Nano-enabled synthetic biology

Biological systems display a functional diversity, density and efficiency that make them a paradigm for synthetic systems. In natural systems, the cell is the elemental unit and efforts to emulate cells, their components, and organization have relied primarily on the use of bioorganic materials. Impressive advances have been made towards assembling simple genetic systems within cellular scale containers. These biological system assembly efforts are particularly instructive, as we gain command over the directed synthesis and assembly of synthetic nanoscale structures. Advances in nanoscale fabrication, assembly, and characterization are providing the tools and materials for characterizing and emulating the smallest scale features of biology. Further, they are revealing unique physical properties that emerge at the nanoscale. Realizing these properties in useful ways will require attention to the assembly of these nanoscale components. Attention to systems biology principles can lead to the practical development of nanoscale technologies with possible realization of synthetic systems with cell-like complexity. In turn, useful tools for interpreting biological complexity and for interfacing to biological processes will result

Monitoring Genetic and Metabolic Potential for In-Situ Bioremediation: Mass Spectrometry

A number of DOE sites are contaminated with dense non-aqueous phase liquids (DNAPLs) such as carbon tetrachloride and trichloroethylene. At many of these sites, microbial bioremediation is an attractive strategy for cleanup, since it has the potential to degrade DNAPLs in situ. A rapid screening method to determine the broad range potential of a site's microbial population for contaminant degradation would greatly facilitate assessment for in situ bioremediation, as well as for monitoring ongoing bioremediation treatment. Current laboratory-based treatability methods are cumbersome and expensive. In this project, we are developing methods based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for rapid and accurate detection of polymerase chain reaction (PCR) products from microbial genes involved in biodegradation of pollutants. PCR primers are being developed to amplify DNA sequences that are amenable to MALDI-MS detection. This work will lay the foundation for development of a field-portable MS-based technique for rapid on site assessment and monitoring of bioremediation processes

A carotenoid-deficient mutant in Pantoea sp. YR343, a bacteria isolated from the Rhizosphere of Populus deltoides, is defective in root colonization

The complex interactions between plants and their microbiome can have a profound effect on the health and productivity of the plant host. A better understanding of the microbial mechanisms that promote plant health and stress tolerance will enable strategies for improving the productivity of economically important plants. Pantoea sp. YR343 is a motile, rod-shaped bacterium isolated from the roots of Populus deltoides that possesses the ability to solubilize phosphate and produce the phytohormone indole-3-acetic acid (IAA). Pantoea sp. YR343 readily colonizes plant roots and does not appear to be pathogenic when applied to the leaves or roots of selected plant hosts. To better understand the molecular mechanisms involved in plant association and rhizosphere survival by Pantoea sp. YR343, we constructed a mutant in which the crtB gene encoding phytoene synthase was deleted. Phytoene synthase is responsible for converting geranylgeranyl pyrophosphate to phytoene, an important precursor to the production of carotenoids. As predicted, the ΔcrtB mutant is defective in carotenoid production, and shows increased sensitivity to oxidative stress. Moreover, we find that the ΔcrtB mutant is impaired in biofilm formation and production of IAA. Finally we demonstrate that the ΔcrtB mutant shows reduced colonization of plant roots. Taken together, these data suggest that carotenoids are important for plant association and/or rhizosphere survival in Pantoea sp. YR343.Work at the University of Notre Dame was supported by DOE grant SC0006642 (RM) and by a subcontract from Oak Ridge National Laboratory (SP).http://www.frontiersin.orgam2016Microbiology and Plant Patholog

Thrombin-Mediated Transcriptional Regulation Using DNA Aptamers in DNA-Based Cell-Free Protein Synthesis

Realizing the potential of cell-free systems will require development of ligand-sensitive gene promoters that control gene expression in response to a ligand of interest. Here, we describe an approach to designing ligand-sensitive transcriptional control in cell-free systems that is based on the combination of a DNA aptamer that binds thrombin and the T7 bacteriophage promoter. Placement of the aptamer near the T7 promoter, and using a primarily single-stranded template, results in up to a 6-fold change in gene expression in a ligand concentration-dependent manner. We further demonstrate that the sensitivity to thrombin concentration and the fold change in expression can be tuned by altering the position of the aptamer. The results described here pave the way for the use of DNA aptamers to achieve modular regulation of transcription in response to a wide variety of ligands in cell-free systems