Difference between revisions of "Part:BBa K1463600"
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<partinfo>BBa_K1463600 short</partinfo> | <partinfo>BBa_K1463600 short</partinfo> | ||
| − | FliC encodes the major flagellar protein of E. coli and is thus fundamental to motility | + | FliC encodes the major flagellar protein of E. coli and is thus fundamental to motility. |
| + | <br>Figure 1 shows this biobrick used in conjunction with various promoters and ribosome binding sites to restore swimming in fliC knockout strains. This data is also shown as a histogram in Figure 2. These show that, with an appropriate promoter and RBS, this biobrick can restore swimming to almost wild-type levels. | ||
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| + | <br> We sequenced our fliC biobrick and found that it had the expected sequence, identical to fliC of the sequenced E. coli strain MG1655 at all positions except for the changes we had made to remove three PstI sites and one SpeI site. However, there were two coding differences between our biobrick and a previous fliC biobrick in the parts registry [https://parts.igem.org/Part:BBa_K777109 K777109]. These differences are due to different source strains (We used MG1655 genomic DNA, they used E. coli strain K-12 substr. DH10B). It is unclear if these nonsynonymous changes alter flagella formation. | ||
| + | <br> | ||
| + | ==Flic Motility Swarm Assay== | ||
https://static.igem.org/mediawiki/2014/thumb/d/d0/GU_Figure_1_swarm_M.png/310px-GU_Figure_1_swarm_M.png | https://static.igem.org/mediawiki/2014/thumb/d/d0/GU_Figure_1_swarm_M.png/310px-GU_Figure_1_swarm_M.png | ||
| + | <p><b>Figure 1: FliC Swarm Motility Assays.</b> <br><b>(A)</b> DS941, <b>(B)</b> DS941 ΔfliC, | ||
| + | <br><b>(C)</b> DS941 ΔfliC + pSB1C3 fliC (no promoter), <b>(D)</b> DS941 ΔfliC + J23100 (mutant promoter) fliC, <br><b>(E)</b> DS941 ΔfliC + J23116-fliC(1), <b>(F)</b> DS941 ΔfliC + J23116-fliC(2), <br><b>(G)</b> DS941 ΔfliC + J23106-fliC(1), <b>(H)</b> DS941 ΔfliC + J23106-fliC(2)</p> | ||
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| + | <br> | ||
| + | ==Flic Motility Histogram== | ||
https://static.igem.org/mediawiki/2014/f/fc/GU_Figure_2_Motility_histogram.png | https://static.igem.org/mediawiki/2014/f/fc/GU_Figure_2_Motility_histogram.png | ||
| + | <br><b>Figure 2 - FliC Motility Histogram</b> The promoters indicated in the histogram were used to drive the fliC biobrick K1463600 with the RBS B0034. Plasmids containing these constructs were used to complement a chromosomal fliC mutation. The diameter of swimming in a 16 hour swarm assay at 37 degrees is shown. The error bars indicate the range or results obtained in two repeats of the experiment. The strong J23100 promoter in this result contained a mutation rendering it inactive [https://parts.igem.org/Part:BBa_K1463604 see K1463604]. | ||
| + | <br><br> | ||
For more information on the biobrick and methods used go to http://2014.igem.org/wiki/index.php?title=Team:Glasgow/Project/Mobility_Proteins#motA | For more information on the biobrick and methods used go to http://2014.igem.org/wiki/index.php?title=Team:Glasgow/Project/Mobility_Proteins#motA | ||
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<partinfo>BBa_K1463600 parameters</partinfo> | <partinfo>BBa_K1463600 parameters</partinfo> | ||
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| + | <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_1413480945739'><table id="AutoAnnotator"><tr><!-- Time stamp in ms since 1/1/1970 1413480945739 --><th id="AutoAnnotatorHeader" colspan="2">Protein data table for BioBrick <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1463600<!------------------------Enter BioBrick number here------------------------>">BBa_K1463600<!