Difference between revisions of "Part:BBa K5246029"

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<partinfo>BBa_K5246029 short</partinfo>
 
<partinfo>BBa_K5246029 short</partinfo>
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===Introduction===
 
===Introduction===
  
 +
Vilnius-Lithuania iGEM 2024 project Synhesion aspires to create biodegradable and environmentally friendly adhesives. We were inspired by bacteria, which naturally produce adhesives made from polysaccharides. Two bacteria from aquatic environments - <I> C. crescentus </I> and <I> H. Baltica </I> - harness 12 protein synthesis pathways to produce sugars anchoring them to the surfaces. We aimed to transfer the polysaccharide synthesis pathway to industrially used <I>E. coli</I> bacteria to produce adhesives. Our team concomitantly focused on creating a novel <I>E. coli</I> strain for more efficient production of adhesives.
  
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===Usage and Biology===
 
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<p><i>Caulobacter crescentus</i> is a common freshwater gram-negative oligotrophic bacterium of the clade <i>Caulobacterales</i>. Its distinguishing feature is its dual lifestyle. Initially, <i>C. crescentus</I> daughter cells are in a “swarmer” cell phase, which has a flagellum, enabling them to perform chemotaxis. After the motile phase, they differentiate into “stalked” cells. This phase features a tubular stalk with an adhesive structure holdfast, allowing them to adhere to surfaces and perform cell division.</p>
 
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<p>Caulobacterales synthesize a polysaccharide-based adhesin known as holdfast at one of their cell poles, enabling tight attachment to external surfaces. It is established that holdfast consists of repeating identical units composed of multiple monomers. Current literature agrees that in Caulobacter crescentus, these units form tetrads composed of glucose, an unidentified monosaccharide (either N-mannosamine uronic acid or xylose), N-acetylglucosamine, and N-glucosamine. These units are polymerized and exported to the outer membrane of the cell, where they function as anchors, securing the bacterium to a surface.
 
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</p>
 
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<p>The <i>C. crescentus</i> holdfast is produced via a polysaccharide synthesis and export pathway similar to the group I capsular polysaccharide synthesis Wzy/Wzx-dependent pathway in <i>Escherichia coli</i>.</p>
 
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<p>The holdfast synthesis (<i>hfs</i>) genes include those encoding predicted glycosyltransferases, carbohydrate modification factors, and components of a wzy-type polysaccharide assembly pathway.</p>
 
  
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<p><b><i>HfsL</i> in particular is responsible for N-acetyl-D-glucosamine transfer to the acceptor molecule.</b></p>
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<html>
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<p style="font-size: 1em;">
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This protein is part of the Tetrad assembly system <a href="https://parts.igem.org/Part:BBa_K5246043">BBa_K5246043</a> and operon responsible for the addition of N-acetyl-D-glucosamine and deacetylation of the former molecule <a href="https://parts.igem.org/Part:BBa_K5246042">BBa_K5246042</a>. </p>
  
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<p>
  
 +
Part was used in Vilnius-Lithuania iGEM 2024 project "Synhesion" <HTML><b><a href="https://2024.igem.wiki/vilnius-lithuania" target="_blank">https://2024.igem.wiki/vilnius-lithuania/</a></b></html>.
  
 
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<p style="font-size: 1em;">
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<p>
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This part also has a non 6xhis-tagged variant <a href="https://parts.igem.org/Part:BBa_K5246012">BBa_K5246012</a>.
 
This part also has a non 6xhis-tagged variant <a href="https://parts.igem.org/Part:BBa_K5246012">BBa_K5246012</a>.
 
</p>
 
</p>
 
</html>
 
</html>
  
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===Sequence and Features===
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__TOC__
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<partinfo>BBa_K5246029 SequenceAndFeatures</partinfo>
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===Biology and usage===
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<h2>Biology</h2>
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<html>
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<p style="font-size: 1em;">
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<i>Caulobacter crescentus</i> is a common freshwater gram-negative oligotrophic bacterium of the clade <i>Caulobacterales</i>. Its distinguishing feature is its dual lifestyle. Initially, <i>C. crescentus</i> daughter cells are in a “swarmer” cell phase, which has a flagellum, enabling them to perform chemotaxis. After the motile phase, they differentiate into “stalked” cells. This phase features a tubular stalk with an adhesive structure called holdfast, allowing them to adhere to surfaces and perform cell division.[1][2]
 +
</p>
 +
<p style="font-size: 1em;">
 +
Caulobacterales synthesize a polysaccharide-based adhesin known as holdfast at one of their cell poles, enabling tight attachment to external surfaces. It is established that holdfast consists of repeating identical units composed of multiple monomers. Current literature agrees that in Caulobacter crescentus, these units form tetrads composed of glucose, an unidentified monosaccharide (either N-mannosamine uronic acid or xylose), N-acetylglucosamine, and N-glucosamine. These units are polymerized and exported to the outer membrane of the cell, where they function as anchors, securing the bacterium to a surface[3][4].
 +
</p>
 +
<p style="font-size: 1em;">
 +
The <i>C. crescentus</i> holdfast is produced via a polysaccharide synthesis and export pathway similar to the group I capsular polysaccharide synthesis Wzy/Wzx-dependent pathway in <i>Escherichia coli</i>.
 +
</p>
 +
<p style="font-size: 1em;">
 +
The holdfast synthesis (<i>hfs</i>) genes include those encoding predicted glycosyltransferases, carbohydrate modification factors, and components of a wzy-type polysaccharide assembly pathway[4][5][6][9].
 +
</p>
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<p style="font-size: 1em;">
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<b>HfsL</b> in particular is responsible for N-acetyl-D-glucosamine transfer to the acceptor molecule.
 +
</p>
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</html>
  
