Having evolved longer than any other group of organisms, bacteria have been perfecting mechanisms of sensing their environments for billions of years. Recent advances in the field of biosensing have enabled miniaturization of existing biosensors, but the number of characterized biosensor elements remains limited. Sensing parts derived from bacteria make promising targets for integration into biosensor devices and could expand the repertoire of easily detectable compounds. Here, RNA sequencing screening was used to identify a novel TetR family 3-ketosteroid inducible transcription factor called SRTF1 (Steroid Responsive Transcription Factor 1) from the Gram-positive soil bacterium Pimelobacter simplex. This is the first transcription factor confirmed to be inducible by these steroids in-vitro. A potential regulon was identified using in-vitro chromatin immunoprecipitation sequencing, revealing a conserved 20 base pair long palindrome within several promoters in a region of the P. simplex genome highly differentially expressed on exposure to steroids. Biolayer interferometry and intrinsic tryptophan fluorescence were used to quantitatively characterize the transcription factor’s DNA binding strength and hormone induction specificity. Circular dichroism study of SRTF1 revealed it is primarily alpha-helical like almost all other TetR family transcription factors. Bases in a core GCCG repeat within the palindrome were identified as important for SRTF1 binding and a disrupted palindrome was generated that greatly increased a quantum-dot based SRTF1 biosensor’s sensitivity for progesterone. As the transcription factor displays cross reactivity to cortisol and aldosterone that is undesirable in a diagnostic device, a fluorescent reporter assay based on the transcription factor was constructed. This reporter assay showed a similar steroid induction profiles as purified SRTF1, and was used to select for mutant SRTF1 variants generated using error-prone polymerase chain reaction with reduced inducibility by these 11-hydroxy steroids. This pipeline for identifying novel transcription factors, characterizing their DNA and ligand binding profiles, and altering them through mutation of DNA and protein sequences could allow for an expanded number of biosensor parts.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/41109 |
| Date | 29 May 2020 |
| Creators | Baer, R. Cooper |
| Contributors | Galagan, James, Fearns, Rachel |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
| Rights | Attribution-NonCommercial-ShareAlike 4.0 International, http://creativecommons.org/licenses/by-nc-sa/4.0/ |
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