Evaluation Of The Effectiveness And Safety Of The Thermo-Treatment Process To Dispose Of Recombinant DNA Waste

Along with the development of modern biological technology, its adversity has drawn increasing attention. The discharge of waste-recombination gene from biological laboratory is one of the pathways for resulting in "gene pollution". As a result, the discharge of waste-recombination gene from biological laboratory should follow some pretreatment steps. Heating at 100°C is the main method for treating waste-recombination gene in the biological laboratory in China. In this study, plasmid pET-28b was used as example to investigate the decay/denaturation regulation of DNA during the thermo-treatment process and the degradation regulation of thermo-treated DNA in a simulated aquatic environment using quantitative PCR plus plasmid transformation and electrophsis technology. The results of this study will not only help us to evaluate the effectiveness and safety of the thermo-treatment process to dispose of recombinant DNA waste, but also provide the establishment of framework for ecological risk assessment of recombinant DNA waste with reference values and technical support in the future.The experimental result shows that with the heating temperature rising and the effect of fluid shear stress, accordingly, the melting and fracture rate of the plasmid pET-28b will increase during the thermo treatment, and the half-life of pET-28b was about 2.7～4min during the thermo-treatment process. But after treating 30 min there still are 3%～5% of pET-28b plasmids with transforming activity, which means that the recombination plasmid DNA cannot be degraded or destroyed effectively by thermo-treatment.The thermo-treated DNA decays fast at low pH condition. NaCl, bovine serum albumin (BSA) and EDTA, they all can protect DNA from being degraded, especially EDTA. The wastewater from biological laboratory usually contains the above-mentioned organic and inorganic materials. So that the half-live of plasmid DNA during thermo-treatment could be longer than 2.7～4min in practical situations, suggesting we must pay attention to this possibility. After being renaturated, denaturized DNA will obtain its transformation activity to a certain degree, and high temperature is helpful for the denaturation of thermo-treated plasmid DNA. As a result, there is certain ecological risk, once these gene fragments were released into environment.The denaturized DNA cannot be degraded effectively in a short period after being put into the simulated aquatic environment. Different pH conditions will change the degradation rate of thermo treated plasmid pET-28b in simulated aquatic environment. The intact plasmid pET-28b cannot be detected after 55min under pH 5, 6 and 9. The plasmid pET-28b cannot be detected after 1.6h under pH 7 and 8. The thermo-treated DNA decays slow at high ionic strength condition. When the ionic strength reaches to 0.5% in simulated aquatic environment, the intact plasmid pET-28b can be detected after 2.2h by real-time PCR.As mentioned above, the common method (thermo treatment) for treating recombinant DNA waste is not effective enough, which means once these gene fragments were released into environment, un-decayed recombinant plasmid during the thermo-treatment process were not degraded completely in a short period, they may have enough time to re-nature and transform, thus resulting in gene diffusion. Therefore, there presented certain Ecological Risk, once these gene fragments were released into environment.Work is under way to validate the renaturation and horizontal gene transfer of thermo-treated recombinant DNA in the aquatic environment.