Difference between revisions of "Part:BBa K2332314"
 
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__NOTOC__
 
 
<partinfo>BBa_K2332314 short</partinfo>
 
<partinfo>BBa_K2332314 short</partinfo>
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{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right"
 
{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right"
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! colspan="2" style="background:#FFBF00;"|E-cadherin_PhoCl Fusion Protein
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! colspan="2" style="background:#FFBF00;"|E-cadherin_PhoCl Fusion Protein, a photosensitive cell adhesion protein
 
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|'''Function'''
 
|'''Function'''
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|Photoactive Cell-Cell Adhesion
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|Photo-sensitive cell-cell adhesion
 
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|'''Use in'''
 
|'''Use in'''
 
|Mammalian cells
 
|Mammalian cells
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|'''Chassis Tested'''
 
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|Chinese Hamster Ovary (CHO)
 
 
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|'''Abstraction Hierarchy'''
 
|'''Abstraction Hierarchy'''
 
|Part
 
|Part
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|-
 
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|'''Related Device'''
 
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|[https://parts.igem.org/Part:BBa_K2332313 BBa_K2332313]
 
 
|-
 
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|'''RFC standard'''
 
|'''RFC standard'''
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|[https://parts.igem.org/Help:Assembly_standard_10 RFC10] & [https://parts.igem.org/Help:Assembly_standard_23 RFC23] compatible  
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|[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]
 
|}
 
|}
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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.
  
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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.
 
  
 
<html>
 
<html>
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</html>
 
</html>
 
__TOC__
 
__TOC__
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===Usage and Biology===
 
===Usage and Biology===
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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:UCL-iGEM17_Light-induced Mammalian_Cell-Adhesion.gif|400px|thumb|right|
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<center>'''Figure 1: Light-activation of E-cadherin.PhoCl fusion protein'''</center> ]]
  
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[[File:Adherens junction (cadherin in action).png|400px|thumb|left|
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As stated in the original paper: "The photoconversion reaction is a violet light (~400 nm)-induced &#946;-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."
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<center>'''Figure 1: Adherens Junction - Cadherin Mediated Cell-Cell Adhesion.'''</center>
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<p>
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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.
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(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)
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</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.
 
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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:
 
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<p>
 
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- E-cadherin is present on many types of epithelial cells;</p>
 
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<p>
 
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- N-cadherin on nerve, muscle and lens cells;</p>
 
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<p>
 
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- P-cadherin on cells in the placenta and epidermis.</p>
 
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All are also found in other tissues.
 
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These and other classical cadherins are closely related in sequence throughout their extracellular and intracellular domains.
 
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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.
 
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[[File:Cadherin Function, Alberts et al. 2015, Figure 19-6.png|500px|thumb|right|
 
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<center>'''Figure 2: Molecular Model of E-cadherin'''</center>
 
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<p>
 
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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.
 
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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.
 
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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.
 
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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===
 
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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).
 
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===Experimental approach===
 
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[[File:pcDNA3 containing E-cadherin Map.png|200px|thumb|right|
 
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<center>'''Figure 3: E-cadherin in pcDNA3 Map'''</center> ]]
 
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=====Vector Considerations=====
 
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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.
 
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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.
 
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=====Chassis Considerations=====
 
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Choosing the correct chassis for your experiments is of equal importance to choosing the correct gene.
 
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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.
 
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=====Experimental Setup=====
 
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Choosi
 
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=====Results=====
 
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Choosi
 
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<center>'''Brightfield Microscopy Images of the Cell Aggregation Experiment'''</center>
 
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<p>
 
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.
 
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</p>
 
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<html>
 
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<head>
 
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<style>
 
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table, th, td {
 
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    text-align: center;
 
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    border: 1px solid black;
 
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    border-collapse: collapse;
 
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}
 
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</style>
 
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</head>
 
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<body>
 
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<table style="width:80%">
 
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<table align="center">
 
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  <tr>
 
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    <th>Well</th>
 
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    <th>Contents</th>
 
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    <th>Single Cells</th>
 
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    <th>Single Cells Average</th>
 
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    <th>Aggregated Cells</th>
 
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    <th>Aggregated Cells Average</th>
 
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    <th>Ratio single:aggregated</th>
 
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  </tr>
 
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  <tr>
 
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    <td>A</td>
 
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    <td>Cells + superfect + plasmid</td>
 