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===Experimental characterization===
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<h3>HfsL</h3>
  
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====Bioinformatic analysis====
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<html>
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Gene HfsL from <i>Caulobacter crescentus</i> encodes a protein of 305aa. HfsL is one of the glucotransferases involved in the holdfast synthesis pathway and is responsible for addition of N-acetyl-D-glucosamine that is fourth monomer on the sugar tetrad in the beginning of holdfast synthesis.
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</html>
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<h2>Usage</h2>
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<p>
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  Proteins of the holdfast synthesis system assemble a short chain of sugar monomers in a specific sequence on a lipid carrier - a glycolipid.
 +
</p>
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 +
<p>
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  Glycolipids are predominantly located on the extracellular surface of eukaryotic cell membranes and are responsible for various functions such as receptors for viruses and other pathogens, allowing them to enter a specific host cell that has unique glycolipid markers. This feature can let us use said glycolipids as labels for a precise and targeted liposome distribution throughout the body, delivering anything from cancer drugs to gene editing systems directly to the target cells.
 +
</p>
 +
<p>
 +
  To create a liposome labeling system, we had to select specific proteins that could be utilized for this purpose. Following bioinformatics analysis using the Conserved Domain Database, Protein BLAST, DeepTMHMM, and AlphaFold 3, we identified five proteins of interest from each strain: HfsG, HfsH, HfsJ, HfsK, and HfsL.
 +
</p>
 +
 
 +
<p>
 +
  To utilize these enzymes, it was essential to develop a suitable purification strategy. For efficient cloning, we chose Golden Gate assembly. For efficient purification, we selected immobilized ion affinity chromatography (IMAC) as our purification method, based on recommendations from one of the few available papers where <i>C. crescentus</i> proteins were expressed and purified from <i>E. coli</i>. We opted for conventional 6x histidine tags (his-tag) to facilitate straightforward purification. It was crucial to determine the appropriate terminus for 6xHis-tag insertion to avoid disrupting the protein conformation and lessening purification efficiency.
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</p>
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 +
===Sequence and Features===
 +
<partinfo>BBa_K5246029 SequenceAndFeatures</partinfo>
 +
 
 +
===Experimental charazterization===
 +
====Bioinformatical analysis====
  
 
CDD analysis showed specific hits in glycosyl transferase family 2. This diverse family transfers sugar from UDP-glucose, UDP-N-acetyl-galactosamine, GDP-mannose, or CDP-abequose to a range of substrates. Protein BLAST further supports these findings and suggests that HfsL is most likely a family 2 glycosyltransferase, which has a domain very similar to the poly-beta-1,6-N-acetyl-D-glucosamine synthase domain of biofilm PGA synthase.
 
CDD analysis showed specific hits in glycosyl transferase family 2. This diverse family transfers sugar from UDP-glucose, UDP-N-acetyl-galactosamine, GDP-mannose, or CDP-abequose to a range of substrates. Protein BLAST further supports these findings and suggests that HfsL is most likely a family 2 glycosyltransferase, which has a domain very similar to the poly-beta-1,6-N-acetyl-D-glucosamine synthase domain of biofilm PGA synthase.
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DeepTMHMM analysis suggests that the protein is likely globular and positioned on the inner side of the cell membrane. The AlphaFold3 structure provides additional evidence supporting its globular shape. A pTM score above 0.5 suggests that the predicted overall structure may closely resemble the true protein fold, while ipTM indicates the accuracy of the subunit positioning within the complex. Values higher than 0.8 represent confident, high-quality predictions (Fig.1).
 
DeepTMHMM analysis suggests that the protein is likely globular and positioned on the inner side of the cell membrane. The AlphaFold3 structure provides additional evidence supporting its globular shape. A pTM score above 0.5 suggests that the predicted overall structure may closely resemble the true protein fold, while ipTM indicates the accuracy of the subunit positioning within the complex. Values higher than 0.8 represent confident, high-quality predictions (Fig.1).
  
