Development of a GFP Fluorescent Bacterial Biosensor for the Detection and Quantification of Silver and Copper Ions

Abstract

18Ionic silver is known to be an effective antimicrobial agent widely used in the cleaning 19 and medical industries, however, there are several concerns regarding the release of silver 20 pollutants into the environment. Presented here are two engineered bacterial biosensors for the 21 detection and quantification of silver. The biosensors contain a silver resistance operon and a 22 GFP gene that is strictly regulated through silver activated regulatory regions that control 23 expression of the sil operons. The two biosensors are responsive to a wide range of silver ion 24 concentrations, and a correlation between silver and GFP signal is seen at select concentration 25 ranges. The biosensors were shown to detect silver ions released from silver nanoparticles, and 26 have the potential to become a method for monitoring ion release rates of different nanoparticles. 27Interestingly, the close homology of the silver resistance and copper resistance genes allowed for 28 the biosensor to also be responsive to copper ions, implying that copper ions activate silver 29 resistance. Further development of this biosensor could lead to commercial applications for 30 environmental monitoring. 31 Importance 32Ionic silver is known to have many harmful environmental effects. Silver pollutants have been 33 found in various environmental settings such as natural waterways and tailings from mining 34 operations, raising concern. In addition, persistent exposure to silver in medical and 35 environmental settings has led to the development of silver resistant bacteria, many of which are 36 also resistant to a wide range of antibiotics. Some of these have the potential to develop into 37 human pathogens. It then becomes important to have standardized methods for detecting and 38 3 monitoring silver concentrations in various environments so that appropriate measures can be 39 taken to prevent further silver ion release. This research shows that bacterial biosensors 40 engineered to detect and quantify silver ions can be developed as effective alternatives to 41 traditional analytical techniques. Further development of such biosensors could result in a 42 commercial system for short and long term environmental monitoring, which is important as 43 products containing silver and other heavy metals become increasingly popular. 44 131 media (LB and modified LB) is expected since sodium chloride is known to form a AgCl 132 precipitate which would decrease the availability of free silver ions. A lower amount of available 133 silver ions in the media could assist the biosensor cells survival at higher absolute silver 134 concentrations, resulting in a greater detection range, as was observed. 135 Correlation between GFP signal and silver ion concentration is detected with 136 the biosensor variants 137 A correlation between GFP signal and silver ion concentration is detected with the biosensor 138 variants. The correlation range varies depending on the media, and in specific segments of the 139 detection range. Both RADEK.1 and RADEK.2...