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    <td>163, 245, 251</td>
 
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    <td>220</td>
 
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    <td>195, 242, 311</td>
 
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    <td>249</td>
 
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    <td>0.88</td>
 
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  </tr>
 
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  <tr>
 
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    <td>B</td>
 
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    <td>Cells + superfect + plasmid + calcium</td>
 
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    <td>239, 237, 213</td>
 
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    <td>230</td>
 
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    <td>251, 342, 477</td>
 
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    <td>356</td>
 
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    <td>0.65</td>
 
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  </tr>
 
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  <tr>
 
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    <td>C</td>
 
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    <td>CaCl<sub>2</sub> only</td>
 
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    <td>/</td>
 
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    <td></td>
 
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    <td>/</td>
 
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    <td></td>
 
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    <td></td>
 
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  </tr>
 
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  <tr>
 
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    <td>D</td>
 
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    <td>Control cells</td>
 
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    <td>236, 173, 290, 185</td>
 
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    <td>221</td>
 
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    <td>157, 145, 116, 258</td>
 
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    <td>169</td>
 
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    <td>1.3</td>
 
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  </tr>
 
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  <tr>
 
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    <td>E</td>
 
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    <td>Control cells + calcium</td>
 
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    <td>187, 220, 142</td>
 
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    <td>183</td>
 
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    <td>137, 219, 136</td>
 
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    <td>164</td>
 
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    <td>1.1</td>
 
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  </tr>
 
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  <tr>
 
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    <td>F</td>
 
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    <td>Untreated cells</td>
 
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    <td>549, 385, 554</td>
 
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    <td>496</td>
 
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    <td>500, 397, 582</td>
 
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    <td>493</td>
 
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    <td>1.0</td>
 
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  </tr>
 
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</table>
 
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</body>
 
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</html>
 
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<center>'''Table 1: Cell Aggregation Table'''</center>
 
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Control cells = treated with superfect + PBS instead of plasmid
 
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]]
 
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===Usability===
 
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A functional,
 
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=====Primer Designs=====
 
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PCR out Primers
 
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5'- -3', Ecadh_BioBrick.FwP
 
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5'- -3', Ecadh_BioBrick.RevP
 
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These
 
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Sequencing Primers
 
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<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>
 
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Since the gene was in pcDNA3 we used the standard primers for pcDNA3.1 for the first round of sequencing:
 
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5'- ctctggctaactagagaac -3', pcDNA3.1-FwP
 
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5'- caaacaacagatggctggc -3', pcDNA3.1-RevP
 
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For the second round of sequencing we designed and synthesized the following primers:
 
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5'- -3', E-cadh.sequ.Round2-FwP
 
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5'- -3', E-cadh.sequ.Round2-RevP
 
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=====Characterisation Opportunities=====
 
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This
 
  
  
  
 
===Sequence and Features===
 
===Sequence and Features===
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=====DNA Features=====
 
  
 
<partinfo>BBa_K2332314 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2332314 SequenceAndFeatures</partinfo>
  
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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, [https://www.ncbi.nlm.nih.gov/nuccore/NM_009864 NCBI].
 
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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.
 
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=====Protein Features=====
 
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[[File:E-cadherin (Preproprotein BLAST).png|700px|thumb|center|'''Figure 10: Protein BLAST Results from E-cadherin''' [https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi?RID=XJK3RWW6014&mode=all (Link)].
 
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<p>
 
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[https://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?ascbin=8&maxaln=10&seltype=2&uid=smart01055 <b><font color="green">Cadherin Prodomain Like</font></b>    ]- Cadherin proteins are activated through cleavage of a prosequence in the late Golgi. This prevents cadherin aggregation in the early stage of the secretory pathway. This domain corresponds to the folded region of the prosequence, and is termed the prodomain. The prodomain shows structural resemblance to the cadherin domain, but lacks all the features known to be important for cadherin-cadherin interactions.
 
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</p>
 
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[https://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?ascbin=8&maxaln=10&seltype=2&uid=cd00031 <b><font color="LightSteelBlue">Cadherin Repeat-Like Domain</font></b>]- The cadherin repeat domains occur as tandem repeats in the extracellular regions, which are thought to mediate cell-cell contact when bound to calcium. They play numerous roles in cell fate, signalling, proliferation, differentiation, and migration; members include E-, N-, P-, T-, VE-, CNR-, proto-, and FAT-family cadherin, desmocollin, and desmoglein, a large variety of domain architectures with varying repeat copy numbers. Cadherin-repeat containing proteins exist as monomers, homodimers, or heterodimers. This family also includes the cadherin-like repeats of extracellular alpha-dystroglycan.
 