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To sum up, HfsL is most probably a globular family 2 glycosyltransferase, responsible for N-acetyl-D-glucosamine transfer to the acceptor molecule, as is further verified by existing research. [1][2][3]
+
To sum up, HfsL is most probably a globular family 2 glycosyltransferase, responsible for N-acetyl-D-glucosamine transfer to the acceptor molecule, as is further verified by existing research. [6][7][8]
  
 
<center> https://static.igem.wiki/teams/5246/registry/hfsl.png </center>
 
<center> https://static.igem.wiki/teams/5246/registry/hfsl.png </center>
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<center> <b> Fig. 1. </b> AlphaFold 3 structure showing  </center>
 
<center> <b> Fig. 1. </b> AlphaFold 3 structure showing  </center>
  
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===Experimental characterization===
  
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===Protein expression===
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<head>
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<h2>CB2 strain</h2>
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    <meta charset="UTF-8">
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<p>We chose the BL21(DE3) strain for adjustable and efficient expression of target proteins since the system's proteins were best expressed in this strain. Given the lack of time, we went with conditions optimized beforehand in earlier experiments for the whole pathway expression: temperature of 37°C, induction with 0.5 mM IPTG concentration, and expression for 3 hours.</p>
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    <meta name="viewport" content="width=device-width, initial-scale=1.0">
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    <title>Protein Expression Results</title>
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<p>After SDS-PAGE gel analysis, we concluded that we successfully expressed all HfsG, HfsH, HfsJ, HfsK, and HfsL proteins from <i>C. crescentus</i> CB2 and CB2A strains. We noticed that HfsJ and HfsL glycosyltransferases were visible in lower quantities compared to the other proteins. Both of these protein expression conditions need to be further investigated and optimized.</p>
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        }
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<p><b>HfsL</b> is clearly visible in the center of the gel (Fig. 2).</p>
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        table, th, td {
 
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            padding: 8px;
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            text-align: center;
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    .container {
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      gap: 5px; /* Space between table and figure */
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    .table-container {
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<body>
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  <div class="container">
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    <!-- Table on the left -->
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    <div class="table-container">
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      <h3>Table 1. <i>C. crescentus</i> protein sizes in kDa</h3>
 +
      <table border="1">
 +
        <tr>
 +
          <th>Protein Name</th>
 +
          <th>Size (kDa)</th>
 +
        </tr>
 +
        <tr>
 +
          <td>HfsG</td>
 +
          <td>34</td>
 +
        </tr>
 +
        <tr>
 +
          <td>HfsH</td>
 +
          <td>27.9</td>
 +
        </tr>
 +
        <tr>
 +
          <td>HfsJ</td>
 +
          <td>34.7</td>
 +
        </tr>
 +
        <tr>
 +
          <td>HfsK</td>
 +
          <td>43.3</td>
 +
        </tr>
 +
        <tr>
 +
          <td>HfsL</td>
 +
          <td>33.3</td>
 +
        </tr>
 +
      </table>
 +
    </div>
  
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      <figure>
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         <div class="center">
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          <img src="https://static.igem.wiki/teams/5246/results/protein-expression/cb2-expression.webp" style="width:500px;">
 +
         </div>
 +
        <figcaption><center><b>Fig. 2.</b> 12% SDS-PAGE analysis of <i>C. crescentus</i> CB2 strain proteins in BL21(DE3) before expression and after induction at 0.5 mM IPTG concentrations for 3 hours at 37°C. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific). </center></figcaption>
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      </figure>
 +
    </div>
 +
  </div>
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</body>
 +
</html>
  
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        figure {
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<h2>CB2A strain</h2>
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            text-align: center;
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<p>We chose the BL21(DE3) strain for adjustable and efficient expression of target proteins since the system's proteins were best expressed in this strain. Given the lack of time, we went with conditions optimized beforehand in earlier experiments for the whole pathway expression: temperature of 37°C, induction with 0.5 mM IPTG concentration, and expression for 3 hours.</p>
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            margin: 20px 0;
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        }
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        img {
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<p>After SDS-PAGE gel analysis, we concluded that we successfully expressed all HfsG, HfsH, HfsJ, HfsK, and HfsL proteins from <i>C. crescentus</i> CB2 and CB2A strains. We noticed that HfsJ and HfsL glycosyltransferases were visible in lower quantities compared to the other proteins. Both of these protein expression conditions need to be further investigated and optimized.</p>
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        }
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        figcaption {
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<p><b>HfsL</b> is clearly visible in the center of the gel (Fig. 3).</p>
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<head>
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  <style>
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    .container {
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      display: flex;
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</head>
 
