Difference between revisions of "Part:BBa K108005"

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==Contribution From CAU_China 2023==
 
==Contribution From CAU_China 2023==
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'''Group''': CAU_China, 2023  https://2023.igem.wiki/cau-china/
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'''Author''': Huang yaohan, Zhang Xiyuan, Sun Qianhui
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'''Summary''':Verified that this basic Part can work in Escherichia coli and we added some experimental conditions and result images .
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===Characterization===
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<p>The FlgH protomer in the L ring has an oblique arrow-like shape and consists of a long N-terminal loop (LN), six central β strands (β1–β6), and three α helices (α1–α3)</i><sup>[1]</sup>.</p>
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We are trying to develop FlgH as a new carrier protein and use its characteristic of being relatively fixed on the cell wall to achieve our special requirements for the spatial location of the display protein.
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In order to exert the display function, we chose to add the passenger protein to a special middle region of FlgH, and the two were fused through a connecting peptide(BBa_K4614106).
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The α1 region of FlgH is exposed on the OM and is a highly variable region</i><sup>[1]</sup>, which is the most suitable region for the insertion of the passenger protein. After considering reducing or not interfering with the interaction between monomeric FlgH</i><sup>[1]</sup>and the interaction between FlgH and LPS</i><sup>[2]</sup>, we chose to insert the passenger protein between T108 and V109.
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We verify cross-linking in two ways: by measuring optical density 600 and microscopy. Limited by our time and energy, we did not specifically characterize whether FlgH successfully displays passenger proteins. However, based on the results of our cross-linking characterization experiments, we believe that FlgH has successfully displayed the passenger protein on the surface of the cell (Fig1). For details, please refer to <a href="https://parts.igem.org/Part:BBa_K4614102">BBa_K4614102</a>. Also, 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 (Fig2). For details, please refer to <a href="https://parts.igem.org/Part:BBa_K4614100">BBa_K4614100</a>
  
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'''Group''': CAU_China, 2023  https://2023.igem.org/Team:CAU_China
 
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'''Author''': Huang yaohan  Zhang xiyuan  Sun qianhui
 
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'''Summary''':Verified that this basic Part can work in Escherichia coli and we added some experimental conditions and result images
 
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===Biology===
 
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<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 <i>Streptococcus pyogenes</i><sup>[1]</sup>.</p>
 
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===Improvement===
 
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<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<sup>[2]</sup>. 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.</>
 
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<p>We attempted to display SpyTag and SpyCatcher on the surface of <i>Escherichia coli</i> BL21(DE3) respectively, using this system to achieve cross-linking between bacteria.</p>
 
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<p>Using fluorescent proteins, we constructed a system for verifying cross-linking, in which the engineered bacteria introduced plasmids and genes as shown in the table below.</p>
 
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<table class="table">
 
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      <th><strong>pET30a</strong></th>
 
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      <th><strong>pJUMP46-2A</strong></th>
 
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    </tr>
 
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  </thead>
 
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  <tbody>
 
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    <tr>
 
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      <td>A</td>
 
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      <td>SpyTag</td>
 
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      <td>sfGFP</td>
 
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    </tr>
 
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    <tr>
 
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      <td>B</td>
 
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      <td>SpyCatcher</td>
 
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      <td>mCherry</td>
 
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    </tr>
 
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    <tr>
 
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      <td>C</td>
 
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      <td>empty plasmid</td>
 
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      <td>sfGFP</td>
 
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    </tr>
 
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    <tr>
 
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      <td>D</td>
 
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      <td>empty plasmid</td>
 
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      <td>mCherry</td>
 
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    </tr>
 
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<p class="figurelegend">Tab1. Plasmids andgenes induced into engineering bacteria.</p>
 
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<p>We verify cross-linking in two ways: by measuring optical density and microscopy.</p>
 
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<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/od.png" width="500" class="centered-image"></p>
 
<p><img src="https://static.igem.wiki/teams/4614/wiki/parts-jl/od.png" width="500" class="centered-image"></p>
 
