Difference between revisions of "Part:BBa K2332312"
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Revision as of 11:25, 9 October 2017
E-cadherin (Preproprotein, Mus Musculus)
E-cadherin (Preproprotein, Mus Musculus) | |
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Function | Cell-Cell Adhesion |
Use in | Mammalian cells |
Chassis Tested | Chinese Hamster Ovary (CHO) |
Abstraction Hierarchy | Part |
Related Device | BBa_K2332313 |
RFC standard | RFC10 & RFC23 compatible |
Backbone | pSB1C3 |
Submitted by | [http://2017.igem.org/Team:UCL UCL iGEM 2017] |
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.
Contents
Usage and Biology
Cell-cell junctions come in many forms and can be regulated by a variety of different mechanisms. The best understood and most common are the two types of cell-cell anchoring junctions which employ cadherins to link the cytoskeleton of one cell with that of its neighbour. Their primary function is to resist the external forces that pull cells apart. At the same time, however, they need to dynamic and adaptable, so that they can be altered or rearranged when tissues are remodelled or repaired or when there are changes in the forces acting on them.
Cadherins are a diverse family of adhesion molecules that fulfil these requirements. They are present in all multicellular animals whose genomes have been analysed. Other eukaryotes, including fungi and plants, lack cadherins, and they are also absent from bacteria and archaea. Cadherins therefore seem to be part of the essence of what it is to be an animal.
The cadherins take their name from their dependence on Ca2+ ions: removing Ca2+ from the extracellular medium causes adhesions mediated by cadherins to come apart. The first three cadherins to be discovered were named according to the main tissues in which they were found: - E-cadherin is present on many types of epithelial cells; - N-cadherin on nerve, muscle and lens cells; - P-cadherin on cells in the placenta and epidermis. All are also found in other tissues. These and other classical cadherins are closely related in sequence throughout their extracellular and intracellular domains.
Binding between cadherins is generally homophilic. That means cadherin molecules of a specific subtype on one cell bind to cadherin moleculs of the same or closely related subtype on adjacent cells. All members of the superfamily have an extracellular portion consisting of several copies of the extracellular cadherin (EC) domain. Homophilic binding occurs at the N-terminal tips of the cadherin molecules - the cadherin domains that lie furthest from the membrane. These terminal domains each form a knob and a nearby pocket, and teh cadheirn molecules protruding from opposite cell membranes bind by insertion of the knob of one domain into the pocket pf the other.
Each cadherin domain forms a more-or-less rigid unit, joined to the next cadherin domain by a hing. Ca2+ ions bind to sites near each hinge and prevent it from flexing, so that the whole string of cadherin domains behaves as a rigid and slightly curved rod. When Ca2+ is removed, the hinges can flex, and the structure becomes floppy. At the same time, the conformation at the N-terminus is thought to change slightly, weakening the binding affinity for the matching cadherin molecule on the opposite cell.
The cadherins form homodimers in the plasma membrane of each interacting cell. The extracellular domain of one cadherin dimer binds to the extracellular domain of an identical cadherin dimer on the adjacent cell. The intracellular tails of the cadherins bind to anchor proteins that tie them to actin filaments. These anchor proteins include α-catenin, β-catenin, γ-catenin (also called plakoglobin), α-actinin, and vinculin.
UCL iGEM 2017 believes that cadherin proteins will be powerful modulators for efficient tissue engineering. We therefore investigated first the properties of one classical cadherin (E-cadherin, BBa K2332312) and then tried to make it light-responsive.
For more information on cell-cell junctions and cadherins see Alberts B., Molecular Biology of the Cell. 6th ed., Ch.19, New York: Garland Science; 2015.
E-Cadherin Entries in the Registry
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).
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 ensure efficient expression of E-cadherin after transfection.
Chassis Considerations
Choosing the correct chassis for your experiments is of equal importance to choosing the correct gene.
Since we wanted to test cell-cell aggregation induced by the E-cadherin gene, we therefore chose a mammalian cell line that naturally does not express E-cadherin and is commonly used in cadherin research, Chinese Hamster Ovary (CHO) cells. Even though they naturally lack E-cadherin expression they still maintain alpha- and beta-catenin expression, the two proteins that are essential for E-cadherin's connection to the actin cortex of the cell.
Experimental Setup
Choosi
Results
Choosi
Well | Contents | Single Cells | Single Cells Average | Aggregated Cells | Aggregated Cells Average | Ratio single:aggregated |
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A | Cells + superfect + plasmid | 163, 245, 251 | 220 | 195, 242, 311 | 249 | 0.88 |
B | Cells + superfect + plasmid + calcium | 239, 237, 213 | 230 | 251, 342, 477 | 356 | 0.65 |
C | CaCl2 only | 163, 245, 251 | / | / | ||
D | Control cells | 236, 173, 290, 185 | 221 | 157, 145, 116, 258 | 169 | 1.3 |
E | Control cells + calcium | 187, 220, 142 | 183 | 137, 219, 136 | 164 | 1.1 |
F | Untreated cells | 549, 385, 554 | 496 | 500, 397, 582 | 493 | 1.0 |
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
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 of the results 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.
Functional Parameters
Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0 | ||||||||||||||||||||||||||||||||||||||||||||||
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Nucleotide sequence in RFC 10: (underlined part encodes the protein) AGCTTGGTACCTCCACCATGGGAGCC ... GAGGACGACTAGA ORF from nucleotide position 18 to 2669 (excluding stop-codon) | ||||||||||||||||||||||||||||||||||||||||||||||
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)
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Sequence features: (with their position in the amino acid sequence, see the list of supported features)
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Amino acid composition:
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Amino acid counting
| Biochemical parameters
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Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges | ||||||||||||||||||||||||||||||||||||||||||||||
Codon usage
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Alignments (obtained from PredictProtein.org) There were no alignments for this protein in the data base. The BLAST search was initialized and should be ready in a few hours. | ||||||||||||||||||||||||||||||||||||||||||||||
Predictions (obtained from PredictProtein.org) | ||||||||||||||||||||||||||||||||||||||||||||||
There were no predictions for this protein in the data base. The prediction was initialized and should be ready in a few hours. | ||||||||||||||||||||||||||||||||||||||||||||||
The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation. If you have any questions, comments or suggestions, please leave us a comment. |
References
1. 2. 3. 4.
5.