Difference between revisions of "Part:BBa K2027000"
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===Characterization=== | ===Characterization=== | ||
| − | We were able to transform this part into <i>E. coli</i> and induce protein synthesis using IPTG. We extracted this protein using Ni-NTA resin columns | + | We were able to transform this part into <i>E. coli</i> and induce protein synthesis using IPTG. We extracted this protein by relying on its HIS-tag and using Ni-NTA resin columns. |
| + | We ran a SDS-PAGE gel and used Lumio-Green staining on the fractions we collected during the protein purification process to verify which fraction contained the isolated protein. In the gel below, the lanes, from left to right, become progressively cleaner and display a singular prominent band. | ||
| − | Using the bicinchoninic acid assay (BCA) to determine the total concentration of protein in our final elution buffer, we found that our purified protein had a concentration of 149.317 mg/mL (shown below from Nanodrop 2000 software). | + | https://static.igem.org/mediawiki/parts/b/b0/T--Stanford-Brown--rAIP_purity_page.png |
| + | |||
| + | After determining which fraction the protein existed in, we continued with a large-scale protein extraction and purification. With our final product, we wanted to measure its purity. Using the bicinchoninic acid assay (BCA) to determine the total concentration of protein in our final elution buffer, we found that our purified protein had a concentration of 149.317 mg/mL (shown below from Nanodrop 2000 software). | ||
https://static.igem.org/mediawiki/parts/4/49/T--Stanford-Brown--raip_BCA_ss.png | https://static.igem.org/mediawiki/parts/4/49/T--Stanford-Brown--raip_BCA_ss.png | ||
Revision as of 10:38, 24 October 2016
Recombinant Apoptosis-Inducing Protein (L-lysine Alpha Oxidase)
This part is a native Scomber japonicus enzyme codon-optimized for Escherichia coli. The conjugated tag should allow for purification and visualization with anti-FLAG antibodies, visualization with Lumio™ Green, and purification with nickel columns. Tani et al. characterized this enzyme in their quest for synthesis of L-pipecolic acid from racemic lysine after nickel column purification, and we verified the function of our part in a similar way. More information can be found on the experience page for this part.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1004
Illegal BglII site found at 1032
Illegal BamHI site found at 767 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 25
Characterization
We were able to transform this part into E. coli and induce protein synthesis using IPTG. We extracted this protein by relying on its HIS-tag and using Ni-NTA resin columns.
We ran a SDS-PAGE gel and used Lumio-Green staining on the fractions we collected during the protein purification process to verify which fraction contained the isolated protein. In the gel below, the lanes, from left to right, become progressively cleaner and display a singular prominent band.
After determining which fraction the protein existed in, we continued with a large-scale protein extraction and purification. With our final product, we wanted to measure its purity. Using the bicinchoninic acid assay (BCA) to determine the total concentration of protein in our final elution buffer, we found that our purified protein had a concentration of 149.317 mg/mL (shown below from Nanodrop 2000 software).