</head>
 
<body>
 
<body>
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  <div class="container">
 +
    <!-- Table on the left -->
 +
    <div class="table-container">
 +
      <h3>Table 1. <i>C. crescentus</i> protein sizes in kDa</h3>
 +
      <table border="1">
 +
        <tr>
 +
          <th>Protein Name</th>
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          <th>Size (kDa)</th>
 +
        </tr>
 +
        <tr>
 +
          <td>HfsG</td>
 +
          <td>34</td>
 +
        </tr>
 +
        <tr>
 +
          <td>HfsH</td>
 +
          <td>27.9</td>
 +
        </tr>
 +
        <tr>
 +
          <td>HfsJ</td>
 +
          <td>34.7</td>
 +
        </tr>
 +
        <tr>
 +
          <td>HfsK</td>
 +
          <td>43.3</td>
 +
        </tr>
 +
        <tr>
 +
          <td>HfsL</td>
 +
          <td>33.3</td>
 +
        </tr>
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      </table>
 +
    </div>
  
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     <h1>Protein Expression</h1>
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     <!-- Figure on the right -->
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    <div class="figure-container">
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      <figure>
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        <div class="center">
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          <img src="https://static.igem.wiki/teams/5246/results/protein-expression/cb2a-expressions.webp" style="width:500px;">
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        </div>
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        <figcaption><center><b>Fig. 3.</b> 12% SDS-PAGE analysis of <i>C. crescentus</i> CB2A strain proteins in BL21(DE3) before expression and after induction at 0.5 mM IPTG concentrations for 3 hours at 37°C. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific). </center></figcaption>
 +
      </figure>
 +
    </div>
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  </div>
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</body>
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</html>
  
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    <p>We chose <strong>BL21(DE3)</strong> strain for adjustable and efficient expression of target proteins since the system’s proteins were best expressed in this strain. Given the lack of time, we proceeded with conditions optimized in earlier experiments for whole pathway expression: a temperature of 37°C, induction with 0.5 mM IPTG concentration, and expression for 3 hours.</p>
+
===Protein purification===
 +
<h2>CB2 strain</h2>
  
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    <p>After SDS-PAGE gel analysis, we concluded that all 5 proteins from <em>C. crescentus</em> CB2 and CB2A strains were successfully expressed (Fig. 41.1-2). We noticed that the glycosyltransferases <strong>HfsJ</strong> and <strong>HfsL</strong> were present in lower quantities compared to the other proteins. The expression conditions of both proteins require further investigation and optimization.</p>
+
<p>After successful expression, we proceeded to work on the purification of his-tagged proteins. We went with immobilized metal ion affinity chromatography (IMAC). Adapting protocols from the little research that was available, we used HisPur Ni-NTA Spin Columns (Thermo Scientific). Equilibration, wash, and elution buffers contained 10 mM Tris pH 7.4, 150 mM NaCl, and 10 mM, 75 mM, and 500 mM imidazole, respectively.</p>
  
−
    <figure>
+
<p><b>HfsL</b> protein was successfully purified and is clearly seen in the elution fraction .</p>
−
        <img src="https://static.igem.wiki/teams/5246/results/protein-expression/cb2-expression.webp" alt="Protein Expression Gel Image">
+
−
        <figcaption>Fig. 41.1. 12% SDS-PAGE analysis of <em>C. crescentus</em> CB2 strain proteins in BL21(DE3) before expression and after induction at 0.5 mM IPTG concentrations for 3h at 37°C. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific).</figcaption>
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    </figure>
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    <h2>Protein Sizes</h2>
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<html>
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    <table>
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</p>
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        <thead>
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<figure>
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            <tr>
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<div class = "center" >
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                <th>Protein Name</th>
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<center>
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                <th>Size (kDa)</th>
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<img src = "https://static.igem.wiki/teams/5246/results/protein-expression/cb2-l-purification.webp" style = "width:300px;"></center>
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            </tr>
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</div>
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        </thead>
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        <tbody>
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            <tr>
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                <td>HfsG</td>
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                <td>34</td>
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            </tr>
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            <tr>
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−
                <td>HfsH</td>
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−
                <td>27.9</td>
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−
            </tr>
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−
            <tr>
+
−
                <td>HfsJ</td>
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−
                <td>34.7</td>
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−
            </tr>
+
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            <tr>
+
−
                <td>HfsK</td>
+
−
                <td>43.3</td>
+
−
            </tr>
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−
            <tr>
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−
                <td>HfsL</td>
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−
                <td>33.3</td>
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−
            </tr>
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−
        </tbody>
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    </table>
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−
<h2>Protein Purification</h2>
+
<figcaption><center>
 +
<b>Fig. 4.</b> 12% SDS-PAGE analysis. <i>C. crescentus</i> CB2 <b>HfsL</b> protein purified using IMAC and HisPur™ Ni-NTA Spin Columns (ThermoFisher Scientific). Expected protein size ~ 33 kDa. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific), S - soluble fraction, FT - flow-through fraction, W - wash fraction, E - elution fraction.</center></figcaption>
 +
</figure>
 +
<p/>
 +
</html>
  
