Plasmid

Part:BBa_K4869000

Designed by: Woo Rin Lee   Group: iGEM23_Korea-HS   (2023-09-08)


BII-CMV-AfmW3A piggyBac vector contains Afamin and Wnt3a genes

General Information & Literature Review:

This involved employing PCR and gel electrophoresis techniques to assess the presence of the proteins in the BII-CMV-AfmW3A vector with a circular DNA form consisting of 10,434 base pairs. Each gene within the vector, including CMV promoter, Afamin, T2A sequence, Wnt3a, puromycin resistance gene, and ampicillin resistance gene, plays a distinct role in producing Afamin-Wnt3a conditioned media.

Application in Our Project:

<img src="screenshot-2023-10-12-at-2-27-27-pm.png">

Figure: BII-CMV-AfmW3A piggyBac vector contains Afamin and Wnt3a genes (vector sequence).

This project aimed to generate Afamin-Wnt3a-producing cells to obtain an FBS-free growth medium for cultivating bovine muscle cells. We utilized a piggyBac vector (BII-CMV-AfmW3A) to express Afamin and Wnt3a and the Super piggyBac transposase expression vector to transfect human embryonic kidney 293 cells (HEK293).

The BII-CMV-AfmW3A vector is known to be effective when inserting foreign DNA (Afamin-Wnt3a) into the host genome (HEK293). The BII-CMV-AfmW3A vector is made up of a circular DNA consisting of 10,434 base pairs. The vector contains various genes such as CMV promoter, T2A sequence, puromycin resistance gene, Afamin, and Wnt3a gene. Our study mainly focused on two genes, which are the Afamin and Wnt3a genes, which are included in the BII-CMV-AfmW3A vector.

Our goal was to transfect the two genes Afamin and Wnt3a into HEK293 cells and insert Afamin and Wnt3a into the genome of the cell. Afamin is a glycoprotein that plays a significant role in binding and stabilizing various growth factors, while Wnt3a is a critical signaling molecule involved in cell proliferation and differentiation. The co-expression of these two factors was expected to create an ideal microenvironment for the growth of bovine muscle cells, as an alternative to the fetal bovine serum (FBS). Therefore, the piggyBac vector was used as a mechanism to easily transfer the Afamin and Wnt3a gene into the HEK293 cells. This vector will later be amplified after being transformed into DH5alpha E.coli.

References:

Loidolt S., Establishment of Afamin-Wnt3a producer cells to generate serum-free growth medium for canine intestinal organoid culture. phaidra.vetmeduni.ac.at/open/o:869.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 7374
    Illegal XbaI site found at 923
    Illegal XbaI site found at 6885
    Illegal SpeI site found at 917
    Illegal SpeI site found at 1813
    Illegal SpeI site found at 6637
    Illegal PstI site found at 1129
    Illegal PstI site found at 1171
    Illegal PstI site found at 2979
    Illegal PstI site found at 3106
    Illegal PstI site found at 3756
    Illegal PstI site found at 3891
    Illegal PstI site found at 4137
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 7374
    Illegal NheI site found at 889
    Illegal SpeI site found at 917
    Illegal SpeI site found at 1813
    Illegal SpeI site found at 6637
    Illegal PstI site found at 1129
    Illegal PstI site found at 1171
    Illegal PstI site found at 2979
    Illegal PstI site found at 3106
    Illegal PstI site found at 3756
    Illegal PstI site found at 3891
    Illegal PstI site found at 4137
    Illegal NotI site found at 3971
    Illegal NotI site found at 4494
    Illegal NotI site found at 4661
    Illegal NotI site found at 4805
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 7374
    Illegal BglII site found at 872
    Illegal BamHI site found at 911
    Illegal BamHI site found at 2253
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 7374
    Illegal XbaI site found at 923
    Illegal XbaI site found at 6885
    Illegal SpeI site found at 917
    Illegal SpeI site found at 1813
    Illegal SpeI site found at 6637
    Illegal PstI site found at 1129
    Illegal PstI site found at 1171
    Illegal PstI site found at 2979
    Illegal PstI site found at 3106
    Illegal PstI site found at 3756
    Illegal PstI site found at 3891
    Illegal PstI site found at 4137
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 7374
    Illegal XbaI site found at 923
    Illegal XbaI site found at 6885
    Illegal SpeI site found at 917
    Illegal SpeI site found at 1813
    Illegal SpeI site found at 6637
    Illegal PstI site found at 1129
    Illegal PstI site found at 1171
    Illegal PstI site found at 2979
    Illegal PstI site found at 3106
    Illegal PstI site found at 3756
    Illegal PstI site found at 3891
    Illegal PstI site found at 4137
    Illegal NgoMIV site found at 1044
    Illegal NgoMIV site found at 2176
    Illegal NgoMIV site found at 6537
    Illegal NgoMIV site found at 7006
    Illegal NgoMIV site found at 7258
    Illegal NgoMIV site found at 8031
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 3168
    Illegal BsaI site found at 9207
    Illegal BsaI.rc site found at 4486
    Illegal BsaI.rc site found at 5879
    Illegal SapI.rc site found at 10289


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