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<p>
 
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[https://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?ascbin=8&maxaln=10&seltype=2&uid=pfam01049 <b><font color="teal">Cadherin Cytoplasmic Region</font></b>]- Cadherins are vital in cell-cell adhesion during tissue differentiation. Cadherins are linked to the cytoskeleton by catenins. Catenins bind to the cytoplasmic tail of the cadherin. Cadherins cluster to form foci of homophilic binding units. A key determinant to the strength of the binding that it is mediated by cadherins is the juxtamembrane region of the cadherin. This region induces clustering and also binds to the protein p120ctn.'''  ]]
 
  
  
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2332314 parameters</partinfo>
 
<partinfo>BBa_K2332314 parameters</partinfo>
 +
<html><!--- Please copy this table containing parameters for BBa_ at the end of the parametrs section ahead of the references. ---><style type="text/css">table#AutoAnnotator {border:1px solid black; width:100%; border-collapse:collapse;} th#AutoAnnotatorHeader { border:1px solid black; width:100%; background-color: rgb(221, 221, 221);} td.AutoAnnotator1col { width:100%; border:1px solid black; } span.AutoAnnotatorSequence { font-family:'Courier New', Arial; } td.AutoAnnotatorSeqNum { text-align:right; width:2%; } td.AutoAnnotatorSeqSeq { width:98% } td.AutoAnnotatorSeqFeat1 { width:3% } td.AutoAnnotatorSeqFeat2a { width:27% } td.AutoAnnotatorSeqFeat2b { width:97% } td.AutoAnnotatorSeqFeat3 { width:70% } table.AutoAnnotatorNoBorder { border:0px; width:100%; border-collapse:collapse; } table.AutoAnnotatorWithBorder { border:1px solid black; width:100%; border-collapse:collapse; } td.AutoAnnotatorOuterAmino { border:0px solid black; width:20% } td.AutoAnnotatorInnerAmino { border:1px solid black; width:50% } td.AutoAnnotatorAminoCountingOuter { border:1px solid black; width:40%;  } td.AutoAnnotatorBiochemParOuter { border:1px solid black; width:60%; } td.AutoAnnotatorAminoCountingInner1 { width: 7.5% } td.AutoAnnotatorAminoCountingInner2 { width:62.5% } td.AutoAnnotatorAminoCountingInner3 { width:30% } td.AutoAnnotatorBiochemParInner1 { width: 5% } td.AutoAnnotatorBiochemParInner2 { width:55% } td.AutoAnnotatorBiochemParInner3 { width:40% } td.AutoAnnotatorCodonUsage1 { width: 3% } td.AutoAnnotatorCodonUsage2 { width:14.2% } td.AutoAnnotatorCodonUsage3 { width:13.8% } td.AutoAnnotatorAlignment1 { width: 3% } td.AutoAnnotatorAlignment2 { width: 10% } td.AutoAnnotatorAlignment3 { width: 87% } td.AutoAnnotatorLocalizationOuter {border:1px solid black; width:40%} td.AutoAnnotatorGOOuter {border:1px solid black; width:60%} td.AutoAnnotatorLocalization1 { width: 7.5% } td.AutoAnnotatorLocalization2 { width: 22.5% } td.AutoAnnotatorLocalization3 { width: 70% } td.AutoAnnotatorGO1 { width: 5% } td.AutoAnnotatorGO2 { width: 35% } td.AutoAnnotatorGO3 { width: 60% } td.AutoAnnotatorPredFeat1 { width:3% } td.AutoAnnotatorPredFeat2a { width:27% } td.AutoAnnotatorPredFeat3 { width:70% } div.AutoAnnotator_trans { position:absolute; background:rgb(11,140,143); background-color:rgba(11,140,143, 0.8); height:5px; top:100px; } div.AutoAnnotator_sec_helix { position:absolute; background:rgb(102,0,102); background-color:rgba(102,0,102, 0.8); height:5px; top:110px; } div.AutoAnnotator_sec_strand { position:absolute; background:rgb(245,170,26); background-color:rgba(245,170,26, 