−
<p>After successful expression, we proceeded to work on the purification of his-tagged proteins. We went with immobilized metal ion affinity chromatography (IMAC). Adapting protocols from the little research that was available, we used HisPur Ni-NTA Spin Columns (Thermo Scientific) [25][5][4]. Equilibration, wash, and elution buffers contained 10mM Tris pH 7.4, 150mM NaCl, and 10mM, 75mM, and 500mM imidazole, respectively. <i>HfsL</i> was successfully purified.</p>
 
  
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<p><a href="https://static.igem.wiki/teams/5246/results/protein-expression/cb2-l-purification.webp" target="_blank">
+
<h2>CB2A strain</h2>
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<img src="https://static.igem.wiki/teams/5246/results/protein-expression/cb2-l-purification.webp" alt="Protein Purification" width="600"></a></p>
+
  
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<figcaption><b>Fig. 43.5.</b> 12% SDS-PAGE analysis. <i>C. crescentus</i> CB2 <i>HfsL</i> protein purified using IMAC and HisPur™ Ni-NTA Spin Columns (ThermoFisher Scientific). Expected protein size ~33 kDa. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific), S - soluble fraction, FT - flow through fraction, W - wash fraction, E - elution fraction.</figcaption>
+
<p>After successful expression, we proceeded to work on the purification of his-tagged proteins. We went with immobilized metal ion affinity chromatography (IMAC). Adapting protocols from the little research that was available, we used HisPur Ni-NTA Spin Columns (Thermo Scientific). Equilibration, wash, and elution buffers contained 10 mM Tris pH 7.4, 150 mM NaCl, and 10 mM, 75 mM, and 500 mM imidazole, respectively.</p>
  
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</body>
+
<p><b>HfsL</b> Surprisingly, in contrast with CB2 strain CB2A HfsL was trickier to purify and it eluded earlier in the wash fraction at 75mM imidazole.  This could be explained by the dynamic nature of the protein. It is possible that when folding the his-tagged end got folded into the globule of the protein wakening the interaction between histidines and metal ions in the column
 +
.</p>
 +
 
 +
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<div class = "center" >
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<img src = "https://static.igem.wiki/teams/5246/results/protein-expression/cb2a-l-purification.webp" style = "width:300px;"></center>
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<figcaption><center>
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<b>Fig. 5.</b> 12% SDS-PAGE analysis. <i>C. crescentus</i> CB2A <b>HfsL</b> protein purified using IMAC and HisPur™ Ni-NTA Spin Columns (ThermoFisher Scientific). Expected protein size ~ 33 kDa. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific), S - soluble fraction, FT - flow-through fraction, W - wash fraction, E - elution fraction.</center></figcaption>
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</html>
 
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===References===
 
===References===
−
1. Hershey, D.M., Fiebig, A. and Crosson, S. (2019) ‘A genome-wide analysis of adhesion in Caulobacter crescentus identifies new regulatory and biosynthetic components for holdfast assembly’, mBio, 10(1). doi:10.1128/mbio.02273-18.  
+
1. Hendrickson, H., & Lawrence, J. G. (2000). Mutational bias suggests that replication termination occurs near the dif site, not at Ter sites. FEMS Microbiology Reviews, 24(2), 177–183. https://doi.org/10.1111/j.1574-6976.2000.tb00539.x
 +
<br>
 +
2. Andrews, S. C., Robinson, A. K., & Rodríguez-Quiñones, F. (2004). Bacterial iron homeostasis. Journal of Bacteriology, 186(5), 1438–1447. https://doi.org/10.1128/jb.186.5.1438-1447.2004
 +
<br>
 +
3.Rabah, A., & Hanchi, S. (2023). Experimental and modeling study of the rheological and thermophysical properties of molybdenum disulfide-based nanofluids. Journal of Molecular Liquids, 384, 123335. https://doi.org/10.1016/j.molliq.2023.123335
 +
<br>
 +
4. Boutte, C. C., & Crosson, S. (2009). Bacterial lifestyle shapes stringent response activation. Journal of Bacteriology, 191(9), 2904-2912. https://doi.org/10.1128/jb.01003-08
 +
<br>
 +
5. Mackie, J., Liu, Y. C., & DiBartolo, G. (2019). The C-terminal region of the Caulobacter crescentus CtrA protein inhibits stalk synthesis during the G1-to-S transition. mBio, 10(2), e02273-18. https://doi.org/10.1128/mbio.02273-18
 +
<br>
 +
6.Thanbichler, M., & Shapiro, L. (2003). MipZ, a spatial regulator coordinating chromosome segregation with cell division in Caulobacter. Journal of Bacteriology, 185(4), 1432-1442. https://doi.org/10.1128/jb.185.4.1432-1442.2003
 +
<br>
 +
7. Hershey, D.M., Fiebig, A. and Crosson, S. (2019) ‘A genome-wide analysis of adhesion in Caulobacter crescentus identifies new regulatory and biosynthetic components for holdfast assembly’, mBio, 10(1). doi:10.1128/mbio.02273-18.  
 
