Coding

Part:BBa_K1985003

Designed by: Jasmine Cornish   Group: iGEM16_Kent   (2016-10-14)
Revision as of 17:47, 27 October 2016 by Avs (Talk | contribs) (→‎Validation)

mamP, his-tagged, signal sequence cleaved

This part is an differentiation on Part:BBa_K1985000. 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 AgeI site found at 517
  • 1000
    COMPATIBLE WITH RFC[1000]


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_K1985000.

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 895 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_K1985003 in pSB1C3 with EcoRI and PstI.

SDS-PAGE images revealed bands in all gels that were not present in the before induction samples and the control. For MamP the band indicated by the arrow equates to the, 26.76 kDa, soluble MamP protein with his-tag.

Figure 2. Reducing 12% SDS-PAGE of soluble mamP 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 mamP 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 magnetite particles were imaged under TEM with solutions of mamP along with mamT and mamX under anaerobic conditions.
Figure 4. EM image of magnetite crystals with hypothesised new crystals nucleated circled in red, these crystals are in the presence of the three proteins mamP, T and X


The his-tagged proteins were purified by nickle affinity chromatography. An absorbance spectra was produced of all three proteins as well as the control. MamP has a significant increase in absorbance to the control both at 280nm displaying an increase in protein and at 407nm showing the heme group of mamX.

[[File:Allprotein graph.jpeg||400px|thumb|centre|Figure 5. UV spectra of nickle affinity chromatography protein fractions from E.coli expressing mamX, P and T as well as the control. For mamP (Purple) a significant peak was seen compared to the control (grey) at 40The his-tagged proteins were purified by nickle affinity chromatography. An absorbance spectra was produced of all three proteins as well as the control. MamP has a significant increase in absorbance to the control at 280nm displaying an increase in protein.

Figure 5. UV spectra of nickle affinity chromatography protein fractions from E.coli expressing mamX, P and T as well as the control. For mamP (Purple) a significant peak was seen compared to the control (grey) at 280nm.


[edit]
Categories
Parameters
None