1); height:5px; top:110px; } div.AutoAnnotator_acc_buried { position:absolute; background:rgb(89,168,15); background-color:rgba(89,168,15, 0.8); height:5px; top:120px; } div.AutoAnnotator_acc_exposed { position:absolute; background:rgb(0, 0, 255); background-color:rgba(0, 0, 255, 0.8); height:5px; top:120px; } div.AutoAnnotator_dis { position:absolute; text-align:center; font-family:Arial,Helvetica,sans-serif; background:rgb(255, 200, 0); background-color:rgba(255, 200, 0, 1); height:16px; width:16px; top:80px; border-radius:50%; } </style><div id='AutoAnnotator_container_1508714936960'><table id="AutoAnnotator"><tr><!-- Time stamp in ms since 1/1/1970 1508714936960 --><th id="AutoAnnotatorHeader" colspan="2">Protein data table for BioBrick <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_<!------------------------Enter BioBrick number here------------------------>">BBa_<!------------------------Enter BioBrick number here------------------------></a> automatically created by the <a href="http://2013.igem.org/Team:TU-Munich/Results/AutoAnnotator">BioBrick-AutoAnnotator</a> version 1.0</th></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Nucleotide sequence</strong> in <strong>RFC 10</strong>: (underlined part encodes the protein)<br><span class="AutoAnnotatorSequence">&nbsp;AGCTTGGTACCTCCACC<u>ATGGGAGCC&nbsp;...&nbsp;GGAGGTACC</u>TAACCTATTCTATAG</span><br>&nbsp;<strong>ORF</strong> from nucleotide position 18 to 3425 (excluding stop-codon)</td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Amino acid sequence:</strong> (RFC 25 scars in shown in bold, other sequence features underlined; both given below)<br><span class="AutoAnnotatorSequence"><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorSeqNum">1&nbsp;<br>101&nbsp;<br>201&nbsp;<br>301&nbsp;<br>401&nbsp;<br>501&nbsp;<br>601&nbsp;<br>701&nbsp;<br>801&nbsp;<br>901&nbsp;<br>1001&nbsp;<br>1101&nbsp;</td><td class="AutoAnnotatorSeqSeq">MGARCRSFSALLLLLQVSSWLCQELEPESCSPGFSSEVYTFPVPEGHLERGHVLGRVRFEGC<b>TG</b>RPRTAFFSEDSRFKVATDGTITVKRHLKLHKLETSF<br>LVRARDSSHRELSTKVTLKSMGHHHHRHHHRDPASESNPELLMFPSVYPGLRRQKRDWVIPPISCPENEKGEFPKNLVQIKSNRDKETKVFYSI<b>TG</b>QGAD<br>KPPVGVFIIERETGWLKVTQPLDREAIAKYILYSHAVSSNGEAVEDPMEIVITVTDQNDNRPEFTQEVFEGSVAEGAVPGTSVMKVSATDADDDVNTYNA<br>AIAYTIVSQDPELPHKNMFTVNRDTGVISVLTSGLDRESYPTYTLVVQAADLQGEGLSTTAKAVITVKDINDNAPVFNPSTYQGQVPENEVNARIATLKV<br>TDDDAPNTPAWKAVYTVVNDPDQQFVVVTDPTTNDGILKTAKGLDFEAKQQYILHVRVENEEPFEGSLVPSTATVTVDVVDVNEAPIFMPAERRVEVPED<br>FGVGQEITSYTAREPDTFMDQKITYRIWRDTANWLEINPETGAIFTRAEMDREDAEHVKNSTYVALIIATDDGSPIATGTGTLLLVLLDVNDNAPIPEPR<br>NMQFCQRNPQPHIITILDPDLPPNTSPFTAELTHGASVNWTIEYNDAAQESLILQPRKDLEIGEYKIHLKLADNQNKDQVTTLDVHVCDCEGTVNNCMKA<br>GIVAAGLQVPAILGILGGILALLILILLLLLFLRRRTVVKEPLLPPDDDTRDNVYYYDEEGGGEEDQDFDLSQLHRGLDARPEVTRNDVAPTLMSVPQYR<br>PRPANPDEIGNFIDENLKAADSDPTAPPYDSLLVFDYEGSGSEAASLSSLNSSESDQDQDYDYLNEWGNRFKKLADMYGGGEDDGGSGGVIPDYFKQSFP<br>EGYSWERSMTYEDGGICIATNDITMEGDSFINKIHFKGTNFPPNGPVMQKRTVGWEASTEKMYERDGVLKGDVKMKLLLKGGGHYRCDYRTTYKVKQKPV<br>KLPDYHFVDHRIEILSHDKDYNKVKLYEHAVARNSTDSMDELYKGGSGGMVSKGEETITSVIKPDMKNKLRMEGNVNGHAFVIEGEGSGKPFEGIQTIDL<br>EVKEGAPLPFAYDILTTAFHYGNRVFTKYPRGGGGT*</td></tr></table></span></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Sequence features:</strong> (with their position in the amino acid sequence, see the <a href="http://2013.igem.org/Team:TU-Munich/Results/Software/FeatureList">list of supported features</a>)<table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorSeqFeat1"></td><td class="AutoAnnotatorSeqFeat2a">RFC25 