Part:BBa_K1985004

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 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_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 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 2. Agarose gel of the restriction digest of BBa_K1985004 in pSCB13, with EcoRI and PstI.

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

An absorbance spectra was produced that shows peaks at xxx compared to the control.

INSERT SPECTRA AND CONTROL.