Engineering and application of two component system biosensors

[摘要] Bacterial sensors are a critical component of the sense-compute-respond framework used by synthetic biologists to engineer bacteria, and better tools are needed to implement and optimize these sensors. Two-component systems (TCSs) are the largest family of signal transduction pathways in biology, and a major source of sensors for biotechnology. However, the input concentrations to which these biosensors respond are often mismatched with application requirements. Here, we utilize a mathematical model to show that TCS detection thresholds increase with the strength of the phosphatase activity of the sensor histidine kinase. We experimentally validate this result by using known phosphatase-altering mutations to rationally tune the detection thresholds of engineered Bacillus subtilis nitrate and E. coli aspartate sensors up to two orders of magnitude. We go on to demonstrate that a widely-conserved residue previously shown to impact phosphatase activity can be mutated to apply our ;;TCS tuning” method to sensors for which no well-characterized mutations exist.In the second portion of this work, we engineer a soil bacterium to measure levels of fertilizer in soil. We utilize synthetic promoters and engineered transcription factors to transfer a nitrate sensor from the model organism E. coli to the soil bacterium B. subtilis to create a high performing nitrate sensor. We next develop a protocol to use this strain as a biosensor of levels of nitrate in soil, and use this capability to report levels of soil fertilization. Lastly, the detection threshold tuning technique developed in the first half of this work is utilized to expand the range of fertilizer concentrations that can be monitored. This work will enable the engineering of tailor-made biosensors for diverse synthetic biology applications.

[发布机构] Rice University

[效力级别] Biology