scar (shown in bold):&nbsp;</td><td class="AutoAnnotatorSeqFeat3">63 to 64, 195 to 196</td></tr></table></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Amino acid composition:</strong><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Ala (A)</td><td class="AutoAnnotatorInnerAmino">69 (6.1%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Arg (R)</td><td class="AutoAnnotatorInnerAmino">55 (4.8%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Asn (N)</td><td class="AutoAnnotatorInnerAmino">53 (4.7%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Asp (D)</td><td class="AutoAnnotatorInnerAmino">88 (7.7%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Cys (C)</td><td class="AutoAnnotatorInnerAmino">11 (1.0%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Gln (Q)</td><td class="AutoAnnotatorInnerAmino">36 (3.2%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Glu (E)</td><td class="AutoAnnotatorInnerAmino">85 (7.5%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Gly (G)</td><td class="AutoAnnotatorInnerAmino">85 (7.5%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">His (H)</td><td class="AutoAnnotatorInnerAmino">29 (2.6%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Ile (I)</td><td class="AutoAnnotatorInnerAmino">58 (5.1%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Leu (L)</td><td class="AutoAnnotatorInnerAmino">87 (7.7%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Lys (K)</td><td class="AutoAnnotatorInnerAmino">59 (5.2%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Met (M)</td><td class="AutoAnnotatorInnerAmino">22 (1.9%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Phe (F)</td><td class="AutoAnnotatorInnerAmino">41 (3.6%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Pro (P)</td><td class="AutoAnnotatorInnerAmino">72 (6.3%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Ser (S)</td><td class="AutoAnnotatorInnerAmino">66 (5.8%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Thr (T)</td><td class="AutoAnnotatorInnerAmino">81 (7.1%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Trp (W)</td><td class="AutoAnnotatorInnerAmino">10 (0.9%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Tyr (Y)</td><td class="AutoAnnotatorInnerAmino">40 (3.5%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Val (V)</td><td class="AutoAnnotatorInnerAmino">89 (7.8%)</td></tr></table></td></tr></table></td></tr><tr><td class="AutoAnnotatorAminoCountingOuter"><strong>Amino acid counting</strong><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Total number:</td><td class="AutoAnnotatorAminoCountingInner3">1136</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Positively charged (Arg+Lys):</td><td class="AutoAnnotatorAminoCountingInner3">114 (10.0%)</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Negatively charged (Asp+Glu):</td><td class="AutoAnnotatorAminoCountingInner3">173 (15.2%)</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Aromatic (Phe+His+Try+Tyr):</td><td class="AutoAnnotatorAminoCountingInner3">120 (10.6%)</td></tr></table></td><td class="AutoAnnotatorBiochemParOuter"><strong>Biochemical parameters</strong><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Atomic composition:</td><td class="AutoAnnotatorBiochemParInner3">C<sub>5583</sub>H<sub>8693</sub>N<sub>1517</sub>O<sub>1759</sub>S<sub>33</sub></td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Molecular mass [Da]:</td><td