Search bioRxivSearch

Biology subjects

Charles, T. C.

Publications and source records attributed to Charles, T. C..

2 recordsLinked to original sources

Designer Sinorhizobium meliloti strains and multi-functional vectors for direct inter-kingdom transfer of high G+C content DNA

Storage and manipulation of large DNA fragments is crucial for synthetic biology applications, yet DNA with high G+C content can be unstable in many host organisms. Here, we report the development of Sinorhizobium meliloti as a new universal host that can store DNA, including high G+C content, and mobilize DNA to Escherichia coli, Saccharomyces cerevisiae, and the eukaryotic microalgae Phaeodactylum tricornutum. We deleted the S. meliloti hsdR restriction-system to enable DNA transformation with up to 1.4 x 105 efficiency. Multi-host and multi-functional shuttle vectors (MHS) were constructed and shown to stably replicate in S. meliloti, E. coli, S. cerevisiae, and P. tricornutum, with a copy-number inducible E. coli origin for isolating plasmid DNA. Crucially, we demonstrated that S. meliloti can act as a universal conjugative donor for MHS plasmids with a cargo of at least 62 kb of G+C rich DNA derived from Deinococcus radiodurans.

synthetic biology

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

Ionic silver is known to be an effective antimicrobial agent widely used in the cleaning and medical industries, however, there are several concerns regarding the release of silver pollutants into the environment. Presented here are two engineered bacterial biosensors for the detection and quantification of silver. The biosensors contain a silver resistance operon and a GFP gene that is strictly regulated through silver activated regulatory regions that control expression of the sil operons. The two biosensors are responsive to a wide range of silver ion concentrations, and a correlation between silver and GFP signal is seen at select concentration ranges. The biosensors were shown to detect silver ions released from silver nanoparticles, and have the potential to become a method for monitoring ion release rates of different nanoparticles. Interestingly, the close homology of the silver resistance and copper resistance genes allowed for the biosensor to also be responsive to copper ions, implying that copper ions activate silver resistance. Further development of this biosensor could lead to commercial applications for environmental monitoring.\n\nImportanceIonic silver is known to have many harmful environmental effects. Silver pollutants have been found in various environmental settings such as natural waterways and tailings from mining operations, raising concern. In addition, persistent exposure to silver in medical and environmental settings has led to the development of silver resistant bacteria, many of which are also resistant to a wide range of antibiotics. Some of these have the potential to develop into human pathogens. It then becomes important to have standardized methods for detecting and monitoring silver concentrations in various environments so that appropriate measures can be taken to prevent further silver ion release. This research shows that bacterial biosensors engineered to detect and quantify silver ions can be developed as effective alternatives to traditional analytical techniques. Further development of such biosensors could result in a commercial system for short and long term environmental monitoring, which is important as products containing silver and other heavy metals become increasingly popular.

synthetic biology