(556c) Fluorescent Microbial Sensors That Discriminate between Different Radionuclides.

Proper detection and monitoring of nuclear fuel cycle, enrichment, and weapon development activities is critical for supporting warfighter preparation in chemical, biological, radiological, nuclear, and explosives (CBRNE) operations, clandestine activities, and nuclear compliance. Field applications of conventional radiation monitoring and detection systems are restricted by several disadvantages: mandatory proximal placement near a radioactive source for detection, being easily identifiable and avoided, and capacity to only report radioactivity at a specific moment in time. The ability to detect trace amounts of ionizing radiation is a paramount concern for CBRNE operations and observation of clandestine activities in the event of source relocation and contamination. Microbes respond to environmental stress such as low-dose ionizing radiation with morphological and behavioral changes. Responses are known to persist after the stressor is removed. We hypothesize that transcriptional changes can monitor these stress responses and be harnessed to develop biosensors capable of discerning radionuclide type and monitor and report on nuclear fuel cycle, enrichment, and weapon development activities in diverse environmental conditions.

Unique responses of environmental microbes to post exposure to nuclear fuel cycle representative radionuclides were identified through mRNA sequencing analysis. Gene expression analysis showed Pseudomonas putida and Escherichia coli responded differently to acute and chronic exposure to plutonium-239, tritium, and iron-55 at a dose rate of 8.7 mGy/d. Highly abundant genes overexpressed by a sole radionuclide were selected for sensor development. Native promoter sequences from candidate genes were inserted into a broad host range plasmid upstream of a fluorescent reporter. Sequence verified in vivo sensors were then transformed into P. putida and subsequently irradiated by each of the three radionuclides for 24 hours before fluorescence was measured. Preliminary testing revealed, when grown in the presence of the inducing radionuclide, expression of GFP increased. Sensors treated with non-inducing radionuclides showed no significant increase in fluorescence. Future sensor development will include engineering circuits of toehold switch sensors permitting detection of time dependent signals for individual radionuclide sources. In addition to developing passive and autonomous monitoring or clandestine activities, the results of this work will expand upon the limited knowledge of low dose radiation effects in microorganisms.