ENGINEERED MICROOGRANISMS FOR IN SITU REMEDIATION

该项目的目标是优化遗传稳定性，并控制生物修复应用的转基因微生物的生存。 使用转基因微生物进行生物补救的一个主要限制是，在补救过程中无法充分控制生物体的突变率和存活率。这引起了对遗传物质环境释放的关注。为了解决这一障碍，需要严格控制的系统。 这种细菌名为恶臭假单胞菌，将用一种基于链霉亲和素的自杀系统进行基因改造以控制存活，再加上一种分解代谢质粒以降解芳烃。我们的初步数据表明，该系统可以实现所需的目标，遗传稳定性和控制的生存在受控的实验室实验。该项目的第一阶段将集中于将质粒携带的控制系统重新定位到染色体上，以进一步提高基因工程生物的稳定性。第二阶段将解决额外的增强这一系统，包括纳入一个双自杀系统，以进一步控制稳定性和生存，增加金属螯合能力的有机体，并通过链霉亲和素示踪剂快速监测环境中的有机体的方法。拟议的商业应用：基因工程微生物具有解决环境中危险废物污染问题的巨大潜力。然而，一个主要的需求是控制这些生物的生存，以减轻对它们不受控制的释放的担忧。该项目将通过使用调控系统来控制与分解代谢活动相关的细胞存活和简易监测能力，以满足对这些生物体进行严格控制的需要。

The goal of this project is to optimize genetic stability, and to control the survival, of genetically modified microorganisms for bioremediation applications. A major limitation with the use of genetically modified microorganisms for bioremediation is the inability to adequately control mutation and survival rates of the organisms during remediation. This leads to concerns with the environmental release of genetic materials. To address this barrier, tightly controlled systems are needed. The bacterium, Pseudomonas putida, will be genetically modified with a streptavidin- based suicide system to control survival, coupled to a catabolic plasmid for aromatic hydrocarbon degradation. Our preliminary data demonstrate that this system can achieve the required goals of genetic stability and control of survival in controlled laboratory experiments. Phase I of this project will focus on relocating the plasmid-borne control system onto the chromosome to further enhance stability of the genetically engineered organism. Phase II will address additional enhancements to this system, including the incorporation of a dual suicide system for further control of stability and survival, the addition of metal sequestration capabilities in the organism, and methods to rapidly monitor the organism in the environment through the streptavidin tracer. PROPOSED COMMERCIAL APPLICATIONS: Genetically engineered microorganisms have strong potential to address hazardous waste contamination problems in the environment. However, a major need is to control the survival of these organisms to mitigate concerns with their uncontrolled release. The project will address the need for tight control of these organisms through the use of a regulatory system to control cell survival tied to catabolic activity and facile monitoring capability.