Difference between revisions of "Part:BBa K2684000"
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2684000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2684000 SequenceAndFeatures</partinfo>
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<h2 id="P1">II. Period 1 - <i>CsgA - SpyTag</i></h2>
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<h2>Period - <i>CsgA - SpyTag</i></h2>
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<p><img src="https://static.igem.org/mediawiki/2018/b/b9/T--SHSBNU_China--21000.png" style="width:100%"/></image></p>
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Gene <i>csgA</i> found in the genome of MG1655 wild type is capable of forming biofilm. Using CRISPR, we knocked out gene <i>csgA</i> on MG1655’s genome creating ΔMG1655 strain. The cell ΔMG1655 would then be used as chassis cell. Gene <i>csgA</i> was fused into plasmid pET28a. A <i>Spytag</i> sequence was then fused after <i>csgA</i> gene, creating <i>csgA-spycatcher</i> (BBa_K2684006).  
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<p><img src="https://static.igem.org/mediawiki/2018/8/87/T--SHSBNU_China--21001.jpg" style="width:100%"/></image></p>
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<img class="pictures" id = "21000" src="https://static.igem.org/mediawiki/2018/b/b9/T--SHSBNU_China--21000.png"/>
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<p class="pic_text">Reaction stock leftover in experiment</p>
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Using sfGFP – spycatcher protein, the combing function of Spytag and spycatcher system on the biofilm was tested. Gene <i>csgA</i> on the plasmid of pET28a was transferred in to ΔMG1655 as control group. Gene <i>csgA – Spytag</i> on the plasmid of pET28a was transferred in to ΔMG1655 as experiment. To verify the function of Spytag on <i>csgA</i>, the experiment was design to compare the combing rate of sf-GFP – spycatcher protein with cells that have csgA – SpyTag (Experiment) or csgA (Control).  
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Gene <i>csgA</i> found in the genome of MG1655 wild type is capable of forming biofilm. Using CRISPR, we knocked out gene <i>csgA</i> on MG1655’s genome creating ΔMG1655 strain. The cell ΔMG1655 would then be used as chassis cell. Gene <i>csgA</i> was fused into plasmid pET28a. A <i>Spytag</i> sequence was then fused after <i>csgA</i> gene, creating <i>csgA-spycatcher</i> (BBa_K2684006).  
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<p><img src="https://static.igem.org/mediawiki/2018/5/57/T--SHSBNU_China--21002.jpg" stule="width:100%"/></image></p>
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<div style="width:14vw" class="content_pic_left">
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Link: Protocol for <a href="http://2018.igem.org/Team:SHSBNU_China/Protocal#SSS">SpyTag-SpyCatcher</a> system verification
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<img class="pictures" id = "21001" src="https://static.igem.org/mediawiki/2018/8/87/T--SHSBNU_China--21001.jpg"/>
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</p>
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<p class="pic_text">Reaction stock leftover in experiment</p>
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<p class="text">
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As can be seen from the result,  
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</p>
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Using sfGFP – spycatcher protein, the combing function of Spytag and spycatcher system on the biofilm was tested. Gene <i>csgA</i> on the plasmid of pET28a was transferred in to ΔMG1655 as control group. Gene <i>csgA – Spytag</i> on the plasmid of pET28a was transferred in to ΔMG1655 as experiment. To verify the function of Spytag on <i>csgA</i>, the experiment was design to compare the combing rate of sf-GFP – spycatcher protein with cells that have csgA – SpyTag (Experiment) or csgA (Control).  
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Thus we can confirm our <i>csgA – SpyTag</i> system is functional.
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<img class="pictures" id = "21002" src="https://static.igem.org/mediawiki/2018/5/57/T--SHSBNU_China--21002.jpg"/>
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Link: Protocol for <a href="http://2018.igem.org/Team:SHSBNU_China/Protocal#SSS">SpyTag-SpyCatcher</a> system verification
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As can be seen from the result,  
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<p class="text">
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Thus we can confirm our <i>csgA – SpyTag</i> system is functional.
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===References===
 
===References===
 
Guan, Z.-B., Luo, Q., Wang, H.-R., Chen, Y., & Liao, X.-R. (2018). Bacterial laccases: promising biological green tools for industrial applications. Cellular and Molecular Life Sciences.
 
Guan, Z.-B., Luo, Q., Wang, H.-R., Chen, Y., & Liao, X.-R. (2018). Bacterial laccases: promising biological green tools for industrial applications. Cellular and Molecular Life Sciences.

Revision as of 09:45, 17 October 2018


CotA Laccase of B.subtilis

CotA Laccase is an endospore type protein secreted from B.subtilis

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 AgeI site found at 1348
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 286

Period - CsgA - SpyTag

<img src="T--SHSBNU_China--21000.png" style="width:100%"/></image>

Gene csgA found in the genome of MG1655 wild type is capable of forming biofilm. Using CRISPR, we knocked out gene csgA on MG1655’s genome creating ΔMG1655 strain. The cell ΔMG1655 would then be used as chassis cell. Gene csgA was fused into plasmid pET28a. A Spytag sequence was then fused after csgA gene, creating csgA-spycatcher (BBa_K2684006). </p>

<img src="T--SHSBNU_China--21001.jpg" style="width:100%"/></image>

Reaction stock leftover in experiment

Using sfGFP – spycatcher protein, the combing function of Spytag and spycatcher system on the biofilm was tested. Gene csgA on the plasmid of pET28a was transferred in to ΔMG1655 as control group. Gene csgA – Spytag on the plasmid of pET28a was transferred in to ΔMG1655 as experiment. To verify the function of Spytag on csgA, the experiment was design to compare the combing rate of sf-GFP – spycatcher protein with cells that have csgA – SpyTag (Experiment) or csgA (Control).

<img src="T--SHSBNU_China--21002.jpg" stule="width:100%"/></image>

Link: Protocol for <a href="http://2018.igem.org/Team:SHSBNU_China/Protocal#SSS">SpyTag-SpyCatcher</a> system verification </p>

As can be seen from the result,

Thus we can confirm our csgA – SpyTag system is functional.

</div>

References

Guan, Z.-B., Luo, Q., Wang, H.-R., Chen, Y., & Liao, X.-R. (2018). Bacterial laccases: promising biological green tools for industrial applications. Cellular and Molecular Life Sciences.