<br>
 
<br>
−
2. Chepkwony, N.K., Hardy, G.G. and Brun, Y.V. (2022) ‘HFAE is a component of the holdfast anchor complex that tethers the holdfast adhesin to the cell envelope’, Journal of Bacteriology, 204(11). doi:10.1128/jb.00273-22.  
+
8. Chepkwony, N.K., Hardy, G.G. and Brun, Y.V. (2022) ‘HFAE is a component of the holdfast anchor complex that tethers the holdfast adhesin to the cell envelope’, Journal of Bacteriology, 204(11). doi:10.1128/jb.00273-22.  
 
<br>
 
<br>
−
3. Chepkwony, N.K., Berne, C. and Brun, Y.V. (2019) ‘Comparative analysis of ionic strength tolerance between freshwater and marine Caulobacterales adhesins’, Journal of Bacteriology, 201(18). doi:10.1128/jb.00061-19.
+
9. Chepkwony, N.K., Berne, C. and Brun, Y.V. (2019) ‘Comparative analysis of ionic strength tolerance between freshwater and marine Caulobacterales adhesins’, Journal of Bacteriology, 201(18). doi:10.1128/jb.00061-19.

Latest revision as of 13:58, 2 October 2024


CB2/CB2A HfsL Glycosyltransferase, 6xHis tag for purification

Introduction

Vilnius-Lithuania iGEM 2024 project Synhesion aspires to create biodegradable and environmentally friendly adhesives. We were inspired by bacteria, which naturally produce adhesives made from polysaccharides. Two bacteria from aquatic environments - C. crescentus and H. Baltica - harness 12 protein synthesis pathways to produce sugars anchoring them to the surfaces. We aimed to transfer the polysaccharide synthesis pathway to industrially used E. coli bacteria to produce adhesives. Our team concomitantly focused on creating a novel E. coli strain for more efficient production of adhesives.


This protein is part of the Tetrad assembly system BBa_K5246043 and operon responsible for the addition of N-acetyl-D-glucosamine and deacetylation of the former molecule BBa_K5246042.

Part was used in Vilnius-Lithuania iGEM 2024 project "Synhesion" https://2024.igem.wiki/vilnius-lithuania/.

This part also has a non 6xhis-tagged variant BBa_K5246012.

Biology and usage

Biology

Caulobacter crescentus is a common freshwater gram-negative oligotrophic bacterium of the clade Caulobacterales. Its distinguishing feature is its dual lifestyle. Initially, C. crescentus daughter cells are in a “swarmer” cell phase, which has a flagellum, enabling them to perform chemotaxis. After the motile phase, they differentiate into “stalked” cells. This phase features a tubular stalk with an adhesive structure called holdfast, allowing them to adhere to surfaces and perform cell division.[1][2]

Caulobacterales synthesize a polysaccharide-based adhesin known as holdfast at one of their cell poles, enabling tight attachment to external surfaces. It is established that holdfast consists of repeating identical units composed of multiple monomers. Current literature agrees that in Caulobacter crescentus, these units form tetrads composed of glucose, an unidentified monosaccharide (either N-mannosamine uronic acid or xylose), N-acetylglucosamine, and N-glucosamine. These units are polymerized and exported to the outer membrane of the cell, where they function as anchors, securing the bacterium to a surface[3][4].

The C. crescentus holdfast is produced via a polysaccharide synthesis and export pathway similar to the group I capsular polysaccharide synthesis Wzy/Wzx-dependent pathway in Escherichia coli.

The holdfast synthesis (hfs) genes include those encoding predicted glycosyltransferases, carbohydrate modification factors, and components of a wzy-type polysaccharide assembly pathway[4][5][6][9].

HfsL in particular is responsible for N-acetyl-D-glucosamine transfer to the acceptor molecule.

HfsL

Gene HfsL from Caulobacter crescentus encodes a protein of 305aa. HfsL is one of the glucotransferases involved in the holdfast synthesis pathway and is responsible for addition of N-acetyl-D-glucosamine that is fourth monomer on the sugar tetrad in the beginning of holdfast synthesis.

