Difference between revisions of "Part:BBa K4509369"
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=Characterization= | =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 [https://parts.igem.org/Part:BBa_J23100 BBa_J23100],RBS [https://parts.igem.org/Part:BBa_B0034 | + | 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 [https://parts.igem.org/Part:BBa_J23100 BBa_J23100],RBS [https://parts.igem.org/Part:BBa_B0034 BBa_B0034],and terminator [https://parts.igem.org/Part:BBa_B0015 BBa_B0015]and characterized it through assays. |
| − | ==Biuret | + | ==Biuret's Test== |
| − | Here we determined the general concentration of the enzyme with the help of Biuret | + | 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. |
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| + | https://static.igem.wiki/teams/4509/wiki/registry/apha-basic-test-tube-biu.jpeg | ||
The absorbance of unknown sample concentrations were determined at 520nm and compared to the standard graph to identify enzyme concentration. | The absorbance of unknown sample concentrations were determined at 520nm and compared to the standard graph to identify enzyme concentration. | ||
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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. | 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. | ||
| + | https://static.igem.wiki/teams/4509/wiki/registry/pnpp-apha-tt.jpeg | ||
| + | https://static.igem.wiki/teams/4509/wiki/registry/pnpp-apha-1.png | ||
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| − | + | The absorbance value was taken at 430nm and a graph was plotted. | |
Latest revision as of 05:57, 13 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 433
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_B0015and characterized it through assays.
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.
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 430nm and a graph was plotted.