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| − | | + | _NOTOC__ |
| − | __NOTOC__
| + | <partinfo>BBa_K2332311 short</partinfo> |
| − | <partinfo>BBa_K2332314 short</partinfo> | + | |
| | {| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right" | | {| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right" |
| − | ! colspan="2" style="background:#FFBF00;"|E-cadherin_PhoCl Fusion Protein | + | ! colspan="2" style="background:#FFBF00;"|PhoCl, a Mammalian Photocleavable Protein |
| | |- | | |- |
| | |'''Function''' | | |'''Function''' |
| − | |Photoactive Cell-Cell Adhesion | + | |Photocleavable Linker |
| | |- | | |- |
| | |'''Use in''' | | |'''Use in''' |
| | |Mammalian cells | | |Mammalian cells |
| | |- | | |- |
| − |
| |
| | |'''Abstraction Hierarchy''' | | |'''Abstraction Hierarchy''' |
| | |Part | | |Part |
| | |- | | |- |
| | |'''RFC standard''' | | |'''RFC standard''' |
| − | |[https://parts.igem.org/Help:Assembly_standard_10 RFC10] & [https://parts.igem.org/Help:Assembly_standard_23 RFC23] compatible | + | |[https://parts.igem.org/Help:Assembly_standard_10 RFC10], [https://parts.igem.org/Help:Assembly_standard_23 RFC23] & [https://parts.igem.org/Help:Assembly_standard_25 RFC25] compatible |
| | |- | | |- |
| | |'''Backbone''' | | |'''Backbone''' |
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| | |[http://2017.igem.org/Team:UCL UCL iGEM 2017] | | |[http://2017.igem.org/Team:UCL UCL iGEM 2017] |
| | |} | | |} |
| | + | As part of the UCL 2017's project "Light-induced Technologies" we investigated light sensitive proteins and their possible applications in synthetic genetic circuits. PhoCl is a novel (April 2017) photocleavable protein engineered from a green-to-red photoconvertible fluorescent protein. |
| | | | |
| − | This gene encodes a fusion protein beteween E-cadherin, a calcium-dependent cell adhesion molecule, and PhoCl, a photocleavable linker. Just like the original E-cadherin the encoded preproprotein undergoes proteolytic processing to generate a mature protein.
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| | <html> | | <html> |
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| | </html> | | </html> |
| | __TOC__ | | __TOC__ |
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| | | | |
| | ===Usage and Biology=== | | ===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.
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| − | [[File:Adherens junction (cadherin in action).png|400px|thumb|left|
| + | As stated in the original paper: "The photoconversion reaction is a violet light (~400 nm)-induced β-elimination reaction that extends the conjugated system of the chromophore with concomitant cleavage of the polypeptide backbone to form an ~66-residue N-terminal fragment and an ~166-residue C-terminal fragment that remain associated." |
| − | <center>'''Figure 1: Adherens Junction - Cadherin Mediated Cell-Cell Adhesion.'''</center>
| + | |
| | | | |
| − | <p>
| + | The original protein has been engineered by Zhang et al. 2017 (Robert E. Campbell lab). The Campbell lab has sent the original plasmid to the UCL iGEM 2017 team as part of a collaboration and the team has made a BioBrick out of the engineered protein. |
| − | (A) Adherens junctions, in the form of adhesion belts, between epithelial cells in the small intestine. The beltlike junction encircles each of the interacting cells. Its most obvious feature is a contractile bundle of actin filaments running along the cytoplasmic surface of the junctional plasma membrane. (B) Some of the molecules that form an adherens junction. The actin filaments are joined from cell to cell by transmembrane adhesion proteins called cadherins. (Alberts B. Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015)
| + | |
| − | </p>''' ]]
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| − | | + | |
| − | | + | |
| − | 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 Ca<sup>2+</sup> ions: removing Ca<sup>2+</sup> 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:
| + | |
| − | <p>
| + | |
| − | - E-cadherin is present on many types of epithelial cells;</p>
| + | |
| − | <p>
| + | |
| − | - N-cadherin on nerve, muscle and lens cells;</p>
| + | |
| − | <p>
| + | |
| − | - P-cadherin on cells in the placenta and epidermis.</p>
| + | |
| − | 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. This 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 the cadheirn molecules protruding from opposite cell membranes bind by insertion of the knob of one domain into the pocket of the other.
| + | |
| − | | + | |
| − | [[File:Cadherin Function, Alberts et al. 2015, Figure 19-6.png|500px|thumb|right|
| + | |
| − | <center>'''Figure 2: Molecular Model of E-cadherin'''</center>
| + | |
| − | <p>
| + | |
| − | After processing in the late Golgi, E-cadherin contains five EC domains. The outermost EC domain forms homophilic connections with the equivalent domain of E-cadherin on the neighbouring cell. The stability of E-cadherin depends on the presence of Ca<sup>2+</sup> in the extracellular space. (Alberts B. Molecular Biology of the Cell. 6th ed. Figure 19-6 New York: Garland Science; 2015)]]
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| − | Each cadherin domain forms a more-or-less rigid unit, joined to the next cadherin domain by a hing. Ca<sup>2+</sup> 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 Ca<sup>2+</sup> 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.