class="AutoAnnotatorBiochemParInner3">126268.5</td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Theoretical pI:</td><td class="AutoAnnotatorBiochemParInner3">4.91</td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Extinction coefficient at 280 nm [M<sup>-1</sup> cm<sup>-1</sup>]:</td><td class="AutoAnnotatorBiochemParInner3">114600 / 115288 (all Cys red/ox)</td></tr></table></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges</strong>&nbsp;<input type='button' id='hydrophobicity_charge_button' onclick='show_or_hide_plot_1508714936960()' value='Show'><span id="hydrophobicity_charge_explanation"></span><div id="hydrophobicity_charge_container" style='display:none'><div id="hydrophobicity_charge_placeholder0" style="width:100%;height:150px"></div><div id="hydrophobicity_charge_placeholder1" style="width:100%;height:150px"></div><div id="hydrophobicity_charge_placeholder2" style="width:100%;height:150px"></div><div id="hydrophobicity_charge_placeholder3" style="width:100%;height:150px"></div><div id="hydrophobicity_charge_placeholder4" style="width:100%;height:150px"></div><div id="hydrophobicity_charge_placeholder5" style="width:100%;height:150px"></div></div></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Codon usage</strong><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorCodonUsage1"></td><td class="AutoAnnotatorCodonUsage2">Organism:</td><td class="AutoAnnotatorCodonUsage3"><i>E. coli</i></td><td class="AutoAnnotatorCodonUsage3"><i>B. subtilis</i></td><td class="AutoAnnotatorCodonUsage3"><i>S. cerevisiae</i></td><td class="AutoAnnotatorCodonUsage3"><i>A. thaliana</i></td><td class="AutoAnnotatorCodonUsage3"><i>P. patens</i></td><td class="AutoAnnotatorCodonUsage3">Mammals</td></tr><tr><td class="AutoAnnotatorCodonUsage1"></td><td class="AutoAnnotatorCodonUsage2">Codon quality (<a href="http://en.wikipedia.org/wiki/Codon_Adaptation_Index">CAI</a>):</td><td class="AutoAnnotatorCodonUsage3">good (0.70)</td><td class="AutoAnnotatorCodonUsage3">good (0.69)</td><td class="AutoAnnotatorCodonUsage3">acceptable (0.57)</td><td class="AutoAnnotatorCodonUsage3">good (0.67)</td><td class="AutoAnnotatorCodonUsage3">excellent (0.85)</td><td class="AutoAnnotatorCodonUsage3">excellent (0.82)</td></tr></table></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Alignments</strong> (obtained from <a href='http://predictprotein.org'>PredictProtein.org</a>)<br>&nbsp;&nbsp;&nbsp;There were no alignments for this protein in the data base. The BLAST search was initialized and should be ready in a few hours.</td></tr><tr><th id='AutoAnnotatorHeader' colspan="2"><strong>Predictions</strong> (obtained from <a href='http://predictprotein.org'>PredictProtein.org</a>)</th></tr><tr><td class="AutoAnnotator1col" colspan="2">&nbsp;&nbsp;&nbsp;There were no predictions for this protein in the data base. The prediction was initialized and should be ready in a few hours.</td><tr><td class="AutoAnnotator1col" colspan="2"> The BioBrick-AutoAnnotator was created by <a href="http://2013.igem.org/Team:TU-Munich">TU-Munich 2013</a> iGEM team. For more information please see the <a href="http://2013.igem.org/Team:TU-Munich/Results/Software">documentation</a>.<br>If you have any questions, comments or suggestions, please leave us a <a href="http://2013.igem.org/Team:TU-Munich/Results/AutoAnnotator">comment</a>.</td></tr></table></div><br><!-- IMPORTANT: DON'T REMOVE THIS LINE, OTHERWISE NOT SUPPORTED FOR IE BEFORE 9 --><!