Difference between revisions of "Part:BBa K1351021"

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<partinfo>BBa_K1351021 short</partinfo>
 
<partinfo>BBa_K1351021 short</partinfo>
  
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Mutant of the Biobrick BBa_E1010 [[https://parts.igem.org/Part:BBa_E1010]], in order to make it compatible with the Freiburg-Standard, two AgeI-Restriction sites were deleted
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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.
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monomeric RFP: Red Fluorescent Protein. Excitation peak: 584 nm Emission peak: 607 nm
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monomeric RFP: Red Fluorescent Protein.  
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Excitation peak: 584 nm Emission peak: 607 nm
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=== Usage and Biology ===
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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>
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[[File:LMU14_K1351021_RFP.jpg|center|800px]]
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'''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.
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=== Design ===
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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:
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{|
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|prefix with EcoRI, NotI, XbaI, SD and NgoMIV:
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|<span style="color:blue">GAATTC</span><span style="color:green">GCGGCCGC</span>T<span style="color:red">TCTAGA</span>GT<u>AAGGAGG</u>A<span style="color:orange">GCCGGC</span>
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|-
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|suffix with AgeI, SpeI, NotI and PstI:
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|<span style="color:orange">ACCGGT</span><u>TAA</u>T<span style="color:red">AC<u>TAG</u><u>T</u></span><u>A</u><span style="color:green"><u>G</u>CGGCCG</span><span style="color:blue">CTGCAG</span>
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|}
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Sites of restriction enzymes generating compatible overhangs have the same color:
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<span style="color:blue">EcoRI</span> and <span style="color:blue">PstI</span> in blue, <span style="color:green">NotI</span> in green, <span style="color:red">XbaI</span> and <span style="color:red">SpeI</span> in red, <span style="color:orange">NgoMIV</span> and <span style="color:orange">AgeI</span> in orange. Shine-Dalgarno sequence and stop codons are underlined.
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This part is used in the 2014 LMU-Munich iGEM project [http://2014.igem.org/Team:LMU-Munich BaKillus].
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=== BioBrick AutoAnnotator ===
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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_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">&nbsp;<u><i>ATGGCCGGC</i>ATGGCTTCC&nbsp;...&nbsp;ACCTGTGCT<i>ACCGGT</i></u></span><br>&nbsp;<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&nbsp;<br>101&nbsp;<br>201&nbsp;</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>&nbsp;<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>&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_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&nbsp;&nbsp;'], [-4.5,'hydro-<br>philic&nbsp;&nbsp;']],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>&nbsp;charge'], [-1,'negative<br>&nbsp;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>&nbsp;<input type=\'checkbox\' id=\'hydrophobicity_checkbox\' checked=\'checked\'>&nbsp;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>&nbsp;<input type=\'checkbox\' id=\'charge_checkbox\' checked=\'checked\'>&nbsp;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>&nbsp;<input type=\'checkbox\' id=\'dis_checkbox\' 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_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] - 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(((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>
 +
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

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.

LMU14 K1351021 RFP.jpg


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)
1 
101 
201 		MAGMASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVM
NFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMRLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDI
KLDITSHNEDYTIVEQYERAEGRHSTCATG*
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
None of the supported features appeared in the sequence
Amino acid composition:
Ala (A)13 (5.7%)
Arg (R)9 (3.9%)
Asn (N)4 (1.7%)
Asp (D)14 (6.1%)
Cys (C)1 (0.4%)
Gln (Q)8 (3.5%)
Glu (E)22 (9.6%)
Gly (G)24 (10.4%)
His (H)5 (2.2%)
Ile (I)9 (3.9%)
Leu (L)12 (5.2%)
Lys (K)22 (9.6%)
Met (M)10 (4.3%)
Phe (F)10 (4.3%)
Pro (P)12 (5.2%)
Ser (S)12 (5.2%)
Thr (T)15 (6.5%)
Trp (W)3 (1.3%)
Tyr (Y)11 (4.8%)
Val (V)14 (6.1%)
Amino acid counting
Total number:230
Positively charged (Arg+Lys):31 (13.5%)
Negatively charged (Asp+Glu):36 (15.7%)
Aromatic (Phe+His+Try+Tyr):29 (12.6%)
Biochemical parameters
Atomic composition:C1152H1778N304O353S11
Molecular mass [Da]:25887.3
Theoretical pI:5.65
Extinction coefficient at 280 nm [M-1 cm-1]:32890 / 32953 (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):excellent (0.84)good (0.72)good (0.68)good (0.74)good (0.79)good (0.72)
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.
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Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]