Difference between revisions of "Part:BBa K4214000"
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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.  
 
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.  
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[[Image:Part-BBa K4214000.png|250px|thumb|right|Fig.: ''Human GIF (BBa_K4214000 '']]
 
[[Image:Part-BBa K4214000.png|250px|thumb|right|Fig.: ''Human GIF (BBa_K4214000 '']]
  
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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 systems. However, since we used bacterial and mammalian expression vectors in parallel, we needed to have compatible parts and decided to work only with '''part BBa_K4214001'''[https://parts.igem.org/Part:BBa_K4214001], which '''includes''' the signal peptide sequence (AA1-18).
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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==
 
==Usage and Biology==
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===Agarose gel image of part===
 
===Agarose gel image of part===
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The following agarose gel image shows the results from restriction digestion of BBa_K4214000 with BglI and SalI in order to continue with ligation to our expression vector.  
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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.
  
 
===Western blot image of part===
 
===Western blot image of part===
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Here, the Anti-Rabbit red fluorescence (1:1000) emitted is from the secondary antibody used against our epitope unspecific Intrinsic Factor(IF) Rabbit primary antibody [2]. The Anti-Mouse green fluorescence (1:1000) is emitted from the secondary antibody used against our specific Myc Mouse primary antibody [3]. The red color in the ladder is because of the PageRuler™ Prestained Protein Ladder [4]. The prestained protein ladder is seen in the 700 nm range under fluorescence and hence they appear red in the blot.
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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
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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. The figure also depicts the control housekeeping gene, GAPDH, at 35 kDa. The samples were also collected at two different time points 24 and 48 hours (transfection time points). The Ladder in kDa is marked along both the sides of the blot. Figure B depicts the same blot in a much enlarged view but without the control GAPDH gene.
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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.  
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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. However, the streak of green bands seen in Figure B can be understood as unspecified myc binding to various other cellular proteins in HEK cells. There is no red streak of bands seen in wells designated for samples thus indicating that our epitope unspecific antibody is extremely specific to the IF and does not bind to any other cellular proteins.
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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.
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It is worthy to note that at the desired size range, we have no visible bands in negative control as well as in Wild Type plasmid in both figures. One of the reasons for no protein production in WT plasmid can be attributed to the stop codon removal step in our mutagenesis. While we succeeded in the removal, we believe that it may have caused some changes in the plasmid which we overlooked.
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[[Image:Western-blot.png|600px|thumb|center|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) '']]
 
[[Image:Western-blot.png|600px|thumb|center|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) '']]

Revision as of 10:10, 9 October 2022

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

  • 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

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.

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