Abstract
Recent advancements in bacterial two-component systems (TCS) have spurred research into TCS-based biosensors, notably for their signal amplification and broad input responsiveness. The CusRS system in Escherichia coli (E. coli), comprising cusS and cusR genes, is a copper-sensing module in E. coli. However, due to insufficient sensing performance, CusRS-based biosensors often cannot meet practical requirements. To address this issue, we made improvements and innovation from several aspects. CusR and CusS expression were adjusted to enhance the Cu(II) biosensor's performance. A copy-number inducible plasmid was used for signal amplification, while removing copper detox genes cueO and cusCFBA improved sensitivity and lowered detection limits. Ultimately, in the optimized biosensor of Cu26, the fold-change (I/I0) increased from 1.5-fold to 18-fold at 1 μM, rising to 100-fold after optimizing the cell culture procedure. The biosensor's high fluorescence enabled rapid, instrument-free detection and an improved analysis strategy reduced the detection limit to 0.01 μM, surpassing traditional methods.