Usage

Proteins of the holdfast synthesis system assemble a short chain of sugar monomers in a specific sequence on a lipid carrier - a glycolipid.

Glycolipids are predominantly located on the extracellular surface of eukaryotic cell membranes and are responsible for various functions such as receptors for viruses and other pathogens, allowing them to enter a specific host cell that has unique glycolipid markers. This feature can let us use said glycolipids as labels for a precise and targeted liposome distribution throughout the body, delivering anything from cancer drugs to gene editing systems directly to the target cells.

To create a liposome labeling system, we had to select specific proteins that could be utilized for this purpose. Following bioinformatics analysis using the Conserved Domain Database, Protein BLAST, DeepTMHMM, and AlphaFold 3, we identified five proteins of interest from each strain: HfsG, HfsH, HfsJ, HfsK, and HfsL.

To utilize these enzymes, it was essential to develop a suitable purification strategy. For efficient cloning, we chose Golden Gate assembly. For efficient purification, we selected immobilized ion affinity chromatography (IMAC) as our purification method, based on recommendations from one of the few available papers where C. crescentus proteins were expressed and purified from E. coli. We opted for conventional 6x histidine tags (his-tag) to facilitate straightforward purification. It was crucial to determine the appropriate terminus for 6xHis-tag insertion to avoid disrupting the protein conformation and lessening purification efficiency.

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 265
    Illegal NgoMIV site found at 408
    Illegal NgoMIV site found at 639
  • 1000
    COMPATIBLE WITH RFC[1000]

Experimental charazterization

Bioinformatical analysis

CDD analysis showed specific hits in glycosyl transferase family 2. This diverse family transfers sugar from UDP-glucose, UDP-N-acetyl-galactosamine, GDP-mannose, or CDP-abequose to a range of substrates. Protein BLAST further supports these findings and suggests that HfsL is most likely a family 2 glycosyltransferase, which has a domain very similar to the poly-beta-1,6-N-acetyl-D-glucosamine synthase domain of biofilm PGA synthase.

DeepTMHMM analysis suggests that the protein is likely globular and positioned on the inner side of the cell membrane. The AlphaFold3 structure provides additional evidence supporting its globular shape. A pTM score above 0.5 suggests that the predicted overall structure may closely resemble the true protein fold, while ipTM indicates the accuracy of the subunit positioning within the complex. Values higher than 0.8 represent confident, high-quality predictions (Fig.1).

To sum up, HfsL is most probably a globular family 2 glycosyltransferase, responsible for N-acetyl-D-glucosamine transfer to the acceptor molecule, as is further verified by existing research. [6][7][8]

hfsl.png
Fig. 1. AlphaFold 3 structure showing

Experimental characterization

Protein expression

CB2 strain

We chose the BL21(DE3) strain for adjustable and efficient expression of target proteins since the system's proteins were best expressed in this strain. Given the lack of time, we went with conditions optimized beforehand in earlier experiments for the whole pathway expression: temperature of 37°C, induction with 0.5 mM IPTG concentration, and expression for 3 hours.

After SDS-PAGE gel analysis, we concluded that we successfully expressed all HfsG, HfsH, HfsJ, HfsK, and HfsL proteins from C. crescentus CB2 and CB2A strains. We noticed that HfsJ and HfsL glycosyltransferases were visible in lower quantities compared to the other proteins. Both of these protein expression conditions need to be further investigated and optimized.

HfsL is clearly visible in the center of the gel (Fig. 2).


Table 1. C. crescentus protein sizes in kDa

Protein Name Size (kDa)
HfsG 34
HfsH 27.9
HfsJ 34.7
HfsK 43.3
HfsL 33.3
Fig. 2. 12% SDS-PAGE analysis of C. crescentus CB2 strain proteins in BL21(DE3) before expression and after induction at 0.5 mM IPTG concentrations for 3 hours at 37°C. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific).

CB2A strain

We chose the BL21(DE3) strain for adjustable and efficient expression of target proteins since the system's proteins were best expressed in this strain. Given the lack of time, we went with conditions optimized beforehand in earlier experiments for the whole pathway expression: temperature of 37°C, induction with 0.5 mM IPTG concentration, and expression for 3 hours.

After SDS-PAGE gel analysis, we concluded that we successfully expressed all HfsG, HfsH, HfsJ, HfsK, and HfsL proteins from C. crescentus CB2 and CB2A strains. We noticed that HfsJ and HfsL glycosyltransferases were visible in lower quantities compared to the other proteins. Both of these protein expression conditions need to be further investigated and optimized.

HfsL is clearly visible in the center of the gel (Fig. 3).


