date: 2021-10-08
HEILONGJIANG NHU BIOTECH CO LTD +1
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0004] The conjugative transfer efficiency of Rhodobacter sphaeroides is generally low

Method used

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  • A conjugative transfer method for Rhodobacter sphaeroides

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] A method for conjugation and transfer of Rhodobacter sphaeroides, the specific steps are as follows:

[0046] (1) Inoculate Rhodobacter sphaeroides CGMCC No.15927 in a test tube containing 10ml of liquid culture medium, place the test tube in a constant temperature oscillator, the culture temperature is 30°C, the rotational speed is 200rpm, the light source adopts a halogen tungsten lamp, and the light intensity is 4000Lx, the wavelength is 870nm, cultured for 50h to obtain Rhodobacter sphaeroides bacterial liquid;

[0047] After 32 hours, inoculate E. coli S17-1 containing pTrc99a plasmid into a test tube containing 10 ml of LB medium, and cultivate overnight at 37°C and 200 rpm; 15 hours later, transfer 100 μl of E. Add 500 μg of ampicillin to a test tube of 5 ml of LB medium, and put it into a 37 °C shaker to culture to the OD of the bacterial solution. 600 be 0.5, obtain Escherichia coli bacterial liquid;

[0048] (2) the Rhodobacter sphaeroides bacterial liquid o...

Embodiment 2～8

[0055] On the basis of Example 1, the parameters shown in the following table were modified, namely: light conditions, light culture time in step 1, plasma treatment conditions, light time for conjugation culture in step 3 and additional supplements of screening medium, and the conjugation rate was calculated.

[0056] The result is as follows:

[0057]

[0058] Note: The light incubation time in step 1 refers to the incubation time of Rhodobacter sphaeroides under near-infrared light irradiation conditions.

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Abstract

The invention discloses a method for conjugative transfer of Rhodobacter sphaeroides, comprising: cultivation of donor bacteria, cultivation of Rhodobacter sphaeroides, mixing of donor bacteria and Rhodobacter sphaeroides, conjugation culture of mixed bacteria, and conjugative transfer Screening of Rhodobacter sphaeroides; before the donor bacteria were mixed with Rhodobacter sphaeroides, the Rhodobacter sphaeroides were treated with plasma, and the Rhodobacter sphaeroides were cultivated under near-infrared light irradiation and the conjugative culture of the mixed bacteria was carried out. The present invention utilizes a near-infrared light source to light-culture Rhodobacter sphaeroides, and through plasma treatment, significantly improves the conjugative transfer efficiency of Rhodobacter sphaeroides, and its transfer rate is increased by 20-40% compared with the relatively mature conjugative transfer method at present. At the same time, the time of conjugation culture and screening culture stage is greatly shortened, which is beneficial to the directional genetic transformation and gene function research of Rhodobacter sphaeroides. The method of the invention has the characteristics of high efficiency, rapidity and convenience.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for conjugation and transfer of Rhodobacter sphaeroides. Background technique [0002] Rhodobacter Sphaeroides is a Gram-negative bacterium belonging to the alpha-proteobacteria of the purple non-sulfur bacteria. Rhodobacter sphaeroides has a variety of uses, such as: as a model bacterium for photosynthetic bacteria and as a membrane protein expression system. In addition, Rhodobacter sphaeroides can synthesize coenzyme Q10, which is an ideal coenzyme Q10 producer. [0003] At present, researchers mainly use the method of biparental conjugation transfer to genetically manipulate the wild-type strain of Rhodobacter sphaeroides. In the biparental conjugation transfer system, the host that provides the plasmid is called the donor strain, and the host that receives the plasmid is called the recipient strain. The efficiency of conjugation transfer is affected by many factors, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N13/00C12N1/20C12R1/01C12R1/19
CPCC12N1/20C12N13/00
Inventor 陈召峰朱永强于凯潘梦垚李永胡伟江
Owner HEILONGJIANG NHU BIOTECH CO LTD
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