Expression and Applications of HriCFP in E. coli: A Novel Biosensing Fluorescent Protein

摘要

Metalloids and heavy metal contamination in the environment have become a global problem. Therefore, there is a dire need to develop effective and inexpensive approaches that can facilitate efficient monitoring of the hazardous level of these environmental pollutants. Microbial cell-based and fluorescent protein-based biosensors offer relatively convenient and inexpensive tools for the analysis of environmental pollutants as opposed to traditional instrumental approaches. Small size fluorescent proteins can withstand exposure to denaturants, high temperature and a wide pH range. These characteristics, along with their potential of sensing different toxic analytes, makes them a suitable candidate for developing on-site detection biosensors. The current study exploits the biosensing potential of a novel fluorescent protein called HriCFP. HriCFP was expressed in the prokaryotic system (gram-negative E. coli), which showed stable and discreet expression in bacterial cells. Whole-cell biosensors (WCB) were developed by immobilization of HriCFP expressing non-pathogenic E. coli via nitrocellulose membrane, low melting agarose and sodium silicate gel. These immobilized biosensors were tested for their sensitivity of detection for environmental pollutants, i.e., heavy metals (Cu(II), Hg(II), As(III)). These WCBs exhibited profound fluorescent quenching when exposed to a range of heavy metals. These biosensors remained active for 12 days at 4°C, demonstrating their potential for long-term stability and storage. This study implies that HriCFP may have a significant advantage over other larger and multimeric proteins as it has a minimal impact on host strain metabolism and hence, increasing its sustainability for a longer period.