<p class="figurelegend">Fig1. Quantitative verification of adherence of bacteria.</p>
 
<p class="figurelegend">Fig1. Quantitative verification of adherence of bacteria.</p>
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<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>
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<p><img src="https://static.igem.wiki/teams/4614/wiki/parts-jl/jl-2.png" width="500" class="centered-image"></p>
 
<p><img src="https://static.igem.wiki/teams/4614/wiki/parts-jl/jl-2.png" width="500" class="centered-image"></p>
 
<p class="figurelegend">Fig2. Observation of bacterial adhesion by laser microscopy Observation of bacterial adhesion by laser microscopy were observed under a laser microscope (1000×).</p>
 
<p class="figurelegend">Fig2. Observation of bacterial adhesion by laser microscopy Observation of bacterial adhesion by laser microscopy were observed under a laser microscope (1000×).</p>
  
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<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>
 
  
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===References of CAU_China===
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References of CAU_China.</p>
  
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[1] Hatlem, Daniel et al. “Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins.” International journal of molecular sciences vol. 20,9 (2019): 1-10. doi:10.3390/ijms20092129</p>
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</p>[1] Hatlem, Daniel et al. “Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins.” International journal of molecular sciences vol. 20,9 (2019): 1-10. doi:10.3390/ijms20092129</p>
  
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[2] Kozlowski, Mark T et al. “Genetically Programmable Microbial Assembly.” ACS synthetic biology vol. 10,6 (2021): 1351-1359. doi:10.1021/acssynbio.0c00616</p>
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</p>[2] Kozlowski, Mark T et al. “Genetically Programmable Microbial Assembly.” ACS synthetic biology vol. 10,6 (2021): 1351-1359. doi:10.1021/acssynbio.0c00616</p>
  
  

Latest revision as of 12:23, 12 October 2023

FlgH

flagellar protein of basal-body outer-membrane L ring of E.coli

Contribution From CAU_China 2023

Group: CAU_China, 2023 https://2023.igem.wiki/cau-china/

Author: Huang yaohan, Zhang Xiyuan, Sun Qianhui

Summary:Verified that this basic Part can work in Escherichia coli and we added some experimental conditions and result images .

Characterization

The FlgH protomer in the L ring has an oblique arrow-like shape and consists of a long N-terminal loop (LN), six central β strands (β1–β6), and three α helices (α1–α3)[1].

We are trying to develop FlgH as a new carrier protein and use its characteristic of being relatively fixed on the cell wall to achieve our special requirements for the spatial location of the display protein. In order to exert the display function, we chose to add the passenger protein to a special middle region of FlgH, and the two were fused through a connecting peptide(BBa_K4614106). The α1 region of FlgH is exposed on the OM and is a highly variable region[1], which is the most suitable region for the insertion of the passenger protein. After considering reducing or not interfering with the interaction between monomeric FlgH[1]and the interaction between FlgH and LPS[2], we chose to insert the passenger protein between T108 and V109. We verify cross-linking in two ways: by measuring optical density 600 and microscopy. Limited by our time and energy, we did not specifically characterize whether FlgH successfully displays passenger proteins. However, based on the results of our cross-linking characterization experiments, we believe that FlgH has successfully displayed the passenger protein on the surface of the cell (Fig1). For details, please refer to BBa_K4614102. Also, 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 (Fig2). For details, please refer to BBa_K4614100

Fig1. Quantitative verification of adherence of bacteria.

Fig2. Observation of bacterial adhesion by laser microscopy Observation of bacterial adhesion by laser microscopy were observed under a laser microscope (1000×).

References of CAU_China.

[1] Hatlem, Daniel et al. “Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins.” International journal of molecular sciences vol. 20,9 (2019): 1-10. doi:10.3390/ijms20092129

[2] Kozlowski, Mark T et al. “Genetically Programmable Microbial Assembly.” ACS synthetic biology vol. 10,6 (2021): 1351-1359. doi:10.1021/acssynbio.0c00616

Sequence and Features BBa_K108005 SequenceAndFeatures