Difference between revisions of "Part:BBa K4509669"
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<partinfo>BBa_K4509669 short</partinfo> | <partinfo>BBa_K4509669 short</partinfo> | ||
| − | This part consists of a cadmium metal sensing promoter | + | This part consists of a cadmium metal sensing promoter [https://parts.igem.org/Part:BBa_K896008 BBa_K896008], strong RBS [https://parts.igem.org/Part:BBa_B0034 BBa_B0034], cell surface tag [https://parts.igem.org/Part:BBa_K103006 BBa_K103006] linked to acid phosphatase gene [https://parts.igem.org/Part:BBa_K4509369 BBa_K4509369],with terminator [https://parts.igem.org/Part:BBa_B0015 BBa_B0015]. This part expresses acid phosphatase as a cell surface protein in the presence of cadmium. |
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===Usage and Biology=== | ===Usage and Biology=== | ||
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=Characterization= | =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. | + | This part only produces aphA in the presence of cadmium (due to the metal sensitive promoter [https://parts.igem.org/Part:BBa_K896008 BBa_K896008]). Transformed cells were grown in media with Cd2+ concentration of 0.001mg/ml to help express acid phosphatase. |
==Biuret's Test== | ==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. | 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. | ||
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| + | https://static.igem.wiki/teams/4509/wiki/registry/apha-metu-biu-tt.jpeg | ||
https://static.igem.wiki/teams/4509/wiki/registry/biuret-metal-1.png | https://static.igem.wiki/teams/4509/wiki/registry/biuret-metal-1.png | ||
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==pNPP Assay== | ==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. | 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. | ||
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| + | https://static.igem.wiki/teams/4509/wiki/registry/pnpp-apha-metal-tt.jpeg | ||
https://static.igem.wiki/teams/4509/wiki/registry/pnpp-metal-sen-1.png | https://static.igem.wiki/teams/4509/wiki/registry/pnpp-metal-sen-1.png | ||
Latest revision as of 12:21, 12 October 2022
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
- 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 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.
