Difference between revisions of "Part:BBa K1985004"

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[[File:T--Kent--mamTsds1.jpg||400px|thumb|left|Figure 2. Reducing 12% SDS-PAGE of soluble mamT including his-tag, PM is protein marker, BI is before induction, AI is after induction, SN is supernatant flow through fraction, and BB is binding buffer flow through fraction. Arrow indicates suspected protein.]] [[File:T--Kent--mamTsds2.jpg||400px|thumb|right|Figure 3. Reducing 12% SDS-PAGE of soluble mamT including his-tag, PM is protein marker, purified refers to the protein sample that was used for the absorbance spectrum and the in vitro iron induction. Arrow indicates suspected protein.]]
 
[[File:T--Kent--mamTsds1.jpg||400px|thumb|left|Figure 2. Reducing 12% SDS-PAGE of soluble mamT including his-tag, PM is protein marker, BI is before induction, AI is after induction, SN is supernatant flow through fraction, and BB is binding buffer flow through fraction. Arrow indicates suspected protein.]] [[File:T--Kent--mamTsds2.jpg||400px|thumb|right|Figure 3. Reducing 12% SDS-PAGE of soluble mamT including his-tag, PM is protein marker, purified refers to the protein sample that was used for the absorbance spectrum and the in vitro iron induction. Arrow indicates suspected protein.]]
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Revision as of 13:20, 25 October 2016

mamT his-tagged, signal sequence cleaved

This part is an differentiation on Part:BBa_K1985001. 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 138
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This part has a his-tag included so was used to purify the expressed mamT protein. MamT 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_K1985001.

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 604 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.

Figure 1. Agarose gel of the restriction digest of BBa_K1985004 in pSB1C3, with EcoRI and PstI.


Figure 2. Reducing 12% SDS-PAGE of soluble mamT including his-tag, PM is protein marker, BI is before induction, AI is after induction, SN is supernatant flow through fraction, and BB is binding buffer flow through fraction. Arrow indicates suspected protein.
Figure 3. Reducing 12% SDS-PAGE of soluble mamT including his-tag, PM is protein marker, purified refers to the protein sample that was used for the absorbance spectrum and the in vitro iron induction. Arrow indicates suspected protein.



















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.

Figure 4. EM image of triple protein reaction solution


The his-tagged proteins were purified by nickle affinity chromatography. An absorbance spectra was produced of multiple elution fractions. There is little difference compared to the control but this is likely a problem with the purification process.

Figure 5. UV spectra of nickle affinity chromatography fractions of E.coli expressing a pET3A plasmid without any mam genes.
Figure 6. UV spectra of nickle affinity chromatography protein fractions from E.coli expressing mamT. Little difference can be see compared to the control.