Development and Characterization of Split Methyl Halide Transferase for Biosensor Applications in Soil
Silberg, Jonathan J.; Bennett, George N.
Master of Science
Soil microorganisms contribute to agricultural productivity by forming symbiosis with plant roots and affecting the solubility of soil nutrients. Microbiological activity in soil can be monitored directly by coupling the expression of a methyl halide transferase (MHT) to a conditional reporter. This unique bioreporter can provide information about microbial sensing and behaviors without disrupting soil. However, MHT reporting has only been used to report on slow transcriptional processes, limiting its utility. To overcome this challenge, I have created a split MHT (SMHT) that consists of two MHT fragments that are only functional when reconstituted. This SMHT was rationally designed using family sequence information and shown to exhibit MHT-fragment complementation upon fusion to different pairs of interacting proteins. Furthermore, a rapamycin biosensor was constructed that links the detection of rapamycin, a secondary metabolite synthesized by a soil microorganism, to SMHT fragment complementation. The SMHT should be generally useful for reporting on conditional protein-protein interactions in hard-to-image soils and sediments.
Methyl halide transferase; Multiple sequence alignment; Protein fragment complementation assay; Gas bioreporter; Protein engineering