Experimental investigations of factors controlling the spread of DNAPL contamination in undisturbed columns of fractured saprolite

A series of laboratory experiments were conducted to further investigate the physical and chemical factors influencing the spread of TCE (trichloroethylene) contamination m fractured saprolite formed from sedimentary rock. The experiments involve simulation of a spill by injection of dyed immiscible phase TCE into two undisturbed columns of fractured saprolite, allowing a period of time for dissolution and diffusion of the TCE, and then dismantling them to map the resulting distribution of contamination. The use of dyed TCE allowed for the identification of preferential flow paths, which were associated with fractures and macropores in the two dominant saprolite lithology types, namely clay-rich limestone saprolite and blocky siltstone/shale saprolite, that comprise the columns. Despite evidence of discrete and erratic flow paths, TCE contamination was found throughout almost all of each column, with only small areas below detection limits. Based on observations and supported by calculations, the variable saprolite lithology and the processes of dissolution and diffusion are responsible for rapidly spreading the contamination. Comparisons were made of fracture aperture and porosity values derived from the cubic law and bulk hydraulic conductivity with DNAPL (dense non-aqueous phase liquid) entry pressure apertures and estimates of residual DNAPL volumes. These comparisons indicate that the cubic law can underestimate fracture aperture and may underestimate residual DNAPL volumes

Regulation of Bacterial Gene Expression by Protease-Alleviated Spatial Sequestration (PASS)

In natural microbial systems, conditional spatial sequestration of transcription factors enables cells to respond rapidly to changes in their environment or intracellular state by releasing presynthesized regulatory proteins. Although such a mechanism may be useful for engineering synthetic biology technologies ranging from cell-based biosensors to biosynthetic platforms, to date it remains unknown how or whether such conditional spatial sequestration may be engineered. In particular, based upon seemingly contradictory reports in the literature, it is not clear whether subcellular spatial localization of a transcription factor within the cytoplasm is sufficient to preclude regulation of cognate promoters on plasmid-borne or chromosomal loci. Here, we describe a modular, orthogonal platform for investigating and implementing this mechanism using protease-alleviated spatial sequestration (PASS). In this system, expression of an exogenous protease mediates the proteolytic release of engineered transcriptional regulators from the inner face of the <i>Escherichia coli</i> cytoplasmic membrane. We demonstrate that PASS mediates robust, conditional regulation of either transcriptional repression, <i>via</i> tetR, or transcriptional activation, by the λ phage CI protein. This work provides new insights into a biologically important facet of microbial gene expression and establishes a new strategy for engineering conditional transcriptional regulation for the microbial synthetic biology toolbox