Radiation Biosensor Based on Promoter of SOS Reaction and Oxidative Stress Reaction

doi:10.13523/j.cb.1903028

China Biotechnology

2020, Vol. 40

Issue (7): 30-40 DOI: 10.13523/j.cb.1903028

Radiation Biosensor Based on Promoter of SOS Reaction and Oxidative Stress Reaction

HAO Xiao-ting,LIU Jun-jie,DENG Yu-lin,ZHANG Yong-qian(

)

Beijing Key Laboratory for Separation and Analysis in Biomedicine and Pharmaceuticals, Life of Science, Beijing Institute of Technology, Beijing 100081, China

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Abstract

The radiation environment exists everywhere in the living space, the results of animal experiments in recent years show that the harm of electromagnetic radiation are mainly concentrated on nervous system toxicity, inducing tumors (especially brain tumors, leukemia) and reproductive system damage. Radiation exposure has the characteristics of wide-area, concealed and cumulative effect. It acts on living organisms, causing a large amount of reactive oxygen species (ROS) in cells. By-products of normal aerobic physiological metabolism in the cells can also generate free radicals, thereby causing damage to the body. In other words, radiation can not only directly act on biological molecules and cause damage to the body, but also indirectly act on the body by acting on biological water and so on to produce free radicals. In order to detect the magnitude of radiation toxicity quickly and easily, there been established radiation biosensors. Engineered bacteria sensors carrying SoxR, RecA, Cda and SulA four promoters and enhanced green fluorescent protein (EGFP) fusion gene related to SOS reaction and oxidative stress reaction were constructed, that is, the promoter-reporter system. First, the four biosensors were treated with chemical damage agents, they all expressed a large amount of green fluorescent protein after stimulation, and then γ-ray irradiation was performed. According to the treatment, the sensor with the highest sensitivity was the RecA promoter engineering bacteria sensor under radiation. The promoter-reporter fusion gene obtained by PCR and overlap PCR, and inserted into the vector PUC19, then transformed into E. coli DH5α. After double-enzyme digestion and sequencing verification, the successful engineered bacteria sensors were disposed of chemical oxidant and physical radiation. The results showed that the four engineering bacteria sensors successfully responded to the oxidant hydrogen peroxide and physical radiation, and the green fluorescence intensity gradually increased with the increase of physical radiation dose (0-30Gy). Among them, the green fluorescence of RecA engineered bacteria sensor was the most obvious after stimulation compared with the other sensors. The use of synthetic biology methods to establish physical radiation sensors based on biological effects successfully, with simple preparation, visibility of results, meeting fast, wide range, online monitoring needs, solving the problem of excessive background value in chemical sensors. It has a good application prospect in the measurement of radiation, radiation on the ground and even in the space.

Key words： Radiation Biosensor SOS reaction Oxidative stress

Received: 12 March 2019 Published: 13 August 2020

ZTFLH:

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Corresponding Authors: Yong-qian ZHANG E-mail: zyq@bit.edu.cn

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	Xiao-ting HAO
	Jun-jie LIU
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	Yong-qian ZHANG

Cite this article:

HAO Xiao-ting,LIU Jun-jie,DENG Yu-lin,ZHANG Yong-qian. Radiation Biosensor Based on Promoter of SOS Reaction and Oxidative Stress Reaction. China Biotechnology, 2020, 40(7): 30-40.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.1903028 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I7/30

Fig.1 Genetic circuit map(the promoter-reporter system)

Table 1 Introduction of genetic components

Table 2 Promoter and reporter gene PCR primers

Fig.2 Schematic diagram of overlap PCR Complementary fragments shall be with ten base pairs considered in intermediate primer design; Up primer of overlap PCR: the upstream primer of the promoter; Down primer of overlap PCR: The downstream primer of the reporter gene

Table 3 Primer of overlap PCR

Fig.3 PCR amplification of the target sequence (a)PCR results of promoters and reporter, RecA(lane1), Cda(lane2), SulA(lane3 and 4), SoxR(lane5), EGFP(lane6) (b) Second PCR results of promoter Cda(lane1) and promoter- reporter fusion gene(lane2 and 3, bands of high molecular weight of DNA) by Overlap PCR (c) Overlap PCR results of SoxR-EGFP fusion gene(lane1) and pSB1C3 expression vector was successfully constructed(lane2)

Fig.4 Growth of transformed plate and fluorescence observation under microscope (a),(b) Engineering bacterium transformed growth plate (c) Fluorescence expression in logarithmic growth phase (d) Fluorescence expression at plateau stage

Fig.5 Validation of plasmid by double enzyme digestion (a)The results of double digestion of expression vector pUC19 by EcoRⅠ and HindⅢ(lane4) (b)The results of double digestion of expression vector pSB1C3 by EcoRⅠ and SpeⅠ(lane4 and 5). Control plasmids were primordial plasmids without the insertion of a target fragment(lane3)

Fig.6 Determination of DH5α Escherichia coli growth curve

Fig.7 Stimulation results by chemical damage reagents (a) Fluorescence of RecA engineering bacteria changes with different MMC concentrations (b) Fluorescence of RecA engineering bacteria changes with different H₂O₂ concentrations (c) Fluorescence of SulA engineering bacteria changes with different MMC concentrations (d) Correlation between fluorescence and H₂O₂ concentration of SoxR engineering bacteria at 3h

Fig.8 Physical radiation treatment engineering bacteria sensor (a) The fluorescence of Cda engineering bacteria changes with radiation dose varies after irradiation (b) The fluorescence of SoxR engineering bacteria changes with radiation dose varies after irradiation (c) The fluorescence of RecA engineering bacteria changes with cultured duration varies after irradiation (d) Correlation between fluorescence and radiation dose of RecA engineering bacteria at 3h after irradiation


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