Detection and Damage-analysis of Bio-Particles and for Safety-Evaluation of Plasma-treated water using DNA-manipulation
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Detection and Damage-analysis of Bio-Particles and for Safety-Evaluation of Plasmatreated water using DNA-manipulation Akira Mizuno, Hachiro Yasuda, Hirofumi Kurita and Kazunori Takashima Dept. Environmental and Life Sciences, Toyohashi University of Technology, Hibarigaoka 1-1, Tenpaku-cho, Toyohashi, 441-8580 Japan ABSTRACT Effect of non-thermal plasma (NTP) on bio-particles has been studied using Bacillus subtilis (B. subtilis), Escherichia coli (E. coli) and bacteriophages. NTP has been used, and states of different biological components were monitored during the course of the exposure. Analysis of green fluorescent protein (GFP), introduced into E.coli cells proved that NTP causes a prominent protein damages without cutting peptide bonds. We have developed a biological assay which evaluates in vivo DNA damage of the bacteriophages. Different doses of the plasma were applied to wet state of λ phages. From the plasma-exposed λ phages, DNA was purified and subjected to in vitro DNA packaging reactions. The re-packaged phages consist of the DNA from discharged phages and brand-new coat proteins. Survival curves of the re-packaged phages showed extremely large D value (D = 25 s) compared to the previous D value (D = 3 s) from the discharged phages. The results indicate that DNA damage hardly contributed to the inactivation, and the damage in coat proteins is responsible for inactivation of the phages. We also report a single-molecule-based analysis of strand breakages on large DNA molecules induced by the plasma exposure. Single-molecule observation of DNA that involved molecular combing was used to measure the length of individual DNA molecules. The measured DNA length showed that plasma exposure caused a marked change in length of DNA molecules. The rate of plasmainduced strand breakage on large random-coiled DNA molecules was determined using a simple mathematical model. The measured rate shows good relation with the plasma exposure time, and could be used for safety evaluation of the plasma treated water. INTRODUCTION Non-thermal atmospheric pressure plasma is effective for destruction of bio-particles (BPs) such as microbes and viruses [1-4] at low gas temperature without using bactericides. Though the study of plasma decontamination is expanding [5-9], the mechanism of inactivation of BPs are still to be studied. We have evaluated the damages to Escherichia coli (E. coli), and bacteriophages exposed to non-thermal plasma (NTP) [10]. Green fluorescent protein (GFP) in E. coli can be damaged quickly by the exposure to the DBD (dielectric barrier discharge), and the intensity of GFP and survivability shows similar trend against the exposure time. Turbidity of Bacillus subtilis (B. subtilis) solution decreases quickly with the exposure to the DBD. We have also developed a biological assay to evaluate gene-specific damage of bacteriophages treated with DBD and corona discharge. It was found that λ phage DNA was strong against the plasma application in comparison with coat proteins. Phages having single stranded DNA
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