Part:BBa_K1420006

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 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 H₂S 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.

the tubes appeared as displayed in Figure 5. The pBBRBB:phsABC K12 cells were the only ones seen to

Figure 3. 1:10 dilution of cell samples in Cline's Reagent used for hydrogen sulfide assays.

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

Figure 5.

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]