Difference between revisions of "Part:BBa K1617000"
 
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<b>Flagellin</b> is the filament forming component of bacterial flagellas. The up to 20 µm long filaments are formed by the self-assembly of flagellin. Flagellin proteins consist of four domains (D0-D3) and have asize of about 20-30kDa. While the D0 and D1 domains are well conserved, the D2 and D3 domains are highly variable in sequence and length. Here, we created an modular flagellin with an easily replacable D3 region. A multi-cloning side flanking the D3 domain allows the design of costume-made flagellins by being compatible to iGEM fusion standards such as RFC25 and RFC21.
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<b>Flagellin</b> is the filament forming component of bacterial flagella. The up to 20 µm long filaments are formed by the self-assembly of flagellin. Flagellin proteins consist of four domains (D0-D3) and have a size of about 20-30kDa. While the D0 and D1 domains are well conserved, the D2 and D3 domains are highly variable in sequence and length [1]. Here, we created a modular flagellin with an easily replacable D3 region. A multiple-cloning site flanking the D3 domains allows for the design of custom-made flagellins by being compatible with iGEM fusion standards such as RFC25 and RFC21.
  
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<br><br><b> You can pick any RFC25 compatible BioBrick from the registry and test its functionality immidiatally via an easy motility assay.</b>
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<br><br><b> You can pick any RFC25 compatible BioBrick from the registry and test its functionality immediately via a facile motility assay.</b>
 
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This system enables the creation of a three-dimensional reactive nanostructure that offers an increased specific surface with high catalytic activity. With this method produced functionalized flagella filaments can be easily harvested via shearing stress following a simple protocol. Flaggela are self-assembling polymer systems. A temperature increase up to 95 °C depolymerizes the flagella and disconnects the flagellin subunits. By cooling down, these subunits assemble back into fully formed flagella filaments. This means that various flagellin subunits with different molecular set-ups can be combined and used to form <b>multifunctional nanostructures</b>.
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One flagella usually carries about  flavone from grapefruits. In plants, it is synthesized from tyrosine and is one of the central metabolites in the flavone biosynthesis. It is able to reduce the oxidative stress and inhibit some P450 enzymes. One of these cytochrome P450 enzymes is involved in the degradation of caffeine and increases the effect of caffeine after the inhibition with naringenin. 
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The resulting flagella may be constructed consisting of various different active sites, which will enable the combination of multiple enzymatic steps in close proximity.  
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<b>FdeR</b> is a homo dimeric protein from <i>Herbaspirillum seropedicae</i>. In the presence of naringenin (or naringenin chalcone), FdeR activates the specific promoter region upstream of the FdeR region and induces a strong gene expression. <br> In  <i>Herbaspirillum seropedicae</i> the FdeR activates the Fde-Operon (Fde: Flavanone degradation) and enables the growth with naringenin and the naringenin chalcone. 
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When GFP or another reporter protein is cloned downstream of this part, it can be used as an <i>in vivo</i> naringenin sensor.
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<b>FliC MCS</b> is a monomer which is stacked in a helical manner forming the filament of flagella. In this part we used the fliC from Escherichia coli str. K-12 substr. MG1655. As the natural self-assembly involves the transportation of fliC thorugh the filament tube there are steric limitations to the flagellin design. If flagellin-hybrids have a higher molecular mass than the wildtype flagellin (about 52 kDa ProtParam) problems may arise due to steric effects. In this case, we suggest using non-canonical aminoacids (ncAAs) for in-vitro bioconjugation via click reaction. You can read more about it in our wiki or in our D3 Biobrick description.
 
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       <p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 1</b></span></a><span lang="EN-US">
 
       <p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 1</b></span></a><span lang="EN-US">
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Video showing the self-assembly of the bacterial flagella. FliC is the main component which makes up most of the filament. Source: https://youtu.be/GnNCaBXL7LY </span>
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Video showing the self-assembly of the bacterial flagellum. FliC is the main component which makes up most of the filament. Source: https://youtu.be/GnNCaBXL7LY </span>
 
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       <p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 2</b></span></a><span lang="EN-US">
 
       <p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 2</b></span></a><span lang="EN-US">
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In the modular flagellin the not conserved D3 domain was replaced with a polylinker coding a multi-cloning site. This part may be used to introduce any standard RFC25 part into the filament forming flagellin.</span></p>
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In the modular flagellin the variable D3 domain was replaced with a polylinker coding a multiple-cloning site. This part may be used to introduce any standard RFC25 part into the flagellin precursor forming novel flagallins.</span></p>
 
