Part:BBa_K1985005

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

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 216
  
• 1000
  INCOMPATIBLE 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 mamX protein. MamX 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.

Figure 1. Agarose gel of the restriction digest of BBa_K1985005 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 MamX the band indicated by the arrow equates to the, 24 kDa, soluble MamX protein with his-tag.

Figure 2. Reducing 12% SDS-PAGE of soluble mamX 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 mamX 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 mamX along with mamT and mamP 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 mamX, T and P

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.

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 mamX. A peak can be seen at 407nm.