Device

Part:BBa_K2332313

Designed by: Camillo Moschner   Group: iGEM17_UCL   (2017-10-07)
Revision as of 21:42, 7 October 2017 by CamilloMoschner (Talk | contribs)


E-cadherin (Preproprotein) with CMV promoter & bGH poly(A) tail

E-cadherin (Preproprotein) with CMV promoter & bGH poly(A) tail
Function Cell-Cell Adhesion
Use in Mammalian cells
Chassis Tested Chinese Hamster Ovary (CHO)
Abstraction Hierarchy Device
RFC standard RFC10 & RFC23 compatible
Backbone pSB1C3
Submitted by [http://2017.igem.org/Team:UCL]


This composite part is a device which efficiently encodes E-cadherin, a calcium-dependent cell adhesion molecule that functions in the establishment and maintenance of epithelial cell morphology during embryongenesis and adulthood. The encoded preproprotein undergoes proteolytic processing to generate a mature protein.



Figure 1: Construct Design. The CMV promoter was fused with the E-cadherin coding part by creating a 5'-UTR (untranslated region) which is essential for efficient translation. The resulting fusion construct contains a T7 promoter according to pcDNA3 standard, a standard mammalian expression plasmid. The CMV promoter and bGH terminator were chosen to ensure efficient gene expression.












Experimental approach

For testing this device we used CHO cells as our chassis of choice, which naturally lack E-cadherin expression but still maintain alpha- and beta-catenin expression. By chosing CHO cells as our chassis we, therefore, eliminated background noise.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 3212
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1414
    Illegal BamHI site found at 1490
    Illegal BamHI site found at 1606
    Illegal BamHI site found at 2530
    Illegal BamHI site found at 2832
    Illegal XhoI site found at 2214
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 870
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 727
    Illegal BsaI site found at 1127
    Illegal BsaI site found at 1534
    Illegal BsaI site found at 2076
    Illegal BsaI.rc site found at 968


[edit]
Categories
//cds/membrane
Parameters
device_type