------------------------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"> <u>ATGGCACAA ... CTCCAGGGT</u>TAA</span><br> <strong>ORF</strong> from nucleotide position 1 to 1494 (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 <br>101 <br>201 <br>301 <br>401 </td><td class="AutoAnnotatorSeqSeq">MAQVINTNSLSLITQNNINKNQSALSSSIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQAARNANDGISVAQTTEGALSEINNNLQRVRELTVQAT<br>TGTNSESDLSSIQDEIKSRLDEIDRVSGQTQFNGVNVLAKNGSMKIQVGANDNQTITIDLKQIDAKTLGLDGFSVKNNDTVTTSAPVTAFGATTTNNIKL<br>TGITLSTEAATDTGGTNPASIEGVYTDNGNDYYAKITGGDNDGKYYAVTVANDGTVTMATGATANATVTDANTTKATTITSGGTPVQIDNTAGSATANLG<br>AVSLVKLQDSKGNDTDTYALKDTNGNLYAADVNETTGAVSVKTITYTDSSGAASSPTAVKLGGDDGKTEVVDIDGKTYDSADLNGGNLQTGLTAGGEALT<br>AVANGKTTDPLKALDDAIASVDKFRSSLGAVQNRLDSAVTNLNNTTTNLSEAQSRIQDADYATEVSNMSKAQIIQQAGNSVLAKANQVPQQVLSLLQG*</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="AutoAnnotatorSeqFeat2b">None of the supported features appeared in the sequence</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">59 (11.8%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Arg (R)</td><td class="AutoAnnotatorInnerAmino">11 (2.2%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Asn (N)</td><td class="AutoAnnotatorInnerAmino">48 (9.6%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Asp (D)</td><td class="AutoAnnotatorInnerAmino">39 (7.8%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Cys (C)</td><td class="AutoAnnotatorInnerAmino">0 (0.0%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Gln (Q)</td><td class="AutoAnnotatorInnerAmino">27 (5.4%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Glu (E)</td><td class="AutoAnnotatorInnerAmino">14 (2.8%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Gly (G)</td><td class="AutoAnnotatorInnerAmino">44 (8.8%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">His (H)</td><td class="AutoAnnotatorInnerAmino">0 (0.0%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Ile (I)</td><td class="AutoAnnotatorInnerAmino">28 (5.6%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Leu (L)</td><td class="AutoAnnotatorInnerAmino">37 (7.4%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Lys (K)</td><td class="AutoAnnotatorInnerAmino">25 (5.0%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Met (M)</td><td class="AutoAnnotatorInnerAmino">4 (0.8%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Phe (F)</td><td class="AutoAnnotatorInnerAmino">5 (1.0%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Pro (P)</td><td class="AutoAnnotatorInnerAmino">6 (1.2%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Ser (S)</td><td class="AutoAnnotatorInnerAmino">43 (8.6%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Thr (T)</td><td class="AutoAnnotatorInnerAmino">65 (13.1%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Trp (W)</td><td class="AutoAnnotatorInnerAmino">0 (0.0%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Tyr (Y)</td><td class="AutoAnnotatorInnerAmino">10 (2.0%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Val (V)</td><td class="AutoAnnotatorInnerAmino">33 (6.6%)</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">498</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Positively charged (Arg+Lys):</td><td class="AutoAnnotatorAminoCountingInner3">36 (7.2%)</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Negatively charged (Asp+Glu):</td><td class="AutoAnnotatorAminoCountingInner3">53 (10.6%)</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Aromatic (Phe+His+Try+Tyr):</td><td class="AutoAnnotatorAminoCountingInner3">15 (3.0%)</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>2163</sub>H<sub>3553</sub>N<sub>631</sub>O<sub>798</sub>S<sub>4</sub></td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Molecular mass [Da]:</td><td class="AutoAnnotatorBiochemParInner3">51295.0</td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Theoretical pI:</td><td class="AutoAnnotatorBiochemParInner3">4.50</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">14900 / 14900 (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> <input type='button' id='hydrophobicity_charge_button' onclick='show_or_hide_plot_1413480945739()' 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></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">excellent (0.83)</td><td class="AutoAnnotatorCodonUsage3">good (0.78)</td><td class="AutoAnnotatorCodonUsage3">good (0.68)</td><td class="AutoAnnotatorCodonUsage3">good (0.74)</td><td class="AutoAnnotatorCodonUsage3">excellent (0.83)</td><td class="AutoAnnotatorCodonUsage3">good (0.70)</td></tr></table></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Alignments</strong> (obtained from <a href='http://predictprotein.org'>PredictProtein.org</a>)<table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorAlignment1"></td><td class="AutoAnnotatorAlignment2">SwissProt:</td><td class="AutoAnnotatorAlignment3"><a href='http://www.uniprot.org/uniprot/P04949'>P04949</a> (100% identity on 498 AAs), <a href='http://www.uniprot.org/uniprot/P06179'>P06179</a> (47% identity on 495 AAs)</td></tr><tr><td class="AutoAnnotatorAlignment1"></td><td class="AutoAnnotatorAlignment2">TrEML:</td><td class="AutoAnnotatorAlignment3"><a href='http://www.uniprot.org/uniprot/L2Z8U7'>L2Z8U7</a> (100% identity on 498 AAs), <a href='http://www.uniprot.org/uniprot/M2PKZ4'>M2PKZ4</a> (100% identity on 498 AAs), <a href='http://www.uniprot.org/uniprot/C6EBD8'>C6EBD8</a> (100% identity on 498 AAs), <a href='http://www.uniprot.org/uniprot/C9QSQ0'>C9QSQ0</a> (100% identity on 498 AAs), <a href='http://www.uniprot.org/uniprot/F9QUT2'>F9QUT2</a> (100% identity on 498 AAs), <a href='http://www.uniprot.org/uniprot/G2EY21'>G2EY21</a> (100% identity on 498 AAs), <a href='http://www.uniprot.org/uniprot/I0ZSG6'>I0ZSG6</a> (100% identity on 498 AAs), <a href='http://www.uniprot.org/uniprot/C4ZQK1'>C4ZQK1</a> (100% identity on 498 AAs), <a href='http://www.uniprot.org/uniprot/Q53ZW4'>Q53ZW4</a> (100% identity on 498 AAs), <a href='http://www.uniprot.org/uniprot/U6N709'>U6N709</a> (100% identity on 498 AAs)</td></tr><tr><td class="AutoAnnotatorAlignment1"></td><td class="AutoAnnotatorAlignment2">PDB:</td><td class="AutoAnnotatorAlignment3"> - </td></tr></table></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="AutoAnnotatorLocalizationOuter"><strong>Subcellular Localization</strong> (reliability in brackets)<table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorLocalization1"></td><td class="AutoAnnotatorLocalization2">Archaea:</td><td class="AutoAnnotatorLocalization3">cytosol (100%)</td></tr><tr><td class="AutoAnnotatorLocalization1"></td><td class="AutoAnnotatorLocalization2">Bacteria:</td><td class="AutoAnnotatorLocalization3">secreted (65%)</td></tr><tr><td class="AutoAnnotatorLocalization1"></td><td class="AutoAnnotatorLocalization2">Eukarya:</td><td class="AutoAnnotatorLocalization3">nucleus (55%)</td></tr></table></td><td class="AutoAnnotatorGOOuter"><strong>Gene Ontology</strong> (reliability in brackets)<br><table class="AutoAnnotatorNoBorder"><tr><td class='AutoAnnotatorGO1'></td><td class='AutoAnnotatorGO2'>Molecular Function Ontology:</td><td class='AutoAnnotatorGO3'><a href='http://amigo.geneontology.org/cgi-bin/amigo/term_details?term=GO:0005515'>GO:0005515</a> (100%)</td></tr><tr><td class='AutoAnnotatorGO1'></td><td class='AutoAnnotatorGO2'>Biological Process Ontology:</td><td class='AutoAnnotatorGO3'><a href='http://amigo.geneontology.org/cgi-bin/amigo/term_details?term=GO:0009405'>GO:0009405</a> (9%), <a href='http://amigo.geneontology.org/cgi-bin/amigo/term_details?term=GO:0001539'>GO:0001539</a> (63%)</td></tr><tr><td class='AutoAnnotatorGO1'> </td><td class='AutoAnnotatorGO2'> </td><td class='AutoAnnotatorGO3'> </td></tr></table></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Predicted features:</strong><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorPredFeat1"></td><td class="AutoAnnotatorPredFeat2a">Disulfid bridges:</td><td class="AutoAnnotatorPredFeat3"> - </td></tr><tr><td class="AutoAnnotatorPredFeat1"></td><td class="AutoAnnotatorPredFeat2a">Transmembrane helices:</td><td class="AutoAnnotatorPredFeat3"> - </td></tr></table></td></tr><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_1413480945739(){hydrophobicity_datapoints = 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= [];trans_datapoints = [];sec_helix_datapoints = [[9,32],[45,69],[72,99],[106,128],[405,428],[430,447],[450,453],[459,485],[490,496]];sec_strand_datapoints = [[2,6],[37,38],[135,139],[144,149],[155,163],[166,177],[184,200],[201,201],[203,221],[233,240],[243,252],[255,287],[294,299],[302,308],[318,323],[326,333],[339,342],[348,350],[358,373],[383,384],[391,400],[401,401]];acc_exposed_datapoints = [[1,1],[16,17],[19,20],[23,24],[26,27],[30,30],[56,56],[59,60],[66,66],[69,69],[87,87],[101,101],[107,107],[110,110],[114,114],[117,118],[121,121],[125,125],[128,128],[133,133],[143,143],[145,145],[151,151],[153,153],[161,162],[164,164],[166,166],[171,171],[174,174],[176,176],[178,178],[191,191],[193,193],[195,197],[199,199],[204,204],[206,206],[208,208],[210,217],[220,220],[228,229],[231,231],[235,235],[237,237],[240,242],[244,244],[259,259],[261,261],[263,263],[265,265],[267,267],[269,273],[275,275],[277,278],[280,281],[291,295],[298,298],[300,301],[303,304],[306,306],[308,309],[311,311],[313,314],[316,316],[321,324],[331,331],[333,333],[336,336],[342,343],[345,345],[348,349],[352,352],[354,354],[364,367],[369,369],[372,372],[374,374],[376,377],[379,380],[382,382],[387,387],[389,389],[391,391],[395,395],[397,397],[405,405],[409,409],[412,412],[416,416],[419,419],[423,423],[440,441],[443,444],[447,447],[450,450],[467,467],[487,487],[490,490],[494,494],[497,498]];acc_buried_datapoints = [[3,15],[18,18],[21,21],[25,25],[28,29],[32,35],[38,38],[41,41],[43,51],[54,54],[58,58],[61,61],[64,65],[67,68],[71,82],[85,85],[88,89],[92,93],[95,100],[102,103],[109,109],[112,113],[116,116],[119,120],[123,123],[126,127],[130,130],[132,132],[134,135],[137,140],[146,146],[148,150],[155,156],[158,158],[160,160],[165,165],[168,168],[170,170],[175,175],[181,182],[184,187],[198,198],[200,200],[203,203],[205,205],[209,209],[225,225],[232,233],[245,246],[248,248],[250,250],[255,255],[258,258],[274,274],[284,284],[286,286],[288,288],[327,328],[332,332],[337,337],[339,339],[341,341],[353,353],[356,356],[358,359],[361,361],[373,373],[383,383],[399,399],[401,403],[407,407],[410,411],[414,414],[417,418],[421,421],[424,426],[428,435],[437,439],[442,442],[445,446],[448,449],[452,452],[454,454],[456,456],[458,462],[465,466],[468,469],[471,471],[473,474],[476,483],[485,485],[489,489],[492,493],[495,496]];flot_plot_options = []; flot_plot_options[0] = {grid: {borderWidth: {top: 0,right: 0,bottom: 0,left: 0}},legend: {show: false},xaxes: [{show: true,min: 0,max: 200,ticks: [[0.5, '1'], [24.5, '25'], [49.5, '50'], [74.5, '75'], [99.5, '100'], [124.5, '125'], [149.5, '150'], [174.5, '175'], [199.5, '200']],tickLength: -5}],yaxes: [{show: true,ticks: [[0, '0'], [4.5,'hydro-<br>phobic '], [-4.5,'hydro-<br>philic ']],min: -4.5,max: +4.5,font: {size: 12,lineHeight: 14,style: 'italic',weight: 'bold',family: 'sans-serif',variant: 'small-caps',color: 'rgba(100,149,237,1)'}},{show: true,ticks: [[0, ''], [1,'positive<br> charge'], [-1,'negative<br> 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 = 3;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_1413480945739 #hydrophobicity_charge_button').val() =='Show' ){jqAutoAnnotator('#AutoAnnotator_container_1413480945739 #hydrophobicity_charge_container').css('display','block');jqAutoAnnotator('#AutoAnnotator_container_1413480945739 #hydrophobicity_charge_button').val('Hide');var description_html = '<div id=\'AutoAnnotator_plot_selectors\'>';description_html = description_html + '<br> <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 + '<br> <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 + '<br> <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 + '<br> <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 + '<br> <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 + '<br> <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 + '<br></div>';jqAutoAnnotator('#AutoAnnotator_container_1413480945739 #hydrophobicity_charge_explanation').html(description_html);plot_according_to_selectors_1413480945739();jqAutoAnnotator('#AutoAnnotator_container_1413480945739 #AutoAnnotator_plot_selectors').find('input').click(plot_according_to_selectors_1413480945739);}else{jqAutoAnnotator('#AutoAnnotator_container_1413480945739 #hydrophobicity_charge_container').css('display','none');jqAutoAnnotator('#AutoAnnotator_container_1413480945739 #hydrophobicity_charge_button').val('Show');jqAutoAnnotator('#AutoAnnotator_container_1413480945739 #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_1413480945739(){try{var plot_datasets = [[],[]];if(jqAutoAnnotator('#AutoAnnotator_container_1413480945739 #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_1413480945739 #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_1413480945739 #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_1413480945739 #dis_checkbox').prop('checked') == true){for ( j = 0 ; j < dis_datapoints.length 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Latest revision as of 14:42, 1 November 2014
FliC
FliC encodes the major flagellar protein of E. coli and is thus fundamental to motility.
Figure 1 shows this biobrick used in conjunction with various promoters and ribosome binding sites to restore swimming in fliC knockout strains. This data is also shown as a histogram in Figure 2. These show that, with an appropriate promoter and RBS, this biobrick can restore swimming to almost wild-type levels.
We sequenced our fliC biobrick and found that it had the expected sequence, identical to fliC of the sequenced E. coli strain MG1655 at all positions except for the changes we had made to remove three PstI sites and one SpeI site. However, there were two coding differences between our biobrick and a previous fliC biobrick in the parts registry K777109. These differences are due to different source strains (We used MG1655 genomic DNA, they used E. coli strain K-12 substr. DH10B). It is unclear if these nonsynonymous changes alter flagella formation.
Flic Motility Swarm Assay
Figure 1: FliC Swarm Motility Assays.
(A) DS941, (B) DS941 ΔfliC,
(C) DS941 ΔfliC + pSB1C3 fliC (no promoter), (D) DS941 ΔfliC + J23100 (mutant promoter) fliC,
(E) DS941 ΔfliC + J23116-fliC(1), (F) DS941 ΔfliC + J23116-fliC(2),
(G) DS941 ΔfliC + J23106-fliC(1), (H) DS941 ΔfliC + J23106-fliC(2)
Flic Motility Histogram
Figure 2 - FliC Motility Histogram The promoters indicated in the histogram were used to drive the fliC biobrick K1463600 with the RBS B0034. Plasmids containing these constructs were used to complement a chromosomal fliC mutation. The diameter of swimming in a 16 hour swarm assay at 37 degrees is shown. The error bars indicate the range or results obtained in two repeats of the experiment. The strong J23100 promoter in this result contained a mutation rendering it inactive see K1463604.
For more information on the biobrick and methods used go to http://2014.igem.org/wiki/index.php?title=Team:Glasgow/Project/Mobility_Proteins#motA
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1224
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 301
Illegal AgeI site found at 709 - 1000COMPATIBLE WITH RFC[1000]
| Protein data table for BioBrick BBa_K1463600 automatically created by the BioBrick-AutoAnnotator version 1.0 | ||||||||||||||||||||||||||||||||||||||||||||||
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| Nucleotide sequence in RFC 10: (underlined part encodes the protein) ATGGCACAA ... CTCCAGGGTTAA ORF from nucleotide position 1 to 1494 (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)
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| Predictions (obtained from PredictProtein.org) | ||||||||||||||||||||||||||||||||||||||||||||||
Subcellular Localization (reliability in brackets)
| Gene Ontology (reliability in brackets)
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Predicted features:
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| 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. | ||||||||||||||||||||||||||||||||||||||||||||||