--[if lte IE 8]><script language="javascript" type="text/javascript" src="http://2013.igem.org/Team:TU-Munich/excanvas.js"></script><![endif]--><script type='text/javascript' src='http://code.jquery.com/jquery-1.10.0.min.js'></script><script type='text/javascript' src='http://2013.igem.org/Team:TU-Munich/Flot.js?action=raw&ctype=text/js'></script><script>var jqAutoAnnotator = jQuery.noConflict(true);function show_or_hide_plot_1508714936960(){hydrophobicity_datapoints = 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checked=\'checked\'>&nbsp;Predicted disulfid bridges (<img src=\'https://static.igem.org/mediawiki/2013/2/28/TUM13_dis_icon.png\' alt=\'yellow circle\' height=\'10\'></img>) with the number of the bridge in the center';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'trans_checkbox\' checked=\'checked\'>&nbsp;Predicted transmembrane helices (<img src=\'https://static.igem.org/mediawiki/2013/7/78/TUM13_trans_icon.png\' alt=\'turquois bars\' height=\'10\'></img>)';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'sec_checkbox\' checked=\'checked\'>&nbsp;Predicted secondary structure: Helices (<img src=\'https://static.igem.org/mediawiki/2013/b/bf/TUM13_helix_icon.png\' alt=\'violet bars\' height=\'10\'></img>) and beta-strands (<img src=\'https://static.igem.org/mediawiki/2013/b/bf/TUM13_strand_icon.png\' alt=\'yellow bars\' height=\'10\'></img>)';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'acc_checkbox\' checked=\'checked\'>&nbsp;Predicted solvent accessability: Exposed (<img src=\'https://static.igem.org/mediawiki/2013/1/16/TUM13_exposed_icon.png\' alt=\'blue bars\' height=\'10\'></img>) and buried (<img src=\'https://static.igem.org/mediawiki/2013/0/0b/TUM13_buried_icon.png\' alt=\'green bars\' height=\'10\'></img>) residues';description_html = description_html + '<br></div>';jqAutoAnnotator('#AutoAnnotator_container_1508714936960 #hydrophobicity_charge_explanation').html(description_html);plot_according_to_selectors_1508714936960();jqAutoAnnotator('#AutoAnnotator_container_1508714936960 #AutoAnnotator_plot_selectors').find('input').click(plot_according_to_selectors_1508714936960);}else{jqAutoAnnotator('#AutoAnnotator_container_1508714936960 #hydrophobicity_charge_container').css('display','none');jqAutoAnnotator('#AutoAnnotator_container_1508714936960 #hydrophobicity_charge_button').val('Show');jqAutoAnnotator('#AutoAnnotator_container_1508714936960 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1)/200)*200*tick_diff).toFixed(0)).toString() + 'px\'></div>');}}if(jqAutoAnnotator('#AutoAnnotator_container_1508714936960 #acc_checkbox').prop('checked') == true){for ( j = 0 ; j < acc_buried_datapoints.length ; j++ ){jqAutoAnnotator('#AutoAnnotator_container_1508714936960 #hydrophobicity_charge_placeholder' + Math.floor((acc_buried_datapoints[j][0] - 1)/200) ).append('<div class=\'AutoAnnotator_acc_buried\' style=\'width:' + (((acc_buried_datapoints[j][1] - acc_buried_datapoints[j][0] + 1)*tick_diff).toFixed(0)).toString() + 'px; left:' + ((pos_of_first_tick + (acc_buried_datapoints[j][0] - 1.5)*tick_diff - Math.floor((acc_buried_datapoints[j][0] - 1)/200)*200*tick_diff).toFixed(0)).toString() + 'px\'></div>');}for ( j = 0 ; j < acc_exposed_datapoints.length ; j++ ){jqAutoAnnotator('#AutoAnnotator_container_1508714936960 #hydrophobicity_charge_placeholder' + Math.floor((acc_exposed_datapoints[j][0] - 1)/200) ).append('<div class=\'AutoAnnotator_acc_exposed\' style=\'width:' + 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 +
===Reference===
  
