Team:Minnesota
From 2014.igem.org
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- | < | + | <h4> Heavy Metal Bioprecipitation Results </h4> |
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+ | In order to measure thiosulfate reducing activity of phsABC of NaS2O3 to H2S, the operon was first inserted into the pBBRBB vector with a constitutive Plac promoter and transformed into E. coli K12. As a negative control, pBBRBB::gfp was tested under the same conditions. The pBBRBB::phsABC K12 and pBBRBB::gfp K12 cells were grown in three test tubes each containing heavy metal tryptone medium as well as 3mM NaS2O3. A third set of test tubes were set up with the same contents except without cells as an additional negative control. After 24 hours of incubation, The exact amount of H2S present in each of three different sets of tubes was then measured using a hydrogen sulfide assay designed by J.D. Cline in 1968 to determine hydrogen sulfide concentrations in natural waters.2 This consisted of adding 1x or 0.5x 30μL of Cline's Reagent (2g Diamine + 3g FeCl3 in 50mL of 50% cool HCl) to 270μL of sample. The results were tested against a known standard curve of various Na2S concentrations. Each sample was allowed 20 minutes for the color to develop before being diluted 1:10 with water for testing (Figure 3). The plate was then read at 670nm with the numerical results displayed in Figure 4. The bar graph shows that the sulfide concentration was considerable higher for the cultures containing pBBRBB:phsABC (380.1 μM ± 13.5) compared to the pBBRBB::gfp negative control (118.9μM ± 1.1). These results are in line with those seen by the Keasling lab.2 Following sulfide measurements, cadmium chloride was added (200 μM), and cells were allowed to incubate without shaking at 37C overnight. Cells were pelleted to look for a color change indicating precipitation of CdS (Figure 7). Cell pellets for K12 expressing phsABS were yellow/brown indicating precipitation of CdS while the vector control cells (pBBRBB::gfp) remained white. | ||
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- | + | Our design is very similar to commonly used fixed-bed adsorption and ion exchange columns in that it involves flow of a fluid down through a packed bed that removes a solute. However, an important difference is those types of columns contain chemical resins or adsorbents that can become saturated (i.e. reach capacity) as they adsorb solute. Since we use encapsulated bacteria whose "capacity" is only limited their lifetime, our device has the added benefit that the mass-transfer zone (the part of the column where the solute is removed) doesn't move down the column due to saturation. This also means that we don't need to alternate between adsorption and regeneration cycles. | |
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Revision as of 01:19, 18 October 2014