Difference between revisions of "Part:BBa K1985005"
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− | The his-tagged proteins were purified by nickle affinity chromatography. An absorbance spectra was produced of multiple elution fractions. There is | + | The his-tagged proteins were purified by nickle affinity chromatography. An absorbance spectra was produced of multiple elution fractions. There is a significant peak at 407nm that indicates the presence of mamX. |
− | [[File:Control uv spectra.jpeg||400px|thumb|left|Figure 1. UV spectra of nickle affinity chromatography fractions of E.coli expressing a pET3A plasmid without any mam genes.]] [[File:MamX uv spectra.jpeg||400px|thumb|right|Figure 2. UV spectra of nickle affinity chromatography protein fractions from E.coli expressing mamX. | + | [[File:Control uv spectra.jpeg||400px|thumb|left|Figure 1. UV spectra of nickle affinity chromatography fractions of E.coli expressing a pET3A plasmid without any mam genes.]] [[File:MamX uv spectra.jpeg||400px|thumb|right|Figure 2. UV spectra of nickle affinity chromatography protein fractions from E.coli expressing mamX. A peak can be seen at 407nm.]] |
Revision as of 16:02, 17 October 2016
mamX his-tagged, signal sequence cleaved
This part is an differentiation on Part:BBa_K1985002. The wild type sequence was altered to remove the membrane anchor so that the part is soluble and a SecS sequence was added to target the protein to the periplasm. A his-tag was added for easier protein purification.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 216
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 783
Usage and Biology
This part has a his-tag included so was used to purify the expressed mamP protein. MamP is usually targeted to the membrane, however in this part the targeting sequence has been cleaved and it should instead be targeted to the periplasm. It was expressed, purified and then exposed to iron. For more information on its biology and usage, see part BBa_K1985002.
Validation
The part was first validated with a diagnostic restriction digest using EcoRI and PstI and agarose gel electrophoresis. The expected band sizes from the digest were: 2029 base pairs for the plasmid backbone and 838 for the insert. A 1kB plus DNA marker was used to verify the sizes of the bands and it was confirmed that the correct plasmid had been produced.
The proteins were then visualised with EM under different conditions: a reducing environment and with magnetite crystals. The samples were concentrated but protein could be seen.
INSERT EM IMAGE
The his-tagged proteins were purified by nickle affinity chromatography. An absorbance spectra was produced of multiple elution fractions. There is a significant peak at 407nm that indicates the presence of mamX.