Difference between revisions of "Part:BBa K1420006"

(Experimental Results)
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== Experimental Results ==
 
== Experimental Results ==
<p>In order to measure the thiosulfate reducing activity of ''phsABC'' of NaS<sub>2</sub>O<sub>3</sub> to H<sub>2</sub>S, the gene was first inserted into the pBBRBB vector with a constitutive P<sub>lac</sub> 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 NaS<sub>2</sub>O<sub>3</sub>. 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 H<sub>2</sub>S 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 FeCl<sub>3</sub> in 50mL of 50% cool HCl) to 270μL of sample. The results were tested against known a standard curve of various H<sub>2</sub>S 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''.</p>
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<p>In order to measure the thiosulfate reducing activity of ''phsABC'' of NaS<sub>2</sub>O<sub>3</sub> to H<sub>2</sub>S, the gene was first inserted into the pBBRBB vector with a constitutive P<sub>lac</sub> 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 NaS<sub>2</sub>O<sub>3</sub>. 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 H<sub>2</sub>S 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 FeCl<sub>3</sub> in 50mL of 50% cool HCl) to 270μL of sample. The results were tested against known a standard curve of various Na<sub>2</sub>S 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''.</p>
<p>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 NaS<sub>2</sub>O<sub>3</sub>tubes, and 2.5mM Fe(II)Cl<sub>2</sub>. Since the ''phsABC'' gene is responsible reducing thiosulfate,  NaS<sub>2</sub>O<sub>3</sub> would be converted to H<sub>2</sub>, which will further react with Fe(II)Cl<sub>2</sub> 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 NaS<sub>2</sub>O<sub>3</sub>tubes and 2.5mM Fe(II)Cl<sub>2</sub>. The results after 24 hours of incubation were similar to the experiments conducted in test tubes (''Figure 6'').
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<p>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 NaS<sub>2</sub>O<sub>3</sub>tubes, and 2.5mM Fe(II)Cl<sub>2</sub>. Since the ''phsABC'' gene is responsible reducing thiosulfate,  NaS<sub>2</sub>O<sub>3</sub> would be converted to H<sub>2</sub>S, which will further react with Fe(II)Cl<sub>2</sub> 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 NaS<sub>2</sub>O<sub>3</sub>tubes and 2.5mM Fe(II)Cl<sub>2</sub>. The results after 24 hours of incubation were similar to the experiments conducted in test tubes (''Figure 6'').
 
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[[File:assay.jpg|950px|center|alt text]]
 
[[File:assay.jpg|950px|center|alt text]]
''Figure 3.'' 1:10 dilution of cell samples in Cline's Reagent used for hydrogen sulfide assays.
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''Figure 3.'' 1:10 dilution of cell samples in Cline's Reagent used for hydrogen sulfide assays. The samples in box 1 contain the Na<sub>2</sub>S 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.
 
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Revision as of 01:53, 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.

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 Plac 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>


alt text

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.


alt text

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


alt text

Figure 5.


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Figure 6.

References

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 135
    Illegal BglII site found at 2197
    Illegal BamHI site found at 141
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 249
    Illegal AgeI site found at 2772
    Illegal AgeI site found at 3555
  • 1000
    COMPATIBLE WITH RFC[1000]