Difference between revisions of "Part:BBa K1351021"
(→Usage and Biology) |
|||
(15 intermediate revisions by 2 users not shown) | |||
Line 2: | Line 2: | ||
<partinfo>BBa_K1351021 short</partinfo> | <partinfo>BBa_K1351021 short</partinfo> | ||
− | Mutant of the Biobrick | + | Mutant of the Biobrick [https://parts.igem.org/Part:BBa_E1010 BBa_E1010]. In order to make it compatible with the Freiburg-Standard RFC25, two AgeI-Restriction sites were deleted. |
monomeric RFP: Red Fluorescent Protein. | monomeric RFP: Red Fluorescent Protein. | ||
Line 11: | Line 11: | ||
=== Usage and Biology === | === Usage and Biology === | ||
− | This part was created in order to enhance the [http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks ''Bacillus'' BioBrick Box] by adding different fluorescent proteins | + | This part was created in order to enhance the [http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks ''Bacillus'' BioBrick Box] by adding different fluorescent proteins to have more reporters available in ''B. subtilis''. |
− | + | Since the Biobrick [https://parts.igem.org/Part:BBa_E1010 BBa_E1010] is already evaluated [https://parts.igem.org/Part:BBa_E1010:Experience very well] and shows a very bright color in ''E. coli'', the rather light coloring in our experimental setup may be due to the used expression vector [https://parts.igem.org/wiki/index.php?title=Part:BBa_K1351040 pBS0K-Pspac]. This vector is optimized for the use in ''B. subtilis'' and shows a rather weak and heterogenous expression in ''E. coli''. <br><br> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
+ | [[File:LMU14_K1351021_RFP.jpg|center|800px]] | ||
+ | '''Picture 1:''' BBa_K1351021 in the ''B. subtilis'' expression vector [https://parts.igem.org/wiki/index.php?title=Part:BBa_K1351040 pBS0K-Pspac], cloned into ''E. coli''. The rather weak color is due to the vector, which shows a rather weak an heterogenous expression. On the right picture, the left eppendorf tube contains a sample of ''E. coli'' containing the Biobrick BBa_K1351021, the right eppendorf tube contains ''E. coli'' with just the empty vector. | ||
=== Design === | === Design === | ||
Line 46: | Line 36: | ||
This part is used in the 2014 LMU-Munich iGEM project [http://2014.igem.org/Team:LMU-Munich BaKillus]. | This part is used in the 2014 LMU-Munich iGEM project [http://2014.igem.org/Team:LMU-Munich BaKillus]. | ||
+ | |||
+ | |||
+ | |||
+ | === BioBrick AutoAnnotator === | ||
+ | |||
+ | <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_1414675305031'><table id="AutoAnnotator"><tr><!-- Time stamp in ms since 1/1/1970 1414675305031 --><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 25</strong>, so ATGGCCGGC and ACCGGT were added (in italics) to the 5' and 3' ends: (underlined part encodes the protein)<br><span class="AutoAnnotatorSequence"> <u><i>ATGGCCGGC</i>ATGGCTTCC ... ACCTGTGCT<i>ACCGGT</i></u></span><br> <strong>ORF</strong> from nucleotide position -8 to 681 (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 </td><td class="AutoAnnotatorSeqSeq">MAGMASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVM<br>NFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMRLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDI<br>KLDITSHNEDYTIVEQYERAEGRHSTCATG*</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">13 (5.7%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Arg (R)</td><td class="AutoAnnotatorInnerAmino">9 (3.9%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Asn (N)</td><td class="AutoAnnotatorInnerAmino">4 (1.7%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Asp (D)</td><td class="AutoAnnotatorInnerAmino">14 (6.1%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Cys (C)</td><td class="AutoAnnotatorInnerAmino">1 (0.4%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Gln (Q)</td><td class="AutoAnnotatorInnerAmino">8 (3.5%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Glu (E)</td><td class="AutoAnnotatorInnerAmino">22 (9.6%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Gly (G)</td><td class="AutoAnnotatorInnerAmino">24 (10.4%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">His (H)</td><td class="AutoAnnotatorInnerAmino">5 (2.2%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Ile (I)</td><td class="AutoAnnotatorInnerAmino">9 (3.9%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Leu (L)</td><td class="AutoAnnotatorInnerAmino">12 (5.2%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Lys (K)</td><td class="AutoAnnotatorInnerAmino">22 (9.6%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Met (M)</td><td class="AutoAnnotatorInnerAmino">10 (4.3%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Phe (F)</td><td class="AutoAnnotatorInnerAmino">10 (4.3%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Pro (P)</td><td class="AutoAnnotatorInnerAmino">12 (5.2%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Ser (S)</td><td class="AutoAnnotatorInnerAmino">12 (5.2%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Thr (T)</td><td class="AutoAnnotatorInnerAmino">15 (6.5%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Trp (W)</td><td class="AutoAnnotatorInnerAmino">3 (1.3%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Tyr (Y)</td><td class="AutoAnnotatorInnerAmino">11 (4.8%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Val (V)</td><td class="AutoAnnotatorInnerAmino">14 (6.1%)</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">230</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Positively charged (Arg+Lys):</td><td class="AutoAnnotatorAminoCountingInner3">31 (13.5%)</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Negatively charged (Asp+Glu):</td><td class="AutoAnnotatorAminoCountingInner3">36 (15.7%)</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Aromatic (Phe+His+Try+Tyr):</td><td class="AutoAnnotatorAminoCountingInner3">29 (12.6%)</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>1152</sub>H<sub>1778</sub>N<sub>304</sub>O<sub>353</sub>S<sub>11</sub></td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Molecular mass [Da]:</td><td class="AutoAnnotatorBiochemParInner3">25887.3</td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Theoretical pI:</td><td class="AutoAnnotatorBiochemParInner3">5.65</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">32890 / 32953 (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_1414675305031()' 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></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 (0.84)</td><td class="AutoAnnotatorCodonUsage3">good (0.72)</td><td class="AutoAnnotatorCodonUsage3">good (0.68)</td><td class="AutoAnnotatorCodonUsage3">good (0.74)</td><td class="AutoAnnotatorCodonUsage3">good (0.79)</td><td class="AutoAnnotatorCodonUsage3">good (0.72)</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> 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"> 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_1414675305031(){hydrophobicity_datapoints = [[2.5,1.40],[3.5,0.86],[4.5,0.34],[5.5,-0.28],[6.5,-1.36],[7.5,-0.88],[8.5,0.18],[9.5,-0.44],[10.5,-0.44],[11.5,0.82],[12.5,0.36],[13.5,-1.44],[14.5,-0.10],[15.5,-0.18],[16.5,0.10],[17.5,-1.18],[18.5,0.10],[19.5,-1.16],[20.5,-0.46],[21.5,-1.46],[22.5,0.28],[23.5,-0.80],[24.5,-0.18],[25.5,-0.74],[26.5,-1.28],[27.5,-1.56],[28.5,-1.56],[29.5,-0.58],[30.5,-0.64],[31.5,-0.02],[32.5,-1.28],[33.5,-0.66],[34.5,-2.26],[35.5,-1.64],[36.5,-2.46],[37.5,-2.08],[38.5,-2.26],[39.5,-2.26],[40.5,-2.26],[41.5,-1.50],[42.5,-1.88],[43.5,-1.76],[44.5,-0.70],[45.5,-1.40],[46.5,-0.50],[47.5,-0.58],[48.5,0.40],[49.5,-0.10],[50.5,-0.10],[51.5,-0.94],[52.5,-0.24],[53.5,-1.40],[54.5,-0.50],[55.5,-0.04],[56.5,0.60],[57.5,1.04],[58.5,1.18],[59.5,-0.28],[60.5,0.94],[61.5,1.14],[62.5,0.62],[63.5,0.48],[64.5,0.48],[65.5,0.14],[66.5,-1.32],[67.5,-1.42],[68.5,-1.18],[69.5,-0.64],[70.5,-1.98],[71.5,-0.92],[72.5,-0.92],[73.5,-0.00],[74.5,-0.62],[75.5,-0.48],[76.5,-1.16],[77.5,-0.54],[78.5,-2.08],[79.5,-0.40],[80.5,-0.08],[81.5,-0.46],[82.5,-1.08],[83.5,0.38],[84.5,-1.30],[85.5,-0.22],[86.5,0.32],[87.5,1.14],[88.5,0.06],[89.5,0.14],[90.5,-0.70],[91.5,0.02],[92.5,-1.32],[93.5,-1.18],[94.5,-1.18],[95.5,-2.00],[96.5,-1.72],[97.5,-0.56],[98.5,-1.08],[99.5,0.18],[100.5,0.38],[101.5,-1.16],[102.5,-1.62],[103.5,-1.00],[104.5,-0.72],[105.5,0.82],[106.5,1.38],[107.5,2.30],[108.5,2.24],[109.5,0.70],[110.5,-0.84],[111.5,-0.86],[112.5,-1.86],[113.5,-0.96],[114.5,-0.96],[115.5,-0.96],[116.5,-0.88],[117.5,-1.42],[118.5,-1.62],[119.5,-0.02],[120.5,0.42],[121.5,-0.28],[122.5,1.26],[123.5,-0.08],[124.5,-0.22],[125.5,-0.86],[126.5,-0.16],[127.5,-1.14],[128.5,-1.06],[129.5,-1.26],[130.5,-0.68],[131.5,-0.76],[132.5,-1.32],[133.5,-0.70],[134.5,-1.58],[135.5,-0.42],[136.5,0.12],[137.5,0.12],[138.5,-0.58],[139.5,-1.04],[140.5,-2.02],[141.5,-2.02],[142.5,-1.40],[143.5,-0.80],[144.5,-0.72],[145.5,-0.22],[146.5,-0.76],[147.5,-0.82],[148.5,-1.34],[149.5,-1.54],[150.5,-1.52],[151.5,-1.62],[152.5,-1.80],[153.5,-1.80],[154.5,-1.60],[155.5,-2.06],[156.5,-1.44],[157.5,-0.36],[158.5,-0.44],[159.5,0.18],[160.5,-0.44],[161.5,0.10],[162.5,-1.44],[163.5,-0.28],[164.5,-1.10],[165.5,0.36],[166.5,-1.32],[167.5,0.22],[168.5,-0.94],[169.5,-0.74],[170.5,-1.58],[171.5,-0.88],[172.5,-2.28],[173.5,-1.76],[174.5,-1.76],[175.5,-1.32],[176.5,-1.94],[177.5,-0.46],[178.5,-0.98],[179.5,-0.42],[180.5,-0.92],[181.5,-0.48],[182.5,-0.94],[183.5,0.20],[184.5,-0.44],[185.5,-1.08],[186.5,-1.14],[187.5,-0.68],[188.5,-1.74],[189.5,-0.20],[190.5,0.26],[191.5,0.50],[192.5,0.02],[193.5,0.46],[194.5,-1.08],[195.5,-0.90],[196.5,-1.52],[197.5,-0.98],[198.5,-1.50],[199.5,0.04],[200.5,-0.52],[201.5,1.08],[202.5,0.04],[203.5,0.66],[204.5,-0.74],[205.5,-0.74],[206.5,-2.34],[207.5,-2.90],[208.5,-3.00],[209.5,-2.50],[210.5,-0.90],[211.5,0.64],[212.5,0.64],[213.5,0.20],[214.5,0.08],[215.5,-1.52],[216.5,-3.26],[217.5,-2.20],[218.5,-2.20],[219.5,-2.02],[220.5,-2.22],[221.5,-1.96],[222.5,-2.48],[223.5,-1.92],[224.5,-1.34],[225.5,-0.08],[226.5,0.42],[227.5,0.50]];charge_datapoints = [[2.5,0.00],[3.5,0.00],[4.5,0.00],[5.5,-0.20],[6.5,-0.40],[7.5,-0.40],[8.5,-0.40],[9.5,-0.20],[10.5,-0.20],[11.5,0.00],[12.5,0.00],[13.5,0.20],[14.5,0.00],[15.5,0.40],[16.5,0.40],[17.5,0.60],[18.5,0.40],[19.5,0.20],[20.5,0.00],[21.5,0.00],[22.5,-0.20],[23.5,-0.20],[24.5,0.00],[25.5,0.10],[26.5,-0.10],[27.5,-0.10],[28.5,-0.30],[29.5,-0.30],[30.5,-0.60],[31.5,-0.40],[32.5,-0.60],[33.5,-0.40],[34.5,-0.60],[35.5,-0.40],[36.5,-0.20],[37.5,0.00],[38.5,0.00],[39.5,0.00],[40.5,0.00],[41.5,-0.20],[42.5,-0.20],[43.5,-0.20],[44.5,0.00],[45.5,0.20],[46.5,0.20],[47.5,0.40],[48.5,0.40],[49.5,0.40],[50.5,0.40],[51.5,0.40],[52.5,0.20],[53.5,0.20],[54.5,0.20],[55.5,0.00],[56.5,0.00],[57.5,0.00],[58.5,0.00],[59.5,-0.20],[60.5,-0.20],[61.5,-0.20],[62.5,-0.20],[63.5,-0.20],[64.5,0.00],[65.5,0.00],[66.5,0.00],[67.5,0.00],[68.5,0.00],[69.5,0.00],[70.5,0.20],[71.5,0.20],[72.5,0.20],[73.5,0.20],[74.5,0.40],[75.5,0.30],[76.5,0.30],[77.5,0.30],[78.5,0.10],[79.5,-0.10],[80.5,-0.20],[81.5,-0.40],[82.5,-0.40],[83.5,-0.20],[84.5,0.00],[85.5,0.00],[86.5,0.20],[87.5,0.20],[88.5,0.20],[89.5,-0.20],[90.5,-0.20],[91.5,-0.20],[92.5,0.00],[93.5,0.00],[94.5,0.00],[95.5,0.20],[96.5,0.20],[97.5,0.00],[98.5,0.00],[99.5,0.20],[100.5,-0.20],[101.5,-0.40],[102.5,-0.40],[103.5,-0.40],[104.5,-0.40],[105.5,-0.20],[106.5,0.00],[107.5,0.00],[108.5,0.00],[109.5,0.00],[110.5,-0.20],[111.5,-0.20],[112.5,-0.20],[113.5,-0.20],[114.5,-0.20],[115.5,-0.20],[116.5,-0.20],[117.5,-0.40],[118.5,-0.40],[119.5,-0.40],[120.5,-0.20],[121.5,0.00],[122.5,0.20],[123.5,0.40],[124.5,0.40],[125.5,0.60],[126.5,0.40],[127.5,0.40],[128.5,0.20],[129.5,0.20],[130.5,0.00],[131.5,0.00],[132.5,-0.20],[133.5,-0.20],[134.5,-0.20],[135.5,-0.20],[136.5,-0.20],[137.5,0.00],[138.5,0.20],[139.5,0.40],[140.5,0.40],[141.5,0.40],[142.5,0.40],[143.5,0.20],[144.5,-0.20],[145.5,-0.20],[146.5,-0.20],[147.5,-0.20],[148.5,-0.40],[149.5,0.00],[150.5,0.00],[151.5,0.00],[152.5,0.00],[153.5,0.00],[154.5,-0.40],[155.5,-0.40],[156.5,-0.40],[157.5,-0.40],[158.5,0.00],[159.5,0.20],[160.5,0.00],[161.5,0.00],[162.5,0.20],[163.5,0.00],[164.5,0.20],[165.5,0.40],[166.5,0.60],[167.5,0.40],[168.5,0.60],[169.5,0.20],[170.5,0.20],[171.5,0.00],[172.5,0.10],[173.5,-0.10],[174.5,-0.10],[175.5,-0.10],[176.5,-0.30],[177.5,-0.40],[178.5,-0.20],[179.5,0.00],[180.5,0.00],[181.5,0.20],[182.5,0.20],[183.5,0.00],[184.5,0.20],[185.5,0.40],[186.5,0.40],[187.5,0.40],[188.5,0.40],[189.5,0.20],[190.5,0.00],[191.5,0.00],[192.5,0.00],[193.5,0.00],[194.5,0.20],[195.5,0.20],[196.5,0.00],[197.5,0.00],[198.5,0.20],[199.5,0.00],[200.5,-0.20],[201.5,0.00],[202.5,0.00],[203.5,-0.20],[204.5,-0.10],[205.5,0.10],[206.5,-0.10],[207.5,-0.30],[208.5,-0.30],[209.5,-0.40],[210.5,-0.40],[211.5,-0.20],[212.5,-0.20],[213.5,-0.20],[214.5,-0.20],[215.5,-0.40],[216.5,-0.20],[217.5,0.00],[218.5,-0.20],[219.5,-0.20],[220.5,0.20],[221.5,0.10],[222.5,0.10],[223.5,0.30],[224.5,0.30],[225.5,0.10],[226.5,0.00],[227.5,0.00]];dis_datapoints = undefined;trans_datapoints = undefined;sec_helix_datapoints = undefined;sec_strand_datapoints = undefined;acc_exposed_datapoints = undefined;acc_buried_datapoints = undefined;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 = 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_1414675305031 #hydrophobicity_charge_button').val() =='Show' ){jqAutoAnnotator('#AutoAnnotator_container_1414675305031 #hydrophobicity_charge_container').css('display','block');jqAutoAnnotator('#AutoAnnotator_container_1414675305031 #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_1414675305031 #hydrophobicity_charge_explanation').html(description_html);plot_according_to_selectors_1414675305031();jqAutoAnnotator('#AutoAnnotator_container_1414675305031 #AutoAnnotator_plot_selectors').find('input').click(plot_according_to_selectors_1414675305031);}else{jqAutoAnnotator('#AutoAnnotator_container_1414675305031 #hydrophobicity_charge_container').css('display','none');jqAutoAnnotator('#AutoAnnotator_container_1414675305031 #hydrophobicity_charge_button').val('Show');jqAutoAnnotator('#AutoAnnotator_container_1414675305031 #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_1414675305031(){try{var plot_datasets = [[],[]];if(jqAutoAnnotator('#AutoAnnotator_container_1414675305031 #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_1414675305031 #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_1414675305031 #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_1414675305031 #dis_checkbox').prop('checked') == true){for ( j = 0 ; j < dis_datapoints.length ; j++ ){jqAutoAnnotator('#AutoAnnotator_container_1414675305031 #hydrophobicity_charge_placeholder' + Math.floor((dis_datapoints[j][0] - 1)/200) ).append('<div class=\'AutoAnnotator_dis\' style=\'left:' + ((pos_of_first_tick - 8 + (dis_datapoints[j][0] - 1)*tick_diff - Math.floor((dis_datapoints[j][0] - 1)/200)*200*tick_diff).toFixed(0)).toString() + 'px;\'><b>' + (j+1) + '</b></div>');jqAutoAnnotator('#AutoAnnotator_container_1414675305031 #hydrophobicity_charge_placeholder' + Math.floor((dis_datapoints[j][1] - 1)/200) ).append('<div class=\'AutoAnnotator_dis\' style=\'left:' + ((pos_of_first_tick - 8 + (dis_datapoints[j][1] - 1)*tick_diff - Math.floor((dis_datapoints[j][1] - 1)/200)*200*tick_diff).toFixed(0)).toString() + 'px;\'><b>' + (j+1) + '</b></div>');}}if(jqAutoAnnotator('#AutoAnnotator_container_1414675305031 #trans_checkbox').prop('checked') == true){for ( j = 0 ; j < trans_datapoints.length ; j++ ){jqAutoAnnotator('#AutoAnnotator_container_1414675305031 #hydrophobicity_charge_placeholder' + Math.floor((trans_datapoints[j][0] - 1)/200) ).append('<div class=\'AutoAnnotator_trans\' style=\'width:' + (((trans_datapoints[j][1] - trans_datapoints[j][0] + 1)*tick_diff).toFixed(0)).toString() + 'px ;left:' + ((pos_of_first_tick + (trans_datapoints[j][0] - 1.5)*tick_diff - Math.floor((trans_datapoints[j][0] - 1)/200)*200*tick_diff).toFixed(0)).toString() + 'px\'></div>');}}if(jqAutoAnnotator('#AutoAnnotator_container_1414675305031 #sec_checkbox').prop('checked') == true){for ( j = 0 ; j < sec_helix_datapoints.length ; j++ ){jqAutoAnnotator('#AutoAnnotator_container_1414675305031 #hydrophobicity_charge_placeholder' + Math.floor((sec_helix_datapoints[j][0] - 1)/200) ).append('<div class=\'AutoAnnotator_sec_helix\' style=\'width:' + (((sec_helix_datapoints[j][1] - sec_helix_datapoints[j][0] + 1)*tick_diff).toFixed(0)).toString() + 'px; left:' + ((pos_of_first_tick + (sec_helix_datapoints[j][0] - 1.5)*tick_diff - Math.floor((sec_helix_datapoints[j][0] - 1)/200)*200*tick_diff).toFixed(0)).toString() + 'px\'></div>');}for ( j = 0 ; j < sec_strand_datapoints.length ; j++ ){jqAutoAnnotator('#AutoAnnotator_container_1414675305031 #hydrophobicity_charge_placeholder' + Math.floor((sec_strand_datapoints[j][0] - 1)/200) ).append('<div class=\'AutoAnnotator_sec_strand\' style=\'width:' + (((sec_strand_datapoints[j][1] - sec_strand_datapoints[j][0] + 1)*tick_diff).toFixed(0)).toString() + 'px; left:' + ((pos_of_first_tick + (sec_strand_datapoints[j][0] - 1.5)*tick_diff - Math.floor((sec_strand_datapoints[j][0] - 1)/200)*200*tick_diff).toFixed(0)).toString() + 'px\'></div>');}}if(jqAutoAnnotator('#AutoAnnotator_container_1414675305031 #acc_checkbox').prop('checked') == true){for ( j = 0 ; j < acc_buried_datapoints.length ; j++ ){jqAutoAnnotator('#AutoAnnotator_container_1414675305031 #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_1414675305031 #hydrophobicity_charge_placeholder' + Math.floor((acc_exposed_datapoints[j][0] - 1)/200) ).append('<div class=\'AutoAnnotator_acc_exposed\' style=\'width:' + (((acc_exposed_datapoints[j][1] - acc_exposed_datapoints[j][0] + 1)*tick_diff).toFixed(0)).toString() + 'px; left:' + ((pos_of_first_tick + (acc_exposed_datapoints[j][0] - 1.5)*tick_diff - Math.floor((acc_exposed_datapoints[j][0] - 1)/200)*200*tick_diff).toFixed(0)).toString() + 'px\'></div>');}}}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> | ||
