Difference between revisions of "Part:BBa K1420006"

Revision as of 02:05, 17 October 2014

phsABC Generator

Summary

The (phsABC) gene from Salmonella enterica serovar Typhimurium LT2 encodes thiosulfate reductase, which catalyzes the stoichiometric production of hydrogen sulfide and sulfite from thiosulfate for heavy metal removal by precipitation.

Figure 1.

Figure 2.

Experimental Results

In order to measure the thiosulfate reducing activity of phsABC of NaS₂O₃ to H₂S, the gene was first inserted into the pBBRBB vector with a constitutive P_lac promoter and transformed into K12 strain E. coli cells. As a 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. 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 known a standard curve of various Na₂S concentrations. Each sample was allowed 20 minutes for the color to develop before adding 30μL of sample into 270μL of water. The samples were then further diluted 1:10 with water before testing (Figure 3). The entire well plate was then read at 670nm with the numerical compiled results displayed in Figure 4.

A second set of experiments was also conducted with pBBRBB:phsABC K12, pBBRBB::GFP K12, and an abiotic control grown in heavy metal tryptone medium, 3mM NaS₂O₃tubes, and 2.5mM Fe(II)Cl₂. Since the phsABC gene is responsible reducing thiosulfate, NaS₂O₃ would be converted to H₂S, which will further react with Fe(II)Cl₂ to produce FeS, a black precipitate. After a 24 hour incubation period, the cell cultures appeared as displayed in Figure 5. The pBBRBB:phsABC K12 cells were the only ones seen to produce FeS, the black precipitate seen in the figure, confirming the role of the phsABC gene in reducing thiosulfate. To affirm that this reaction is also successful under non-enclosed systems, the same sets of samples were also tested on 0.2% plates containing 3mM NaS₂O₃tubes and 2.5mM Fe(II)Cl₂. The results after 24 hours of incubation were similar to the experiments conducted in test tubes (Figure 6). <p>

Figure 3. 1:10 dilution of cell samples in Cline's Reagent used for hydrogen sulfide assays. The samples in box 1 contain the Na₂S standards (left to right, 1mM, 0.5mM, 0.25mM, 0.125mM, 0.0613mM, and 0.025mM), box 3 contains pBBRBB:GFP K12 cells, while box 3 contains pBBRBB:phsABC K12 cells. Boxes 4, 5, and 6 are the same as boxes 1, 2, and 3 aside from the fact that 0.5x Cline's Reagent was used as opposed to 1x Cline's Reagent that was used in box 1, 2, and 3. Box 7 contains the abiotic sample (left) and water (right) in 1x Cline's reagent. Samples from box 7 are the same as box 8 aside from using 0.5x Cline's Reagent for the latter.

Figure 4. Hydrogen sulfide assay results for pBBRBB:phsABC K12 cells compared to controls.

Figure 5.

Figure 6.

References

Sequence and Features