<div  id='write'  class = 'is-node'><h1><a name='header-n0' class='md-header-anchor '></a><strong>Source</strong></h1><p>The SpyCatcher-SpyTag system was developed by the Howarth laboratory based on the internal isopeptide bond of the CnaB2 domain of FbaB, a fibronectin-binding MSCRAMM and virulence factor of <em>Streptococcus pyogenes</em><span class="MathJax_Preview"></span><span class="MathJax_SVG" id="MathJax-Element-11-Frame" tabindex="-1" style="font-size: 100%; display: inline-block;"><svg xmlns:xlink="http://www.w3.org/1999/xlink" width="1.967ex" height="2.344ex" viewBox="0 -956.9 846.7 1009.2" role="img" focusable="false" style="vertical-align: -0.121ex;"><defs><path stroke-width="0" id="E15-MJMAIN-5B" d="M118 -250V750H255V710H158V-210H255V-250H118Z"></path><path stroke-width="0" id="E15-MJMAIN-31" d="M213 578L200 573Q186 568 160 563T102 556H83V602H102Q149 604 189 617T245 641T273 663Q275 666 285 666Q294 666 302 660V361L303 61Q310 54 315 52T339 48T401 46H427V0H416Q395 3 257 3Q121 3 100 0H88V46H114Q136 46 152 46T177 47T193 50T201 52T207 57T213 61V578Z"></path><path stroke-width="0" id="E15-MJMAIN-5D" d="M22 710V750H159V-250H22V-210H119V710H22Z"></path></defs><g stroke="currentColor" fill="currentColor" stroke-width="0" transform="matrix(1 0 0 -1 0 0)"><g transform="translate(0,362)"><use transform="scale(0.707)" xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#E15-MJMAIN-5B" x="0" y="0"></use><use transform="scale(0.707)" xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#E15-MJMAIN-31" x="278" y="0"></use><use transform="scale(0.707)" xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#E15-MJMAIN-5D" x="778" y="0"></use></g></g></svg></span><script type="math/tex" id="MathJax-Element-11">^{[1]}</script>.</p><p> </p><p> </p><h1><a name='header-n19' class='md-header-anchor '></a>Characterization</h1><p>SpyTag is a short peptide consisting of 13 amino acids. The aspartic acid side chain in SpyTag can form isopeptide bonds with the lysine side chain of SpyCatcher<span class="MathJax_Preview"></span><span class="MathJax_SVG" id="MathJax-Element-5-Frame" tabindex="-1" style="font-size: 100%; display: inline-block;"><svg xmlns:xlink="http://www.w3.org/1999/xlink" width="1.967ex" height="2.344ex" viewBox="0 -956.9 846.7 1009.2" role="img" focusable="false" style="vertical-align: -0.121ex;"><defs><path stroke-width="0" id="E7-MJMAIN-5B" d="M118 -250V750H255V710H158V-210H255V-250H118Z"></path><path stroke-width="0" id="E7-MJMAIN-32" d="M109 429Q82 429 66 447T50 491Q50 562 103 614T235 666Q326 666 387 610T449 465Q449 422 429 383T381 315T301 241Q265 210 201 149L142 93L218 92Q375 92 385 97Q392 99 409 186V189H449V186Q448 183 436 95T421 3V0H50V19V31Q50 38 56 46T86 81Q115 113 136 137Q145 147 170 174T204 211T233 244T261 278T284 308T305 340T320 369T333 401T340 431T343 464Q343 527 309 573T212 619Q179 619 154 602T119 569T109 550Q109 549 114 549Q132 549 151 535T170 489Q170 464 154 447T109 429Z"></path><path stroke-width="0" id="E7-MJMAIN-5D" d="M22 710V750H159V-250H22V-210H119V710H22Z"></path></defs><g stroke="currentColor" fill="currentColor" stroke-width="0" transform="matrix(1 0 0 -1 0 0)"><g transform="translate(0,362)"><use transform="scale(0.707)" xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#E7-MJMAIN-5B" x="0" y="0"></use><use transform="scale(0.707)" xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#E7-MJMAIN-32" x="278" y="0"></use><use transform="scale(0.707)" xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#E7-MJMAIN-5D" x="778" y="0"></use></g></g></svg></span><script type="math/tex" id="MathJax-Element-5">^{[2]}</script>.In particular, the size of SpyTag is equivalent to many epitope tags,which can be produced as fusion proteins and can be applied in the direction of antigen delivery, modification of protein hydrogels, etc.</p><p> </p><p>We attempted to display SpyTag and SpyCatcher on the surface of Escherichia coliBL21 (De3) respectively, using this system to achieve cross-linking between bacteria.</p><p> </p><p>Using fluorescentproteins, we constructed a system for verifying cross-linking, in which theengineered bacteria introduced plasmids and genes as shown in the table below.</p><figure><table><thead><tr><th>      <strong>pET30a</strong> <br/></th><th>  <strong>pJUMP46-2A</strong> <br/></th><th>&nbsp;</th></tr></thead><tbody><tr><td>  A<br/></td><td>  SpyTag<br/></td><td>  sfGFP<br/></td></tr><tr><td>  B<br/></td><td>  SpyCatcher<br/></td><td>  mCherry<br/></td></tr><tr><td>  C<br/></td><td>  empty plasmid<br/></td><td>  sfGFP<br/></td></tr><tr><td>  D<br/></td><td>  empty plasmid<br/></td><td>  mCherry<br/></td></tr></tbody></table></figure><p><strong>Tab1. Plasmids andgenes induced into engineering bacteria.</strong></p><p> </p><p>We verify cross-linkingin two ways: by measuring optical density and microscopy.</p><p> </p><p>Due to the cross-linking between bacteria, the buoyancy increases, and after standing for a period of time, fewer bacteria settle down, and the remaining rate of bacteria is greater.</p><p><img src='https://static.igem.wiki/teams/4614/wiki/parts-jl/jl-1.png></p><p><strong>Fig1 Quantitative verification of adherence of bacteria</strong></p><p>Fluorescence microscopy and confocal microscopy were used to verify the cross-linking, and four groups of experiments were set up, namely the control group (AD, BC, CD) and the experimental group (AB). The observation results were shown in the figures below.</p><p><img src='https://static.igem.wiki/teams/4614/wiki/parts-jl/jl-2.png/></p><p><strong>Fig2. Observationof bacterial adhesion by laser microscopy</strong></p><p>Observation of bacterial adhesion by laser microscopy were observed under a laser microscope(1000×).</p><p>&nbsp;</p><p>It can be seen from the above figure that the bacteria in the experimental group have obvious aggregation phenomenon, and the fluorescence in them can be seen that the aggregated bacteria express SpyTag and SpyCatcher respectively, which shows that the system can work.</p><p>&nbsp;</p><h1><a name='header-n97' class='md-header-anchor '></a>References</h1><p>[1] Hatlem, Daniel et al. “Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins.” <em>International journal of molecular sciences</em> vol. 20,9 2129. 30 Apr. 2019, doi:10.3390/ijms20092129</p><p>[2]Kozlowski, Mark T et al. “Genetically Programmable Microbial Assembly.” <em>ACS synthetic biology</em> vol. 10,6 (2021): 1351-1359. doi:10.1021/acssynbio.0c00616</p></div>