(596d) Enriching the Cell-Free System Based Biosensor Toolbox to Increase Dynamic Range While Retaining Accuracy

Since the cell-free system (CFS) has emerged as an alternative protein synthesis platform to overcome the limitation of the in vivo system, in the synthetic biology era, the CFS receives a spotlight for its inherent versatile characteristics that make the scientists and engineers can repurpose the system to achieve their own research goals outside the cells. With the extraordinary flexibility of the CFS, the system has been expanded to developing biosensors to detect diverse targets from toxic molecules in drinking water to cancer biomarkers in liquid biopsies to viruses. However, these target molecules exist in minuscule amounts in these environmental samples, requiring biosensors to have a low limit of detection, with considerable sacrificing sensing accuracy to reach these low levels. Here, we present ways we have enhanced the output signal of cell-free biosensors while retaining accuracy to achieve low limits of detection. We have improved the output signal by introducing a new fluorescent protein mNeonGreen which shows a significantly brighter output signal compared to commonly used fluorescent proteins such as deGFP, eGFP, and sfGFP. Next, we introduce the use of robust non-native sigma factors, which allow the user more modulation of parts using a cognate promoter compared to a bacteriophage RNAP, and a large reduction in background signal, compared to native E. coli sigma factors. Also, we present optimized conditions to enhance the activity of endogenous RNA polymerase to perform analogously to the robust bacteriophage T7 RNAP. Ongoing work in the laboratory seeks to apply this cell-free biosensor toolbox to sense various target molecules. We anticipate that our early findings on the toolbox will accelerate the development of cell-free based biosensors with increasing sensitivity while retaining sensing accuracy.