Coding

Part:BBa_K2332311

Designed by: Camillo Moschner   Group: iGEM17_UCL   (2017-09-27)
Revision as of 14:36, 8 October 2017 by CamilloMoschner (Talk | contribs)


PhoCl, a mammalian photocleavable protein

PhoCl, a Mammalian Photocleavable Protein
Function Photocleavable Linker
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 (FP).

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.



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Protein data table for BioBrick <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_">BBa_</a> automatically created by the <a href="http://2013.igem.org/Team:TU-Munich/Results/AutoAnnotator">BioBrick-AutoAnnotator</a> version 1.0
Nucleotide sequence in RFC 10: (underlined part encodes the protein)
 ATGGGGGGT ... GGAGGTACCTAA
 ORF from nucleotide position 1 to 849 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)
1 
101 
201 		MGGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPSSSVIPDYFKQSFPEGYSWERSMTYEDGGICIATNDITMEGDSFINKIHFKGTNFPPNGPVMQKRTV
GWEASTEKMYERDGVLKGDVKMKLLLKGGGHYRCDYRTTYKVKQKPVKLPDYHFVDHRIEILSHDKDYNKVKLYEHAVARNSTDSMDELYKGGSGGMVSK
GEETITSVIKPDMKNKLRMEGNVNGHAFVIEGEGSGKPFEGIQTIDLEVKEGAPLPFAYDILTTAFHYGNRVFTKYPRGGGGT*
Sequence features: (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>)
His6-tag: 5 to 10
Enterokinase cleavage site: 27 to 31
Amino acid composition:
Ala (A)9 (3.2%)
Arg (R)11 (3.9%)
Asn (N)10 (3.5%)
Asp (D)22 (7.8%)
Cys (C)2 (0.7%)
Gln (Q)6 (2.1%)
Glu (E)18 (6.4%)
Gly (G)35 (12.4%)
His (H)14 (4.9%)
Ile (I)14 (4.9%)
Leu (L)13 (4.6%)
Lys (K)25 (8.8%)
Met (M)13 (4.6%)
Phe (F)11 (3.9%)
Pro (P)13 (4.6%)
Ser (S)17 (6.0%)
Thr (T)17 (6.0%)
Trp (W)2 (0.7%)
Tyr (Y)15 (5.3%)
Val (V)16 (5.7%)
Amino acid counting
Total number:283
Positively charged (Arg+Lys):36 (12.7%)
Negatively charged (Asp+Glu):40 (14.1%)
Aromatic (Phe+His+Try+Tyr):42 (14.8%)
Biochemical parameters
Atomic composition:C1398H2143N387O429S15
Molecular mass [Da]:31716.6
Theoretical pI:6.39
Extinction coefficient at 280 nm [M-1 cm-1]:33350 / 33475 (all Cys red/ox)
Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges <input type='button' id='hydrophobicity_charge_button' onclick='show_or_hide_plot_1507473177058()' value='Show'>
Codon usage
Organism:E. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
Codon quality (<a href="http://en.wikipedia.org/wiki/Codon_Adaptation_Index">CAI</a>):good (0.70)good (0.65)acceptable (0.53)good (0.63)excellent (0.90)excellent (0.91)
Alignments (obtained from <a href='http://predictprotein.org'>PredictProtein.org</a>)
   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 <a href='http://predictprotein.org'>PredictProtein.org</a>)
   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 <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>.
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>.