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| − | | + | |
| − | | + | |
| − | ===E-Cadherin Entries in the Registry===
| + | |
| − | | + | |
| − | UCSF iGEM 2011 has created a BioBrick of only the extracellular domain of E-Cadherin (Mouse) [https://parts.igem.org/Part:BBa_K644000:Design 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===
| + | |
| − | [[File:pcDNA3 containing E-cadherin Map.png|200px|thumb|right|
| + | |
| − | <center>'''Figure 3: E-cadherin in pcDNA3 Map'''</center> ]]
| + | |
| − | | + | |
| − | =====Vector Considerations=====
| + | |
| − | For testing this coding part we used pcDNA3 [http://www.snapgene.com/resources/plasmid_files/basic_cloning_vectors/pcDNA3/ (SnapGene File)], a standard mammalian expression plasmid, as a vector. We, thereby, created the coding device [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 well characterised strong promoter, efficient poly(A) tail and the pre-existing 5'- and 3'-UTR ensure efficient expression of E-cadherin after transfection.
| + | |
| − | | + | |
| − | There are many ways to express mammalian genes. Using a standard mammalian expression plasmid saves time and reduces the risk of low expression due to variations in 5'- and 3'- UTR.
| + | |
| − | | + | |
| − | =====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
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | ===Usability===
| + | |
| − | A functional,
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | =====Primer Designs=====
| + | |
| − | | + | |
| − | PCR out Primers
| + | |
| − | 5'- -3', Ecadh_BioBrick.FwP
| + | |
| − | 5'- -3', Ecadh_BioBrick.RevP
| + | |
| − | | + | |
| − | These
| + | |
| − | | + | |
| − | | + | |
| − | Sequencing Primers
| + | |
| − | <p>We used Sanger sequencing for the sequencing of our E-cadherin gene. However, since Sanger sequencing only ensures correct results for up to around 800 bp we needed to use 2 sequencing steps ('primer walking') with two primers in each step:</p>
| + | |
| − | | + | |
| − | Since the gene was in pcDNA3 we used the standard primers for pcDNA3.1 for the first round of sequencing:
| + | |
| − | 5'- ctctggctaactagagaac -3', pcDNA3.1-FwP
| + | |
| − | 5'- caaacaacagatggctggc -3', pcDNA3.1-RevP
| + | |
| − | | + | |
| − | For the second round of sequencing we designed and synthesized the following primers:
| + | |
| − | 5'- -3', E-cadh.sequ.Round2-FwP
| + | |
| − | 5'- -3', E-cadh.sequ.Round2-RevP
| + | |
| − | | + | |
| − | =====Characterisation Opportunities=====
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| − | | + | |
| − | This
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| | ===Sequence and Features=== | | ===Sequence and Features=== |
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| − | =====DNA Features=====
| + | <partinfo>BBa_K2332311 SequenceAndFeatures</partinfo> |
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| − | <partinfo>BBa_K2332314 SequenceAndFeatures</partinfo>
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| − |
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| − |
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| − | =====Protein Features=====
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| | ===Functional Parameters=== | | ===Functional Parameters=== |
| − | <partinfo>BBa_K2332314 parameters</partinfo> | + | <partinfo>BBa_K2332311 parameters</partinfo> |
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| | + | ===Reference=== |
| | | | |
| − | ===References===
| + | Zhang W, Lohman AW, Zhuravlova Y, Lu X, Wiens MD, Hoi H, Yaganoglu S, Mohr MA, Kitova EN, Klassen JS, Pantazis P, Thompson RJ, Campbell RE. Optogenetic control with a photocleavable protein, PhoCl. Nat Methods. 14(4):391-394 (2017) [https://www.ncbi.nlm.nih.gov/pubmed/28288123 NCBI] |
| − | 1.
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| − | 2.
| + | |
| − | 3.
| + | |
| − | 4. | + | |
| − | 5.
| + | |
As part of the UCL 2017's project "Light-induced Technologies" we investigated light sensitive proteins and their possible applications in synthetic genetic circuits. PhoCl is a novel (April 2017) photocleavable protein engineered from a green-to-red photoconvertible fluorescent protein.
As stated in the original paper: "The photoconversion reaction is a violet light (~400 nm)-induced β-elimination reaction that extends the conjugated system of the chromophore with concomitant cleavage of the polypeptide backbone to form an ~66-residue N-terminal fragment and an ~166-residue C-terminal fragment that remain associated."
The original protein has been engineered by Zhang et al. 2017 (Robert E. Campbell lab). The Campbell lab has sent the original plasmid to the UCL iGEM 2017 team as part of a collaboration and the team has made a BioBrick out of the engineered protein.
Zhang W, Lohman AW, Zhuravlova Y, Lu X, Wiens MD, Hoi H, Yaganoglu S, Mohr MA, Kitova EN, Klassen JS, Pantazis P, Thompson RJ, Campbell RE. Optogenetic control with a photocleavable protein, PhoCl. Nat Methods. 14(4):391-394 (2017) NCBI