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You can use the reporters for measuring naringenin concentrations in your samples.  
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You can use the motility assay to screen for functional flagellin formation.  
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Depending on which fluorophor you want to detect, you can use one of three biosensors:
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   <li class="block-10vi">A: with CFP response use         <a href="/Part:BBa_K1497022">BBa_K1497022</a></li>
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   <li class="block-10vi">A: MG1655 z1 ΔFliC      
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   <li class="block-10vi">B: with mKate response use <a href="/Part:BBa_K1497021">BBa_K1497021</a></li>
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   <li class="block-10vi">B: B = MG1655 z1
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   <li class="block-10vi">C: with no reporter               <a href="/Part:BBa_K1497019">BBa_K1497019</a></li>
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   <li class="block-10vi">C: C = Prom110_FliC_MCS               <a href="/Part:BBa_K1617003">BBa_K1617003</a></li>
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  <li class="block-10vi">D: with GFP response use          <a href="/Part:BBa_K1497020">BBa_K1497020</a></li>  
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src="https://static.igem.org/mediawiki/2014/8/89/Petridischnaringenin.png"></p>
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src="https://static.igem.org/mediawiki/2015/3/35/Team_Berlin_Results.png"></p>
 
        
 
        
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       <p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 2</b></span></a><span lang="EN-US">
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       <p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 3</b></span></a><span lang="EN-US">
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<i>E. coli</i> Top10 with different naringenin biosensors. Left: On agar plate without naringenin no colour is visible. Middle: On agar plate with 100 µM naringenin colour is visible, except of negative sample <a href="/Part:BBa_K1497019">BBa_K1497019</a> without fluorophor. Right: On agar plate with 100 µM naringenin under UV light. The fluorescence of GFP, CFP and mKate is visible. <br></span></p>
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Comparison of the motility between the FliC MCS carrying strain (right) and a negative (left) and positive (middle) control. Since our construct was transformed into E. coli strain MG1655 z1 ΔFliC, we used the highly motile wildtype strain MG1655 z1 as positive control and as negative control the FliC knockout strain MG1655 z1 ΔFliC. <br></span></p>
 
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You can create your own naringenin sensor or your own naringenin dependent gene expression device as well. For these reasons use the Biobrick <html><a href="/Part:BBa_K1497019">K1497019</a></html> and clone your parts of interest (without RBS!) behind the device.
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You can create your own functional flagellin. Just use an RFC25 compatible domain, gene region and clone it into the standard FliC MCS using AgeI and NgoMIV. You need to check for the correct alignment though...
  
 
===Functional Parameters===
 
===Functional Parameters===
  
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The Biobrick <html><a href="/Part:BBa_K1497019">BBa_K1497019</a></html> produces in <i>E. coli</i> B and K strains the FdeR Protein. The iGEM Team TU Darmstadt 2014 measured the fluorescense of GFP and mKate after the incubation with diffrent conentrations of naringenin. The results are shown in Figure 3.  
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Design of a functional flaggelin variant which does not contain  the variable d3 flagellin domain anymore but instead carries a polylinker region (MCS). This polylinker region is the product of an inserted multi cloning side in place of the endogeneous d3 coding sequence.
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       <p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 4</b></span></a><span lang="EN-US">
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<b>Left:</b> Characterization of  <a href="/Part:BBa_K1497020">BBa_K1497020</a>. GFP fluorescence depends on the concentration of naringenin. We measured the GFP fluorescence after 16 h incubation with different concentrations of naringenin. By setting higher concentrations of naringenin, we gained higher fluorescence of GFP as well. <b>Right:</b> Characterization of <a href="/Part:BBa_K1497021">BBa_K1497021</a>. mKate (<a href="/Part:BBa_K1055000">BBa_K1055000</a>) fluorescence depends on the concentration of naringenin. We measured the mKate (<a href="/Part:BBa_K1055000">BBa_K1055000</a>) fluorescence after 16 h incubation with different concentrations of naringenin. By setting higher concentrations of naringenin, we gained higher fluorescence of mKate as well.</span></p>
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Characterization of  <a href="/Part:BBa_K1617003">BBa_K1617003</a>. Through the motility assay it could be shown that our BioBricks work. With cloning the BioBrick into a delta flick deficient strand the motility could be regained. There is no significant difference according the motility between the wildtype and our transformed consturuct in delta flic deficient clones. </span></p>
 
