Developing a Point-of-Need Cell-Free Hexavalent Chromium Biosensor

Hexavalent Chromium contaminants have large impacts on ecological and agricultural environments across the world. However, sensitive, and specific detection of Hexavalent Chromium is currently cost- and skill- prohibitive, impeding environmental monitoring and remediation efforts. Utilizing cell-free protein expression systems to develop a hexavalent chromium biosensor could allow broader utilization of sensing technology, especially in point-of-need sensing applications. Cell-free systems extract the primary biochemical components from cells to create a microenvironment for more controlled protein expression. Cell-free systems are uniquely suited for point-of-need sensing as they can be freeze dried, allowing for simple transportation, without the costs of maintaining cell-growth or transporting heavy fragile equipment.

Here, we report the design of a cell-free, hexavalent chromium biosensor. We first constructed a single reporter plasmid coding for green fluorescent protein (GFP) expression under the control of a hexavalent chromium responsive promoter (Pchr) regulated by its cognate transcription factor, ChrB. In the presence of hexavalent chromium, ChrB preferentially binds to hexavalent chromium, allowing for promoter de-repression and GFP production. We first validated ChrB repression of GFP production from its cognate promoter Pchr. We then found that constant expression of ChrB repressor in cell-free reactions significantly impacted sensitive de-repression of hexavalent chromate. However, our efforts to purify the ChrB transcription factor for direct reaction supplementation have resulted in large quantities of misfolded proteins in the insoluble fraction. Going forward, we are exploring different protein purification methods to extract soluble ChrB transcription factors for hexavalent chromium detection.