Table 1. C. crescentus protein sizes in kDa

Protein Name Size (kDa)
HfsG 34
HfsH 27.9
HfsJ 34.7
HfsK 43.3
HfsL 33.3
Fig. 3. 12% SDS-PAGE analysis of C. crescentus CB2A strain proteins in BL21(DE3) before expression and after induction at 0.5 mM IPTG concentrations for 3 hours at 37°C. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific).

Protein purification

CB2 strain

After successful expression, we proceeded to work on the purification of his-tagged proteins. We went with immobilized metal ion affinity chromatography (IMAC). Adapting protocols from the little research that was available, we used HisPur Ni-NTA Spin Columns (Thermo Scientific). Equilibration, wash, and elution buffers contained 10 mM Tris pH 7.4, 150 mM NaCl, and 10 mM, 75 mM, and 500 mM imidazole, respectively.

HfsL protein was successfully purified and is clearly seen in the elution fraction .

Fig. 4. 12% SDS-PAGE analysis. C. crescentus CB2 HfsL protein purified using IMAC and HisPur™ Ni-NTA Spin Columns (ThermoFisher Scientific). Expected protein size ~ 33 kDa. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific), S - soluble fraction, FT - flow-through fraction, W - wash fraction, E - elution fraction.


CB2A strain

After successful expression, we proceeded to work on the purification of his-tagged proteins. We went with immobilized metal ion affinity chromatography (IMAC). Adapting protocols from the little research that was available, we used HisPur Ni-NTA Spin Columns (Thermo Scientific). Equilibration, wash, and elution buffers contained 10 mM Tris pH 7.4, 150 mM NaCl, and 10 mM, 75 mM, and 500 mM imidazole, respectively.

HfsL Surprisingly, in contrast with CB2 strain CB2A HfsL was trickier to purify and it eluded earlier in the wash fraction at 75mM imidazole. This could be explained by the dynamic nature of the protein. It is possible that when folding the his-tagged end got folded into the globule of the protein wakening the interaction between histidines and metal ions in the column .

Fig. 5. 12% SDS-PAGE analysis. C. crescentus CB2A HfsL protein purified using IMAC and HisPur™ Ni-NTA Spin Columns (ThermoFisher Scientific). Expected protein size ~ 33 kDa. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific), S - soluble fraction, FT - flow-through fraction, W - wash fraction, E - elution fraction.

References

1. Hendrickson, H., & Lawrence, J. G. (2000). Mutational bias suggests that replication termination occurs near the dif site, not at Ter sites. FEMS Microbiology Reviews, 24(2), 177–183. https://doi.org/10.1111/j.1574-6976.2000.tb00539.x
2. Andrews, S. C., Robinson, A. K., & Rodríguez-Quiñones, F. (2004). Bacterial iron homeostasis. Journal of Bacteriology, 186(5), 1438–1447. https://doi.org/10.1128/jb.186.5.1438-1447.2004
3.Rabah, A., & Hanchi, S. (2023). Experimental and modeling study of the rheological and thermophysical properties of molybdenum disulfide-based nanofluids. Journal of Molecular Liquids, 384, 123335. https://doi.org/10.1016/j.molliq.2023.123335
4. Boutte, C. C., & Crosson, S. (2009). Bacterial lifestyle shapes stringent response activation. Journal of Bacteriology, 191(9), 2904-2912. https://doi.org/10.1128/jb.01003-08
5. Mackie, J., Liu, Y. C., & DiBartolo, G. (2019). The C-terminal region of the Caulobacter crescentus CtrA protein inhibits stalk synthesis during the G1-to-S transition. mBio, 10(2), e02273-18. https://doi.org/10.1128/mbio.02273-18
6.Thanbichler, M., & Shapiro, L. (2003). MipZ, a spatial regulator coordinating chromosome segregation with cell division in Caulobacter. Journal of Bacteriology, 185(4), 1432-1442. https://doi.org/10.1128/jb.185.4.1432-1442.2003
7. Hershey, D.M., Fiebig, A. and Crosson, S. (2019) ‘A genome-wide analysis of adhesion in Caulobacter crescentus identifies new regulatory and biosynthetic components for holdfast assembly’, mBio, 10(1). doi:10.1128/mbio.02273-18.
8. Chepkwony, N.K., Hardy, G.G. and Brun, Y.V. (2022) ‘HFAE is a component of the holdfast anchor complex that tethers the holdfast adhesin to the cell envelope’, Journal of Bacteriology, 204(11). doi:10.1128/jb.00273-22.
9. Chepkwony, N.K., Berne, C. and Brun, Y.V. (2019) ‘Comparative analysis of ionic strength tolerance between freshwater and marine Caulobacterales adhesins’, Journal of Bacteriology, 201(18). doi:10.1128/jb.00061-19.