Development and Amplification of a Zinc Biosensor in a Cell-Free Transcription-Translation System
Biosensors are important tools for detecting substances such as pathogens and heavy metals using biologically derived molecules. While living cells are often used, cell-free systems like the cell-free transcription-translation (TXTL) system offer advantages for biosensor prototyping due to high protein synthesis rates. However, current biosensors struggle with detecting trace compounds. To address this limitation, we attempted to create a biosensor sensitive in a range of zinc concentrations as low as 1 µM and up to the EPA’s recommended limit of 5 mg/L in drinking water. Zinc-dependent transcriptional activator ZntR from E. coli was incorporated into both amplified and non-amplified gene circuits using the zntA operon and T7 transcriptional cascade for amplification. The performance of each circuit was assessed by measuring the expression of fluorescent reporter protein deGFP. The linear DNA constructs exhibited notably low expression for the TXTL system, so none of the gene circuits produced a positive signal above background noise at typical DNA construct concentrations. However, at higher DNA concentration, the amplification cascade improved signal output, yielding a positive signal in a majority of the trials. Optimization of DNA concentrations in the amplified circuit allowed for detection of zinc signals as low as 1 µM, though consistency varied. Further improvements in relative DNA concentration and DNA stock concentration could enhance sensitivity and robustness. The results suggest that exploring multi-amplified circuits in TXTL could have potential for developing cost-effective and sensitive biosensors.