Project description:Bacteria are an enormous and largely untapped reservoir of biosensing proteins. We describe an approach to identify and isolate bacterial allosteric transcription factors (aTFs) that recognize a target analyte and to develop these TFs into biosensor devices. Our approach utilizes a combination of genomic screens and functional assays to identify and isolate biosensing TFs, and a quantum-dot Förster Resonance Energy Transfer (FRET) strategy for transducing analyte recognition into real-time quantitative measurements. We use this approach to identify a progesterone-sensing bacterial aTF and to develop this TF into an optical sensor for progesterone. The sensor detects progesterone in artificial urine with sufficient sensitivity and specificity for clinical use, while being compatible with an inexpensive and portable electronic reader for point-of-care applications. Our results provide proof-of-concept for a paradigm of microbially-derived biosensors adaptable to inexpensive, real-time sensor devices.
Project description:In this work, carbon dots (CDs) were synthesized by a one-step hydrothermal method using citric acid and ethylene diamine, and covalently functionalized with antibodies for the sensing of progesterone hormone. The structural and morphological analysis reveals that the synthesized CDs are of average size (diameter 8-10 nm) and the surface functionalities are confirmed by XPS, XRD and FT-IR. Further graphene oxide (GO) is used as a quencher due to the fluorescence resonance energy transfer (FRET) mechanism, whereas the presence of the analyte progesterone turns on the fluorescence because of displacement of GO from the surface of CDs effectively inhibiting FRET efficiency due to the increased distance between donor and acceptor moieties. The linear curve is obtained with different progesterone concentrations with 13.8 nM detection limits (R2 = 0.974). The proposed optical method demonstrated high selectivity performance in the presence of structurally resembling interfering compounds. The PL intensity increased linearly with the increased progesterone concentration range (10-900 nM) under the optimal experimental parameters. The developed level-free immunosensor has emerged as a potential platform for simplified progesterone analysis due to the high selectivity performance and good recovery in different samples of spiked water.
Project description:Progesterone receptor (PR)-interacting compounds in the environment are associated with serious health hazards. However, methods for their detection in environmental samples are cumbersome. We report a sensitive activity-based biosensor for rapid and reliable screening of progesterone receptor (PR)-interacting endocrine disrupting chemicals (EDCs). The biosensor is a cell line which expresses nuclear mCherry-NF1 and a green fluorescent protein (GFP)-tagged chimera of glucocorticoid receptor (GR) N terminus fused to the ligand binding domain (LBD) of PR (GFP-GR-PR). As this LBD is shared by the PRA and PRB, the biosensor reports on the activation of both PR isoforms. This GFP-GR-PR chimera is cytoplasmic in the absence of hormone and translocates rapidly to the nucleus in response to PR agonists or antagonists in concentration- and time-dependent manner. In live cells, presence of nuclear NF1 label eliminates cell fixation and nuclear staining resulting in efficient screening. The assay can be used in screens for novel PR ligands and PR-interacting contaminants in environmental samples. A limited screen of river water samples indicated a widespread, low-level contamination with PR-interacting contaminants in all tested samples.
Project description:Biocatalytic cyclization is highly desirable for efficient synthesis of biologically derived chemical substances, such as the commodity chemicals ε-caprolactam and δ-valerolactam. To identify biocatalysts in lactam biosynthesis, we develop a caprolactam-detecting genetic enzyme screening system (CL-GESS). The Alcaligenes faecalis regulatory protein NitR is adopted for the highly specific detection of lactam compounds against lactam biosynthetic intermediates. We further systematically optimize the genetic components of the CL-GESS to enhance sensitivity, achieving 10-fold improvement. Using this highly sensitive GESS, we screen marine metagenomes and find an enzyme that cyclizes ω-amino fatty acids to lactam. Moreover, we determine the X-ray crystal structure and catalytic residues based on mutational analysis of the cyclase. The cyclase is also used as a helper enzyme to sense intracellular ω-amino fatty acids. We expect this simple and accurate biosensor to have wide-ranging applications in rapid screening of new lactam-synthesizing enzymes and metabolic engineering for lactam bio-production.