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====In vivo characterisation of the naringenin biosynthesis operon (<html><a href="/Part:BBa_K1497007">BBa_K1497007</a></html>)====
 
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iGEM TU Darmstadt 2014 reconstitute the naringenin biosynthesis in <i>E. coli</i> by construction of a operon polycistronic gene cluster (<a href="/Part:BBa_K1497007">BBa_K1497007</a>) under control of the strong T7 promoter (<a href="/Part:BBa_K1497017">BBa_K1497017</a>). They used the naringenin biosensor with GFP response <a href="/Part:BBa_K1497020">K1497020</a> to characterize the naringenin biosynthesis operon in <i>E. coli</i> BL21(DE3). <br><br> The result are shown in figure 4. The GFP fluorescene is only in the cells with the T7 naringenin operon visible and detectable. The team determined for this operon a naringenin production yield of 3 µmol naringenin per liter.   
 
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      <p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 4</b></span></a><span lang="EN-US">
 
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<b>Left: </b>Cell pellets with and without T7-Naringenin operon from <i>E. coli</i> BL21(DE3)-pSB1C3-<i>fdeR-gfp</i>. By using ultraviolet light the pellet containing the naringenin operon shows a GFP fluorescence. <b>Right: </b>Measurement of the GFP fluorescence in the<i>E. coli</i> BL21(DE3)-pSB1C3-<i>fdeR-gfp</i> strain containing and not containing the T7-Naringenin operon.
 
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Design of a functional flaggelin variant which does not contain  the variable d3 flagellin domain anymore but instead carries a polylinker region (MCS). This polylinker region is the product of an inserted multi cloning side in place of the endogeneous d3 coding sequence.
 
  
  
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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===Auto annotator===
 
