Honors College Thesis
 

Development of a Bioluminescent Sensor of Isothiocyanates through Genetic Modification of Pseudomonas fluorescens and Validation on Crude Plant Materials

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  • Isothiocyanates (ITCs) are produced by roots in a variety of agricultural plants, such as those within the Brassicaceae family. ITCs act as fumigants, and are used to control microbial plant pathogens by adding brassica-origin seed meal to soil prior to replanting. However, there is suggestion that the presence of residual ITCs in soil may inhibit seed germination in recently planted crops. These chemical compounds are difficult to detect, where a biological sensor may be a strategic indicator for when the soil is safe to replant in. Some microbes, including Pseudomonas fluorescens, exhibit resistance to ITCs mediated by an induced biochemical pathway. P. fluorescens is a root-colonizing non-pathogenic microbe, making it an ideal and safe biological sensor for agricultural use. To accomplish this, we linked an ITC-responsive promoter (saxA promoter) to the ilux operon, an enhanced version of the lux operon with an additional FMN reductase. The resulting biosensor was responsive in a dose-dependent manner to the aliphatic ITC, sulforaphane, in the concentration range of 1-100 μM. Further tests were conducted using extracted fluid from plant material of broccoli and daikon for the experimental group, while utilizing clover, alfalfa, and mung bean as a negative control group. The biosensor was responsive to the Brassica-related plants, and did not luminesce in response to the negative control extracts. The biosensor was also responsive to the seed meal of Brassica juncea and Sinapis alba, which are common seed meals used in biofumigation. Future experiments will determine the biosensor’s responsiveness to other ITCs and their precursors, glucosinolates, and detection in seed meal amended soils.
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  • Ongoing Research
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  • 2020-06-02 to 2021-07-03

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