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 charge']],position: 'right',min: -1,max: 1,font: {size: 12,lineHeight: 14,style: 'italic',weight: 'bold',family: 'sans-serif',variant: 'small-caps',color: 'rgba(255,99,71,1)'}}]};number_of_plots = 2;for ( plot_num = 1 ; plot_num < number_of_plots ; plot_num ++){flot_plot_options[plot_num] = jqAutoAnnotator.extend(true, {} ,flot_plot_options[0]);flot_plot_options[plot_num].xaxes = [{min: plot_num*200,max: (plot_num + 1)*200,ticks: [ [plot_num*200 + 0.5, (plot_num*200 + 1).toString()], [plot_num*200 + 24.5, (plot_num*200 + 25).toString()], [plot_num*200 + 49.5, (plot_num*200 + 50).toString()], [plot_num*200 + 74.5, (plot_num*200 + 75).toString()], [plot_num*200 + 99.5, (plot_num*200 + 100).toString()], [plot_num*200 + 124.5, (plot_num*200 + 125).toString()], [plot_num*200 + 149.5, (plot_num*200 + 150).toString()], [plot_num*200 + 174.5, (plot_num*200 + 175).toString()], [plot_num*200 + 199.5, (plot_num*200 + 200).toString()] ],tickLength: -5}];};try {if( jqAutoAnnotator('#AutoAnnotator_container_1507473177058 #hydrophobicity_charge_button').val() =='Show' ){jqAutoAnnotator('#AutoAnnotator_container_1507473177058 #hydrophobicity_charge_container').css('display','block');jqAutoAnnotator('#AutoAnnotator_container_1507473177058 #hydrophobicity_charge_button').val('Hide');var description_html = '
';description_html = description_html + '
 <input type=\'checkbox\' id=\'hydrophobicity_checkbox\' checked=\'checked\'> Moving average over 5 amino acids for hydrophobicity (<img src=\'https://static.igem.org/mediawiki/2013/e/e9/TUM13_hydrophobicity_icon.png\' alt=\'blue graph\' height=\'10\'></img>)';description_html = description_html + '
 <input type=\'checkbox\' id=\'charge_checkbox\' checked=\'checked\'> Moving average over 5 amino acids for charge (<img src=\'https://static.igem.org/mediawiki/2013/3/3e/TUM13_charge_icon.png\' alt=\'red graph\' height=\'10\'></img>)';description_html = description_html + '
 <input type=\'checkbox\' id=\'dis_checkbox\' checked=\'checked\'> 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 + '
 <input type=\'checkbox\' id=\'trans_checkbox\' checked=\'checked\'> 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 + '
 <input type=\'checkbox\' id=\'sec_checkbox\' checked=\'checked\'> 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 + '
 <input type=\'checkbox\' id=\'acc_checkbox\' checked=\'checked\'> 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 + '
';jqAutoAnnotator('#AutoAnnotator_container_1507473177058 #hydrophobicity_charge_explanation').html(description_html);plot_according_to_selectors_1507473177058();jqAutoAnnotator('#AutoAnnotator_container_1507473177058 #AutoAnnotator_plot_selectors').find('input').click(plot_according_to_selectors_1507473177058);}else{jqAutoAnnotator('#AutoAnnotator_container_1507473177058 #hydrophobicity_charge_container').css('display','none');jqAutoAnnotator('#AutoAnnotator_container_1507473177058 #hydrophobicity_charge_button').val('Show');jqAutoAnnotator('#AutoAnnotator_container_1507473177058 #hydrophobicity_charge_explanation').html();}}catch(err){txt='There was an error with the button controlling the visibility of the plot.\n';txt=txt+'The originating error is:\n' + err + '\n\n';alert(txt);}};function plot_according_to_selectors_1507473177058(){try{var plot_datasets = [[],[]];if(jqAutoAnnotator('#AutoAnnotator_container_1507473177058 #hydrophobicity_checkbox').prop('checked') == true){plot_datasets[0] = { color: 'rgba(100,149,237,1)',data: hydrophobicity_datapoints,label: 'Hydrophobicity',lines: { show: true, fill: true, fillColor: 'rgba(100,149,237,0.1)' },yaxis: 1};}if(jqAutoAnnotator('#AutoAnnotator_container_1507473177058 #charge_checkbox').prop('checked') == true){plot_datasets[1] = {color: 'rgba(255,99,71,1)',data: charge_datapoints,label: 'Charge',lines: { show: true, fill: true, fillColor: 'rgba(255,99,71,0.1)' },yaxis: 2};}for (plot_num = 0 ; plot_num < number_of_plots ; plot_num ++){jqAutoAnnotator.plot('#AutoAnnotator_container_1507473177058 #hydrophobicity_charge_placeholder'+ plot_num.toString(), plot_datasets, flot_plot_options[plot_num] );}var screen_width = jqAutoAnnotator('canvas.flot-base').width(); var pos_of_first_tick = 46;var pos_of_last_tick = screen_width - 51;var tick_diff = (screen_width - 97)/199;if(jqAutoAnnotator('#AutoAnnotator_container_1507473177058 #dis_checkbox').prop('checked') == true){for ( j = 0 ; j < dis_datapoints.length ; j++ ){jqAutoAnnotator('#AutoAnnotator_container_1507473177058 #hydrophobicity_charge_placeholder' + Math.floor((dis_datapoints[j][0] - 1)/200) ).append('
' + (j+1) + '
');jqAutoAnnotator('#AutoAnnotator_container_1507473177058 #hydrophobicity_charge_placeholder' + Math.floor((dis_datapoints[j][1] - 1)/200) ).append('
' + (j+1) + '
');}}if(jqAutoAnnotator('#AutoAnnotator_container_1507473177058 #trans_checkbox').prop('checked') == true){for ( j = 0 ; j < trans_datapoints.length ; j++ ){jqAutoAnnotator('#AutoAnnotator_container_1507473177058 #hydrophobicity_charge_placeholder' + Math.floor((trans_datapoints[j][0] - 1)/200) ).append('
');}}if(jqAutoAnnotator('#AutoAnnotator_container_1507473177058 #sec_checkbox').prop('checked') == true){for ( j = 0 ; j < sec_helix_datapoints.length ; j++ ){jqAutoAnnotator('#AutoAnnotator_container_1507473177058 #hydrophobicity_charge_placeholder' + Math.floor((sec_helix_datapoints[j][0] - 1)/200) ).append('
');}for ( j = 0 ; j < sec_strand_datapoints.length ; j++ ){jqAutoAnnotator('#AutoAnnotator_container_1507473177058 #hydrophobicity_charge_placeholder' + Math.floor((sec_strand_datapoints[j][0] - 1)/200) ).append('
');}}if(jqAutoAnnotator('#AutoAnnotator_container_1507473177058 #acc_checkbox').prop('checked') == true){for ( j = 0 ; j < acc_buried_datapoints.length ; j++ ){jqAutoAnnotator('#AutoAnnotator_container_1507473177058 #hydrophobicity_charge_placeholder' + Math.floor((acc_buried_datapoints[j][0] - 1)/200) ).append('
');}for ( j = 0 ; j < acc_exposed_datapoints.length ; j++ ){jqAutoAnnotator('#AutoAnnotator_container_1507473177058 #hydrophobicity_charge_placeholder' + Math.floor((acc_exposed_datapoints[j][0] - 1)/200) ).append('
');}}}catch(err){txt='There was an error while drawing the selected elements for the plot.\n';txt=txt+'The originating error is:\n' + err + '\n\n';throw(txt);}}</script></html>



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

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Parameters
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