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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]
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===References===
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Latest revision as of 00:35, 23 October 2017

E-cadherin_PhoCl Fusion Protein, a photosensitive cell adhesion protein

E-cadherin_PhoCl Fusion Protein, a photosensitive cell adhesion protein
Function Photo-sensitive cell-cell adhesion
Use in Mammalian cells
Abstraction Hierarchy Part
RFC standard RFC10, RFC23 & RFC25 compatible
Backbone pSB1C3
Submitted by [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.



Usage and Biology

Figure 1: Light-activation of E-cadherin.PhoCl fusion 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.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 2550
  • 21
    INCOMPATIBLE 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
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 208
  • 1000
    INCOMPATIBLE 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
    Illegal BsaI.rc site found at 3183



Functional Parameters

Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0
Nucleotide sequence in RFC 10: (underlined part encodes the protein)
 AGCTTGGTACCTCCACCATGGGAGCC ... GGAGGTACCTAACCTATTCTATAG
 ORF from nucleotide position 18 to 3425 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)
1 
101 
201 
301 
401 
501 
601 
701 
801 
901 
1001 
1101 		MGARCRSFSALLLLLQVSSWLCQELEPESCSPGFSSEVYTFPVPEGHLERGHVLGRVRFEGCTGRPRTAFFSEDSRFKVATDGTITVKRHLKLHKLETSF
LVRARDSSHRELSTKVTLKSMGHHHHRHHHRDPASESNPELLMFPSVYPGLRRQKRDWVIPPISCPENEKGEFPKNLVQIKSNRDKETKVFYSITGQGAD
KPPVGVFIIERETGWLKVTQPLDREAIAKYILYSHAVSSNGEAVEDPMEIVITVTDQNDNRPEFTQEVFEGSVAEGAVPGTSVMKVSATDADDDVNTYNA
AIAYTIVSQDPELPHKNMFTVNRDTGVISVLTSGLDRESYPTYTLVVQAADLQGEGLSTTAKAVITVKDINDNAPVFNPSTYQGQVPENEVNARIATLKV
TDDDAPNTPAWKAVYTVVNDPDQQFVVVTDPTTNDGILKTAKGLDFEAKQQYILHVRVENEEPFEGSLVPSTATVTVDVVDVNEAPIFMPAERRVEVPED
FGVGQEITSYTAREPDTFMDQKITYRIWRDTANWLEINPETGAIFTRAEMDREDAEHVKNSTYVALIIATDDGSPIATGTGTLLLVLLDVNDNAPIPEPR
NMQFCQRNPQPHIITILDPDLPPNTSPFTAELTHGASVNWTIEYNDAAQESLILQPRKDLEIGEYKIHLKLADNQNKDQVTTLDVHVCDCEGTVNNCMKA
GIVAAGLQVPAILGILGGILALLILILLLLLFLRRRTVVKEPLLPPDDDTRDNVYYYDEEGGGEEDQDFDLSQLHRGLDARPEVTRNDVAPTLMSVPQYR
PRPANPDEIGNFIDENLKAADSDPTAPPYDSLLVFDYEGSGSEAASLSSLNSSESDQDQDYDYLNEWGNRFKKLADMYGGGEDDGGSGGVIPDYFKQSFP
EGYSWERSMTYEDGGICIATNDITMEGDSFINKIHFKGTNFPPNGPVMQKRTVGWEASTEKMYERDGVLKGDVKMKLLLKGGGHYRCDYRTTYKVKQKPV
KLPDYHFVDHRIEILSHDKDYNKVKLYEHAVARNSTDSMDELYKGGSGGMVSKGEETITSVIKPDMKNKLRMEGNVNGHAFVIEGEGSGKPFEGIQTIDL
EVKEGAPLPFAYDILTTAFHYGNRVFTKYPRGGGGT*
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
RFC25 scar (shown in bold): 63 to 64, 195 to 196
Amino acid composition:
Ala (A)69 (6.1%)
Arg (R)55 (4.8%)
Asn (N)53 (4.7%)
Asp (D)88 (7.7%)
Cys (C)11 (1.0%)
Gln (Q)36 (3.2%)
Glu (E)85 (7.5%)
Gly (G)85 (7.5%)
His (H)29 (2.6%)
Ile (I)58 (5.1%)
Leu (L)87 (7.7%)
Lys (K)59 (5.2%)
Met (M)22 (1.9%)
Phe (F)41 (3.6%)
Pro (P)72 (6.3%)
Ser (S)66 (5.8%)
Thr (T)81 (7.1%)
Trp (W)10 (0.9%)
Tyr (Y)40 (3.5%)
Val (V)89 (7.8%)
Amino acid counting
Total number:1136
Positively charged (Arg+Lys):114 (10.0%)
Negatively charged (Asp+Glu):173 (15.2%)
Aromatic (Phe+His+Try+Tyr):120 (10.6%)
Biochemical parameters
Atomic composition:C5583H8693N1517O1759S33
Molecular mass [Da]:126268.5
Theoretical pI:4.91
Extinction coefficient at 280 nm [M-1 cm-1]:114600 / 115288 (all Cys red/ox)
Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges 
Codon usage
Organism:E. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
Codon quality (CAI):good (0.70)good (0.69)acceptable (0.57)good (0.67)excellent (0.85)excellent (0.82)
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.

Reference

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