Difference between revisions of "Part:BBa K2332313"

Latest revision as of 14:42, 8 October 2017

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
Related Parts	BBa_I712004, BBa_K2332312, BBa_K678019
RFC standard	RFC10 & RFC23 compatible
Backbone	pSB1C3
Submitted by	[http://2017.igem.org/Team:UCL UCL iGEM 2017]

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

@@ Line 10: / Line 10: @@
 |'''Use in'''
 |Mammalian cells
+|-
+|'''Chassis Tested'''
+|Chinese Hamster Ovary (CHO)
 |-
 |'''Abstraction Hierarchy'''
 |Device
+|-
+|'''Related Parts'''
+|[https://parts.igem.org/wiki/index.php?title=Part:BBa_I712004 BBa_I712004], [https://parts.igem.org/Part:BBa_K2332312 BBa_K2332312], [https://parts.igem.org/wiki/index.php?title=Part:BBa_K678019 BBa_K678019]
 |-
 |'''RFC standard'''
@@ Line 21: / Line 27: @@
 |-
 |'''Submitted by'''
-|[http://2017.igem.org/Team:UCL]
+|[http://2017.igem.org/Team:UCL UCL iGEM 2017]
 |}
 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.
@@ Line 29: / Line 36: @@
-[[File:CMV Promoter - 5'-UTR - E-cadherin.png|700px|]]
+[[File:CMV Promoter - 5'-UTR - E-cadherin.png|700px|thumb|left|'''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.
-'''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.
 <!-- Add more about the biology of this part here