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Autonomously illuminated Acinetobacter baumannii and construction method and application thereof

An Acinetobacter baumannii, self-luminous technology, applied in the field of genetic engineering, can solve problems such as not yet seen in Acinetobacter baumannii, and achieve the effects of strong stability, simple and fast operation, and high luminous intensity

Active Publication Date: 2019-05-03
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the Xer-cise system for excision of resistance genes has only been reported in recent years, and has been used for genetic manipulation of Mycobacterium, but has not yet been seen in Acinetobacter baumannii

Method used

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  • Autonomously illuminated Acinetobacter baumannii and construction method and application thereof
  • Autonomously illuminated Acinetobacter baumannii and construction method and application thereof
  • Autonomously illuminated Acinetobacter baumannii and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Construction of transfer plasmid pUC18T-mini-Tn7T-lux-Ab-dif-Apr

[0047] (1) According to the process image 3 The transfer plasmid pUC18T-mini-Tn7T-lux-Ab-dif-Apr was constructed. Such as figure 2 As shown, the plasmid pUC18T-mini-Tn7T-lux-Ab-dif-Apr contains the origin of replication (ori), ampicillin resistance gene (AmpR), and apramycin resistance gene (Apr) in a clockwise direction , the luciferase gene (LuxCDABE), the direct repeat sequences DifR and DifL located at both ends of the AprR gene. The functions of each component are as follows:

[0048] LuxCDABE: the luciferase gene, the enzyme gene required for luminescence, the expression of this gene allows the host bacteria to emit light autonomously.

[0049] Apramycin resistance gene (AprR): Apramycin resistance gene (Apr) is a selectable marker for screening to obtain the target strain; Apramycin resistance gene (Apr) in Acinetobacter baumannii After being expressed in Escherichia coli, the hos...

Embodiment 2

[0060] Example 2 A method for constructing an autonomous luminescent Acinetobacter baumannii without a resistance screening marker

[0061] 1. Electroconversion

[0062] (1) To prepare Acinetobacter baumannii competent cells, inoculate a single colony (Acinetobacter baumannii) to be transformed into 15mL LB medium, culture at 37°C with shaking at 200rpm until OD 600 At 0.4-0.8, room temperature, 12000g, centrifuge for 5min, collect the bacteria. Resuspend and wash the cells with 10 mL of 10% glycerol, centrifuge at 12000 g for 5 min at room temperature, pour off the residual liquid, and repeat twice. Resuspend bacteria in 500 μL of 10% glycerol and mix well. Aliquot the competent cells into 100uL / tubes, put them in a -80°C refrigerator, and save them for later use (it is best to prepare new competent cells each time).

[0063] (2) Transfer 100 μL of Acinetobacter baumannii competent cells to a 2 mm electroporation cuvette, add 50 ng of the transfer plasmid pUC18T-mini-Tn7T-...

Embodiment 3

[0085] Embodiment 3 verifies the luminescence stability of no resistance AlAb

[0086] 1) Subculture the non-resistant AlAb prepared in Example 2

[0087] a. Inoculate 5 mL of AlAb into LB liquid medium at a ratio of 1:10000, and culture it in a constant temperature shaker at 37°C at 200 rpm;

[0088] b. When the bacteria solution OD 600 When it reaches 0.7, inoculate 5 mL of the bacterial solution into LB liquid medium at a ratio of 1:10000, and place it in a constant temperature shaker at 37°C and 200 rpm for cultivation;

[0089] c. After repeating step b several times, spread the bacterial solution on a non-resistant LB plate, and place it in a constant temperature incubator at 37°C for cultivation, and observe after 24 hours.

[0090] 2) Statistical proportion of AlAb

[0091] When colonies grew on the plate, 200 single colonies were randomly picked and RLUs were detected with a luminescence detector. If the RLUs of the detected single colony are more than 5 times the...

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Abstract

The invention discloses a transfer plasmid for transforming Acinetobacter baumannii, which comprises, in clockwise order, a replicon ori, an ampicillin resistance gene AmpR, a transposon sequence, a junction transfer initiation site oriT, the transposon sequence contains a base sequence capable of autonomously emitting Acinetobacter baumannii and a resistance gene, and both ends of the resistancegene are provided with DifR and DifL sequences; also disclosed is an autonomously illuminated Acinetobacter baumannii without a resistance marker, the gene of Acinetobacter baumannii containing a genecapable of expressing autonomous light-emitting protein, and no resistance screening gene is in the genome of Acinetobacter baumannii. In the present invention, the autonomously illuminated Acinetobacter baumannii, which is constructed by the transfer plasmid pUC18T-mini-Tn7T-lux-Ab-dif-Apr and the auxiliary plasmid pTNS3, can emit light without adding any substrate; and luminescent colonies canbe seen with the naked eye in a dark environment.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an autonomous luminescent Acinetobacter baumannii and its construction method and application. Background technique [0002] Acinetobacter baumannii (Acinetobacter baumannii) is a non-fermenting gram-negative bacillus, which widely exists in nature and belongs to conditional pathogenic bacteria. This bacterium is an important pathogen of nosocomial infection, mainly causing respiratory tract infection, and can also cause bacteremia, urinary tract infection, secondary meningitis, surgical site infection, ventilator-associated pneumonia, etc. Usually, the drugs that have a strong effect on Acinetobacter baumannii mainly include penicillins against Pseudomonas aeruginosa, third- and fourth-generation cephalosporins (mainly ceftazidime, cefepime, etc.), carbapenems, etc. β-lactam antibiotic compound preparations (cefoperazone / sulbactam, piperacillin / tazobactam, etc.), fl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/66C12N1/21C12Q1/02C12R1/01
CPCY02A50/30
Inventor 张天宇高亚敏王帅郭玲敏卢智黎蔡晓吟方翠婷
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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