Difference between revisions of "Part:BBa K2332312"
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===Experimental approach=== | ===Experimental approach=== | ||
| − | =Vector Considerations:= | + | ====Vector Considerations:==== |
For testing this coding part we used pcDNA3, a standard mammalian expression plasmid, as a vector. We, thereby, created [https://parts.igem.org/Part:BBa_K2332313 BBa_K2332313], our E-cadherin gene flanked by a CMV promoter and a bGH poly(A) tail. The pre-existing 5'- and 3'-UTR and the strong promoter ensured efficient expression of E-cadherin during our experiments. | For testing this coding part we used pcDNA3, a standard mammalian expression plasmid, as a vector. We, thereby, created [https://parts.igem.org/Part:BBa_K2332313 BBa_K2332313], our E-cadherin gene flanked by a CMV promoter and a bGH poly(A) tail. The pre-existing 5'- and 3'-UTR and the strong promoter ensured efficient expression of E-cadherin during our experiments. | ||
Revision as of 21:42, 7 October 2017
E-cadherin (Preproprotein, Mus Musculus)
| E-cadherin (Preproprotein, Mus Musculus) | |
|---|---|
| Function | Cell-Cell Adhesion |
| Use in | Mammalian cells |
| Chassis Tested | Chinese Hamster Ovary (CHO) |
| Abstraction Hierarchy | Part |
| RFC standard | RFC10 & RFC23 compatible |
| Backbone | pSB1C3 |
| Submitted by | [http://2017.igem.org/Team:UCL] |
This gene 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.
UCSF iGEM 2011 has created a BioBrick of only the extracellular domain of E-Cadherin (Mouse) BBa_K644000 but no BioBrick encoding the full E-cadherin protein has been submitted until now. BBa_K644000 also lacked detailed characterisation and the source was imprecise. Furthermore, we know now that E-cadherin requires interaction of its cytosolic domain for the production of stable cell-cell connections. (see Alberts 6th Ed. 2015, Ch. 19, p. 1040).
This gene was given to the UCL iGEM team 2017 by Prof. Stephen Price (UCL, not part of iGEM) after we searched for cadherin proteins suitable for our project. However, no sequence was known of the plasmid we were given and we sequenced the plasmid ourselves. Consecutive BLAST analysis showed a 99% similarity with Mus musculus cadherin 1 (Cdh1), mRNA: NCBI Reference Sequence: NM_009864.3, NCBI.
Three silent mutations were added into the sequence via side directed mutagenesis in order to remove one EcoRI and two PstI sites. Afterwards we sequence confirmed the entire gene.
Experimental approach
Vector Considerations:
For testing this coding part we used pcDNA3, a standard mammalian expression plasmid, as a vector. We, thereby, created BBa_K2332313, our E-cadherin gene flanked by a CMV promoter and a bGH poly(A) tail. The pre-existing 5'- and 3'-UTR and the strong promoter ensured efficient expression of E-cadherin during our experiments.
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2550
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 752
Illegal BamHI site found at 828
Illegal BamHI site found at 944
Illegal BamHI site found at 1868
Illegal BamHI site found at 2170
Illegal XhoI site found at 1552 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 208
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 65
Illegal BsaI site found at 465
Illegal BsaI site found at 872
Illegal BsaI site found at 1414
Illegal BsaI.rc site found at 306