|Ph.D Student||Barger Natalia|
|Subject||A Bioluminescence-Sensing System for Synthetic Metabolic|
Computation in Whole-cell Bacterial Biosensors
|Department||Department of Biomedical Engineering||Supervisor||ASSOCIATE PROF. Ramez Daniel|
|Full Thesis text|
Whole-cell bacterial biosensors often consist of genetically engineered bacteria, integrated with an electronic detector. Bacterial cells are usually genetically designed to include biological sensory components fused with a reporter. A variety of detection methods have been developed for biological response detection. These methods include colorimetric, fluorescent, bioluminescent, electrochemical detection. Bioluminescence is visible light produced and emitted by living cells using various biological systems (e.g. luxCDABE cassette). Today, this phenomenon is widely exploited in biological research, biotechnology, and medical applications as a quantitative technique for the detection of biological signals.
While significant progress has been made in the development of whole-cell bacterial biosensors, only a few devices are commercialized or are used as a characterization tool in the field. Several reasons make whole-cell biosensors challenging to function properly in real-world samples. Many of these reasons are belong to the physiology and genetic features of the genetically engineered bacteria, such as the reliability and repeatability of measured signals and lack of selectivity over analytes. Hence, with the given significant advancements in genome engineering and DNA assembly techniques, transferring engineering design principles into the natural biological design to achieve the desired function in living cells termed synthetic biology, become a powerful tool to overcome technological challenges in improving the performance of bacterial biosensors. Synthetic biology is a multidisciplinary field that combines the knowledge and techniques of biology, chemistry, computer science, and engineering. Synthetic bio-devices are implemented in diverse applications, including disease therapy, environmental remediation, and biosynthesis of commodity chemicals.
In this work, we re-engineered the complex genetic structure of the luxCDABE cassette to build a biological unit that can detect multi-inputs, process cellular information, and report the computation results. We first split the luxCDABE operon into several parts to create a genetic circuit that can compute a soft minimum in living cells. Then, we used the new design to implement an AND logic function with better performance as compared to AND logic functions based on protein-protein interactions. Furthermore, by controlling the reverse reaction of the luxCDABE cassette independently from the forward reaction, we built a comparator with a programmable detection threshold. Then, we applied the redesigned cassette to build an incoherent feedforward loop that reduced the unwanted crosstalk between stress-responsive promoters (recA, katG).
Based on our new system, we designed an ultrasensitive whole-cell biosensor interfaced with an optoelectronic measurement module for the detection of heme, a red-blood-cell (RBC) component, in urine. The bacterial biosensor includes a synthetic gene circuit that comprises a heme-sensitive system and a re-designed form of bacterial luciferase. We constructed the light detection setup based on commercial optical measurement single-avalanche photodiode (SPAD), which is capable of detecting low-level light. Photons emitted from bacteria detected on the sensor generate a digital voltage pulse, which is transferred through the Arduino USB port to the PC for display. The whole-cell biosensor performance was tested in a synthetic urine diluent, to demonstrate our system as a low-cost, portable, and easy-to-use biosensor for sensitive detection of blood levels in urine.