Part:BBa_K4509769

Cell surface aphA with constitutive promoter

This construct consists of strong promoter and strong RBS combination BBa_K880005, cell surface tag BBa_K103006 linked to aphA gene BBa_K4509369 and double terminator. OmpA Linker tag helps to express acid phosphatase enzyme(aphA) on the cell surface.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 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 966
  

Characterization

The speciality of this composite part is that the periplasmic enzyme aphA is displayed on the cell surface with the help of OmpA linker. This part is made up of the promoter BBa_J23100 which helps in the effective transcription of the recombinant genes. The strong RBS BBa_B0034 helps in the translation process.

Biuret's Test

We have performed a biuret test to confirm the presence of the enzyme. For the biuret test, we have prepared working standard and test sample solutions. Upon addition of the biuret reagent, colour development is observed which was measured at 520 nm. We plotted a graph between the protein concentration and optical density. This helped us in identifying the protein concentration in the test solution.

pNPP Assay

To check the activity of acid phosphatase, we conducted pNPP assay. Keeping the enzyme concentration fixed, we varied the concentration of the substrate pNPP and noted down the change in absorbance. The absorbance value was taken at 430 nm and graph was plotted.