Latest revision as of 16:35, 30 October 2014
Monomeric Red Fluorescent Protein from Discosoma striata
Mutant of the Biobrick BBa_E1010. In order to make it compatible with the Freiburg-Standard RFC25, two AgeI-Restriction sites were deleted.
monomeric RFP: Red Fluorescent Protein.
Excitation peak: 584 nm Emission peak: 607 nm
Usage and Biology
This part was created in order to enhance the [http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks Bacillus BioBrick Box] by adding different fluorescent proteins to have more reporters available in B. subtilis.
Since the Biobrick BBa_E1010 is already evaluated very well and shows a very bright color in E. coli, the rather light coloring in our experimental setup may be due to the used expression vector pBS0K-Pspac. This vector is optimized for the use in B. subtilis and shows a rather weak and heterogenous expression in E. coli.
Picture 1: BBa_K1351021 in the B. subtilis expression vector pBS0K-Pspac, cloned into E. coli. The rather weak color is due to the vector, which shows a rather weak an heterogenous expression. On the right picture, the left eppendorf tube contains a sample of E. coli containing the Biobrick BBa_K1351021, the right eppendorf tube contains E. coli with just the empty vector.
Design
This part was generated in a modified version of RFC25, where a strong Shine Dalgarno Sequence (SD) is included, and has the following prefix and suffix:
prefix with EcoRI, NotI, XbaI, SD and NgoMIV: | GAATTCGCGGCCGCTTCTAGAGTAAGGAGGAGCCGGC |
suffix with AgeI, SpeI, NotI and PstI: | ACCGGTTAATACTAGTAGCGGCCGCTGCAG |
Sites of restriction enzymes generating compatible overhangs have the same color:
EcoRI and PstI in blue, NotI in green, XbaI and SpeI in red, NgoMIV and AgeI in orange. Shine-Dalgarno sequence and stop codons are underlined.
This part is used in the 2014 LMU-Munich iGEM project [http://2014.igem.org/Team:LMU-Munich BaKillus].
BioBrick AutoAnnotator
Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0 | ||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Nucleotide sequence in RFC 25, so ATGGCCGGC and ACCGGT were added (in italics) to the 5' and 3' ends: (underlined part encodes the protein) ATGGCCGGCATGGCTTCC ... ACCTGTGCTACCGGT ORF from nucleotide position -8 to 681 (excluding stop-codon) | ||||||||||||||||||||||||||||||||||||||||||||||
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)
| ||||||||||||||||||||||||||||||||||||||||||||||
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
| ||||||||||||||||||||||||||||||||||||||||||||||
Amino acid composition:
| ||||||||||||||||||||||||||||||||||||||||||||||
Amino acid counting
| Biochemical parameters
| |||||||||||||||||||||||||||||||||||||||||||||
Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges | ||||||||||||||||||||||||||||||||||||||||||||||
Codon usage
| ||||||||||||||||||||||||||||||||||||||||||||||
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. |
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]