Revision as of 06:30, 12 October 2022

Acid phosphatase (aphA)

Acid phosphatase coding sequence without promoter, RBS or terminator from E.coli K12. Some neutral point mutations have been made to this sequence to ensure its compatibility with all assembly standards. This bacterium's aphA gene was chosen to make sure it is effectively expressed in E.coli. When expressed, aphA can be assayed using phosphate substrates like glycerol-2-phosphate, disodium phenyl phosphate, phytic acid etc. The quantity of substrate consumed and free phosphate produced is directly proportional to the quantity of the enzyme present.

Sequence and Features

Characterization

This basic part codes for acid phosphatase enzyme and was isolated from Escherichia Coli K12. In its native form, it secretes aphA into the periplasmic region. The enzyme, when purified, is able to dephosphorylate many phosphate containing substrates. We attempted to characterize the enzyme with substrates pNPP. We constructed a plasmid with promoter BBa_J23100,RBS BBa_B0034, and terminator BBa_B0015 and characterized it through assays.

Biuret Assay

Here we determined the general concentration of the enzyme with the help of Biuret Assay. A standard graph with known concentrations of the enzyme were prepared.

The absorbance of unknown sample concentrations were determined at 520nm and compared to the standard graph to identify enzyme concentration.

pNPP Assay

Here, we characterized acid phosphatase activity using pNPP assay. The transformed cells were lysed and homogenized to prepare enzyme sample. The pH was maintained at 5.5 and substrate concentration was varied in the samples.

The absorbance value was taken at 405nm and a graph was plotted.