Browsing by Subject "Logic gate implementation"

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Open Access
Multiplexed cell-based diagnostic devices for the detection of kidney disease pathological markers
(2020-12) Köse, Sıla
Development of accurate, inexpensive and fast-screening devices has been a big vacancy in the field of medical diagnostics. Close monitoring and diagnosis of various diseases are generally conducted by typical analytical techniques which require intensive efforts and qualified personnel. Therefore, there is an urgent need for more convenient and reliable alternatives that are highly specific, cost-efficient and rapid. One of the approaches in solving this problem is using naturally-derived proteins and nucleic acid molecules that can respond to various types of metabolites. Synthetic biology utilizes these components to assemble biological systems that can probe and control metabolic state of a host. In this context, biosensors can be considered highly specific, costeffective and can be implemented in point-of-care bioanalytical tools for effective healthcare. Here, we have developed biosensors that are responsive to medically relevant biomarkers namely; urea and uric acid. Furthermore, a multi-input version that can sense and respond to both biomarkers simultenously and another multiplexed biosensor that can mimic the AND-logic were developed. Biosensors were designed to respond to their respective target analyte presence through an increase in the fluorescence intensity which can be measured with spectrophotometric devices. To do so, native promoter and transcription factors from different organisms were assembled inside a gene circuit to express a fluorescent protein in the presence of the respective biomarker. For urea biosensor, UreR transcriptional activator and the complete intergenic region inside the urease operon of the organism Proteus mirabilis were utilized. The system was optimized to have the desired dose- response curve using post trancriptional regulation elements and protein engineering. For the uric acid biosensor, transcriptional repressor HucR and the transcription factor binding site inside the uricase operon from the organism Deinococcus radiodurans were assembled in a gene circuit as well as a Uric Acid Transporter (UACT). Using promoter engineering and copy number modifications the response curve of the system was optimized. Next, biological components of the biomarkers were assembled in a multiplexed system to respond both molecules simultenously. Furthermore, a logic gate operating system was developed using promoter engineering that performs AND-logic to be implemented in a medically relevant algorithm A framework for stabilization of biosensors on low-cost portable paper discs through biofilmcellulose interactions, and entrapment of whole-cell biosensors inside biocompatible, biodegradable and mechanically strong gelatin beads was provided for remote detection of the pathological biomarkers. Finally, the robustness of the developed whole cell biosensors was tested with human clinical samples.