Part:BBa_K4214000

Human Gastric Intrinsic Factor (hGIF)

This part codes for human Gastric Intrinsic Factor protein (AA19-417). The gene is located in chromosome 11q12.1. This gene is a part of cobalamin transporters group. It is essential for the absorption of vitamin B12. Mutations in this gene may lead to pernicious anemia.

Fig.: Human GIF (BBa_K4214000)

We removed the native signal peptide (AA1-18) from the N-terminal in order to obtain a sequence that can be worked with more easily in bacterial and mammalian systems.

Usage and Biology

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• NCBI page of the human gastric intrinsic factor [1]
• This part was used by us to create mutants of GIF, which were used for surface display screening of autoantibody binding in our BiG-IF project
• This part of hGIF does not contain the native signal peptide (AA1-18)

Characterization

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Agarose gel image of part

The following agarose gel image shows the results from restriction digestion of BBa_K4214000 with Bgl-II and SalI in order to continue with ligation to our expression vector.

Fig.: Agarose gel image showing the results from restriction digestion of BBa_K4214000 with Bgl-II and SalI in order to continue with ligation to our expression vector

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Western blot image of part

The BBa_K4214000 part is our wild-type intrinsic factor gene and we inserted it in our expression vector. We transfected the HEK293 cells and expressed our protein in the surface of the cells. We detected our protein by using an uspecific Figure A depicts the Western Blot image (fluorescent image) of our samples- Wild type plasmid, mutant 20201 plasmid and Negative control (without any plasmid) loaded at two different concentrations-15 µg and 25 µg. From both Figure A and B, we observed that our protein (marked by green arrows) is being produced in mutant 20201 by the presence of light yellow bands in between 70 kDa to 55 kDa bands. The light yellow bands indicate the binding of both Myc antibody (green) and epitope unspecific IF primary antibody (red). Myc is present in the plasmid backbone and is one of the tags for confirmation for our protein.

Fig.: Western blot image of our samples- Wild Type, mutant 20201 and Negative control loaded with two different time points (24, 48) and concentrations (15 µg and 25 µg)

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
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 265
  Illegal BsaI.rc site found at 945
  Illegal SapI site found at 935