<partinfo>BBa_K1617000 parameters</partinfo>
 
<partinfo>BBa_K1617000 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_1443053505986'><table id="AutoAnnotator"><tr><!-- Time stamp in ms since 1/1/1970 1443053505986 --><th id="AutoAnnotatorHeader" colspan="2">Protein data table for BioBrick <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_<!------------------------Enter BioBrick number here------------------------>">BBa_<!------------------------Enter BioBrick number here------------------------></a> automatically created by the <a href="http://2013.igem.org/Team:TU-Munich/Results/AutoAnnotator">BioBrick-AutoAnnotator</a> version 1.0</th></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Nucleotide sequence</strong> in <strong>RFC 10</strong>: (underlined part encodes the protein)<br><span class="AutoAnnotatorSequence">&nbsp;<u>ATGGCACAA&nbsp;...&nbsp;CTGCAAGGT</u>TAA</span><br>&nbsp;<strong>ORF</strong> from nucleotide position 1 to 1293 (excluding stop-codon)</td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Amino acid sequence:</strong> (RFC 25 scars in shown in bold, other sequence features underlined; both given below)<br><span class="AutoAnnotatorSequence"><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorSeqNum">1&nbsp;<br>101&nbsp;<br>201&nbsp;<br>301&nbsp;<br>401&nbsp;</td><td class="AutoAnnotatorSeqSeq">MAQVINTNSLSLITQNNINKNQSALSSSIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQAARNANDGISVAQTTEGALSEINNNLQRVRELTVQAT<br><b>TG</b>TNSESDLSSIQDEIKSRLDEIDRVSGQTQFNGVNVLAKNGSAKIQVGANDNQTITIDLKQIDAKTLGLDGFSVKNNDTVTTSAPVTAFGAGSAGVDMG<br>GSMGGSMKLRSTGSGGTPVQIDNTAGSATANLGAVSLVKLQDSKGNDTDTYALKDTNGNLYAADVNETTGAVSVKTITYTDSSGAASSPTAVKLGGDDGK<br>TEVVDIDGKTYDSADLNGGNLQTGLTAGGEALTAVANGKTTDPLKALDDAIASVDKFRSSLGAVQNRLDSAVTNLNNTTTNLSEAQSRIQDADYATEVSN<br>GSKAQIIQQAGNSVLAKANQVPQQVLSLLQG*</td></tr></table></span></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Sequence features:</strong> (with their position in the amino acid sequence, see the <a href="http://2013.igem.org/Team:TU-Munich/Results/Software/FeatureList">list of supported features</a>)<table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorSeqFeat1"></td><td class="AutoAnnotatorSeqFeat2a">RFC25 scar (shown in bold):&nbsp;</td><td class="AutoAnnotatorSeqFeat3">101 to 102</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">49 (11.4%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Arg (R)</td><td class="AutoAnnotatorInnerAmino">12 (2.8%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Asn (N)</td><td class="AutoAnnotatorInnerAmino">39 (9.0%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Asp (D)</td><td class="AutoAnnotatorInnerAmino">33 (7.7%)</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 (6.3%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Glu (E)</td><td class="AutoAnnotatorInnerAmino">12 (2.8%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Gly (G)</td><td class="AutoAnnotatorInnerAmino">42 (9.7%)</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">23 (5.3%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Leu (L)</td><td class="AutoAnnotatorInnerAmino">36 (8.4%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Lys (K)</td><td class="AutoAnnotatorInnerAmino">22 (5.1%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Met (M)</td><td class="AutoAnnotatorInnerAmino">4 (0.9%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Phe (F)</td><td class="AutoAnnotatorInnerAmino">5 (1.2%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Pro (P)</td><td class="AutoAnnotatorInnerAmino">5 (1.2%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Ser (S)</td><td class="AutoAnnotatorInnerAmino">45 (10.4%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Thr (T)</td><td class="AutoAnnotatorInnerAmino">43 (10.0%)</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">5 (1.2%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Val (V)</td><td class="AutoAnnotatorInnerAmino">29 (6.7%)</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">431</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Positively charged (Arg+Lys):</td><td class="AutoAnnotatorAminoCountingInner3">34 (7.9%)</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Negatively charged (Asp+Glu):</td><td class="AutoAnnotatorAminoCountingInner3">45 (10.4%)</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Aromatic (Phe+His+Try+Tyr):</td><td class="AutoAnnotatorAminoCountingInner3">10 (2.3%)</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>1859</sub>H<sub>3077</sub>N<sub>555</sub>O<sub>681</sub>S<sub>4</sub></td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Molecular mass [Da]:</td><td class="AutoAnnotatorBiochemParInner3">44227.4</td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Theoretical pI:</td><td class="AutoAnnotatorBiochemParInner3">4.67</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">7450 / 7450 (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_1443053505986()' 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 (0.67)</td><td class="AutoAnnotatorCodonUsage3">good (0.71)</td><td class="AutoAnnotatorCodonUsage3">excellent (0.82)</td><td class="AutoAnnotatorCodonUsage3">good (0.71)</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_1443053505986(){hydrophobicity_datapoints = 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Latest revision as of 00:05, 24 September 2015

Modular Flagellin - FliC_MCS

Flagellin is the filament forming component of bacterial flagella. The up to 20 µm long filaments are formed by the self-assembly of flagellin. Flagellin proteins consist of four domains (D0-D3) and have a size of about 20-30kDa. While the D0 and D1 domains are well conserved, the D2 and D3 domains are highly variable in sequence and length [1]. Here, we created a modular flagellin with an easily replacable D3 region. A multiple-cloning site flanking the D3 domains allows for the design of custom-made flagellins by being compatible with iGEM fusion standards such as RFC25 and RFC21.

You can pick any RFC25 compatible BioBrick from the registry and test its functionality immediately via a facile motility assay.

This system enables the creation of a three-dimensional reactive nanostructure that offers an increased specific surface with high catalytic activity. With this method produced functionalized flagella filaments can be easily harvested via shearing stress following a simple protocol. Flaggela are self-assembling polymer systems. A temperature increase up to 95 °C depolymerizes the flagella and disconnects the flagellin subunits. By cooling down, these subunits assemble back into fully formed flagella filaments. This means that various flagellin subunits with different molecular set-ups can be combined and used to form multifunctional nanostructures. The resulting flagella may be constructed consisting of various different active sites, which will enable the combination of multiple enzymatic steps in close proximity.

FliC MCS is a monomer which is stacked in a helical manner forming the filament of flagella. In this part we used the fliC from Escherichia coli str. K-12 substr. MG1655. As the natural self-assembly involves the transportation of fliC thorugh the filament tube there are steric limitations to the flagellin design. If flagellin-hybrids have a higher molecular mass than the wildtype flagellin (about 52 kDa ProtParam) problems may arise due to steric effects. In this case, we suggest using non-canonical aminoacids (ncAAs) for in-vitro bioconjugation via click reaction. You can read more about it in our wiki or in our D3 Biobrick description.


