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Conjugation transfer method of rhodobacter sphaeroides

A technology of Rhodobacter sphaeroides and conjugation transfer, which is applied in the field of conjugation and transfer of Rhodobacter sphaeroides, can solve the problem of generally low efficiency of conjugation and transfer of Rhodobacter sphaeroides, and achieves the goal of improving conjugation and transfer efficiency, promoting energy absorption, and reducing demand. Effect

Active Publication Date: 2020-04-28
HEILONGJIANG NHU BIOTECH CO LTD +1
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  • Abstract
  • Description
  • Claims
  • 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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  • Conjugation transfer method of rhodobacter sphaeroides

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Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

[0047] After 32 hours, inoculate Escherichia coli S17-1 containing the pTrc99a plasmid into a test tube containing 10ml LB medium, and culture overnight at 37°C and 200rpm; after 15 hours, transfer 100μl Escherichia coli S17-1 bacteria solution Add 500μg ampicillin to a test tube of 5ml LB medium, and culture it on a shaker at 37°C until the bacterial solution OD 600 It is 0.5, obtains Escherichia coli bacterial liquid;

[0048] (2) The Rhodobacter sphaeroides bacterium li...

Embodiment 2～8

[0055] On the basis of Example 1, the parameters shown in the following tables were modified, namely: light conditions, light culture time in step 1, plasma treatment conditions, light time in step 3 conjugation culture and additional additions to the screening medium to calculate the conjugation rate.

[0056] The result is as follows:

[0057]

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

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Abstract

The invention discloses a conjugation transfer method of rhodobacter sphaeroides. The conjugation transfer method comprises the following steps: culturing donor bacteria, culturing rhodobacter sphaeroides, mixing the donor bacteria and the rhodobacter sphaeroides, performing conjugation culturing on the mixed bacteria, and screening conjugation transfers; and before the donor bacteria and the rhodobacter sphaeroides are mixed, carrying out plasma treatment on the rhodobacter sphaeroides, and culturing the rhodobacter sphaeroides and performing conjugation culturing on the mixed bacteria underirradiation of near infrared light. According to the invention, a near-infrared light source is used for carrying out illumination culture on rhodobacter sphaeroides, and plasma treatment is carried out, so that conjugation transfer efficiency of the rhodobacter sphaeroides is obviously improved, and the transfer rate of the rhodobacter sphaeroides is improved by 20-40 times compared with the transfer rate by using a relatively mature conjugation transfer method at present. Meanwhile, the time of conjugation culture and screening culture stages is greatly shortened, and directional genetic transformation and gene function research on rhodobacter sphaeroides are facilitated. The method has the characteristics of high efficiency, rapidness and convenience.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a conjugation transfer method of Rhodobacter sphaeroides. Background technique [0002] Rhodobacter Sphaeroides is a Gram-negative bacterium belonging to the α-proteobacteria group of purple non-sulfur bacteria. Rhodobacter sphaeroides have many uses, such as: as a model bacteria for photosynthetic bacteria and as a membrane protein expression system. In addition, Rhodobacter sphaeroides can synthesize coenzyme Q10, so it is an ideal coenzyme Q10-producing bacterium. [0003] At present, researchers mainly use the method of parental conjugation transfer to genetically manipulate the wild-type strain of Rhodobacter sphaeroides. In the dual-parental conjugative 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 conjugative transfer is affected by many factors, for ex...

Claims

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Application Information

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Patent Type & Authority Applications(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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