Part:BBa_K4509669

Cell surface aphA with Cd2+ sensing promoter

This part consists of a cadmium metal sensing promoter BBa_K896008, strong RBS BBa_B0034, cell surface tag BBa_K103006 linked to acid phosphatase gene BBa_K4509369,with terminator BBa_B0015. This part expresses acid phosphatase as a cell surface protein in the presence of cadmium.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1012
  

Characterization

This part only produces aphA in the presence of cadmium (due to the metal sensitive promoter BBa_K896008). Transformed cells were grown in media with Cd2+ concentration of 0.001mg/ml to help express acid phosphatase.

Biuret's Test

Here we determined the general concentration of the enzyme with the help of Biuret's test. A standard graph with known concentrations of the enzyme were prepared (working standard). Then, two test solutions with unknown concentrations of acid phosphatase were prepared.

Their absorbance was taken at 520 nm and was compared with the standard graph to determine enzyme concentrations.

pNPP Assay

Activity of acid phosphatase was determined with pNPP assay. The pH was maintained at 5.5 and substrate concentration was varied in the samples. Keeping the enzyme concentration fixed, the concentration of the substrate pNPP was varied and a graph was plotted based on the change in absorbance at 430nm.