Figure 1 Video showing the self-assembly of the bacterial flagellum. FliC is the main component which makes up most of the filament. Source: https://youtu.be/GnNCaBXL7LY


Figure 2 In the modular flagellin the variable D3 domain was replaced with a polylinker coding a multiple-cloning site. This part may be used to introduce any standard RFC25 part into the flagellin precursor forming novel flagallins.



Usage and Biology

You can use the motility assay to screen for functional flagellin formation.




  • A: MG1655 z1 ΔFliC
  • B: B = MG1655 z1
  • C: C = Prom110_FliC_MCS BBa_K1617003

Figure 3 Comparison of the motility between the FliC MCS carrying strain (right) and a negative (left) and positive (middle) control. Since our construct was transformed into E. coli strain MG1655 z1 ΔFliC, we used the highly motile wildtype strain MG1655 z1 as positive control and as negative control the FliC knockout strain MG1655 z1 ΔFliC.

You can create your own functional flagellin. Just use an RFC25 compatible domain, gene region and clone it into the standard FliC MCS using AgeI and NgoMIV. You need to check for the correct alignment though...

Functional Parameters

Design of a functional flaggelin variant which does not contain the variable d3 flagellin domain anymore but instead carries a polylinker region (MCS). This polylinker region is the product of an inserted multi cloning side in place of the endogeneous d3 coding sequence.



Figure 4 Characterization of BBa_K1617003. Through the motility assay it could be shown that our BioBricks work. With cloning the BioBrick into a delta flick deficient strand the motility could be regained. There is no significant difference according the motility between the wildtype and our transformed consturuct in delta flic deficient clones.





Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 628
    Illegal BamHI site found at 577
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 583
    Illegal AgeI site found at 634
  • 1000
    COMPATIBLE WITH RFC[1000]


Auto annotator

Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0
Nucleotide sequence in RFC 10: (underlined part encodes the protein)
 ATGGCACAA ... CTGCAAGGTTAA
 ORF from nucleotide position 1 to 1293 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)
1 
101 
201 
301 
401 		MAQVINTNSLSLITQNNINKNQSALSSSIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQAARNANDGISVAQTTEGALSEINNNLQRVRELTVQAT
TGTNSESDLSSIQDEIKSRLDEIDRVSGQTQFNGVNVLAKNGSAKIQVGANDNQTITIDLKQIDAKTLGLDGFSVKNNDTVTTSAPVTAFGAGSAGVDMG
GSMGGSMKLRSTGSGGTPVQIDNTAGSATANLGAVSLVKLQDSKGNDTDTYALKDTNGNLYAADVNETTGAVSVKTITYTDSSGAASSPTAVKLGGDDGK
TEVVDIDGKTYDSADLNGGNLQTGLTAGGEALTAVANGKTTDPLKALDDAIASVDKFRSSLGAVQNRLDSAVTNLNNTTTNLSEAQSRIQDADYATEVSN
GSKAQIIQQAGNSVLAKANQVPQQVLSLLQG*
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
RFC25 scar (shown in bold): 101 to 102
Amino acid composition:
Ala (A)49 (11.4%)
Arg (R)12 (2.8%)
Asn (N)39 (9.0%)
Asp (D)33 (7.7%)
Cys (C)0 (0.0%)
Gln (Q)27 (6.3%)
Glu (E)12 (2.8%)
Gly (G)42 (9.7%)
His (H)0 (0.0%)
Ile (I)23 (5.3%)
Leu (L)36 (8.4%)
Lys (K)22 (5.1%)
Met (M)4 (0.9%)
Phe (F)5 (1.2%)
Pro (P)5 (1.2%)
Ser (S)45 (10.4%)
Thr (T)43 (10.0%)
Trp (W)0 (0.0%)
Tyr (Y)5 (1.2%)
Val (V)29 (6.7%)
Amino acid counting
Total number:431
Positively charged (Arg+Lys):34 (7.9%)
Negatively charged (Asp+Glu):45 (10.4%)
Aromatic (Phe+His+Try+Tyr):10 (2.3%)
Biochemical parameters
Atomic composition:C1859H3077N555O681S4
Molecular mass [Da]:44227.4
Theoretical pI:4.67
Extinction coefficient at 280 nm [M-1 cm-1]:7450 / 7450 (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 (0.83)good (0.78)good (0.67)good (0.71)excellent (0.82)good (0.71)
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.
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