Difference between revisions of "Part:BBa K2684005"
 
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We added the <i>SpyCatcher</i> sequence after <i>cotA</i> gene so that we can produce laccase protein with spycatcher (BBa_K2684005).  
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This part could be used to produce CotA laccase fused with SpyCatcher (BBa_K2684005). Team SHSBNU_China designed this part to improve the the enzyme activity of traditional CotA. When applying the gene <i>CotA</i> without SpyCatcher, the laccase cannot be suspended onto the biofilm created by CsgA-Spytag. Therefore, there will not be significant improvement in enzyme activity. However, while applying with CsgA-Spytag, the enzyme activity dramatically increased, as shown in the graph below.
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<img class="pictures" id = "22001" src="https://static.igem.org/mediawiki/2018/7/72/T--SHSBNU_China--10002.png"/>
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<img src="https://static.igem.org/mediawiki/2018/4/4a/T--SHSBNU_China--23000.jpg" style="width:150%"/>
 
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The more *, the larger difference between each group
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Some experiments showed that, while using singly, the existence of SpyCatcher actually result in a decrease in enzyme activity compared to CotA wild type, as shown in the graph below.
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<img src="https://static.igem.org/mediawiki/2018/d/d5/T--SHSBNU_China--23011.jpg" style="width:150%"/>
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<img src="https://static.igem.org/mediawiki/2018/a/af/T--SHSBNU_China--23012.jpg" style="width:150%"/>
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The problem might be:<br>
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1. The inserted gene <i>Spycatcher</i> affected expression of laccase. <br>
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2. The extra structure of CotA laccase result in a decrease in enzyme activity<br>
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<br>
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For more experimental details, please visit http://2018.igem.org/Team:SHSBNU_China/Experiments
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Latest revision as of 01:35, 18 October 2018


SpyCatcher-CotA

SpyCatcher fused with CotA by 2xGGGGS linker

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 514
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1903
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 841


This part could be used to produce CotA laccase fused with SpyCatcher (BBa_K2684005). Team SHSBNU_China designed this part to improve the the enzyme activity of traditional CotA. When applying the gene CotA without SpyCatcher, the laccase cannot be suspended onto the biofilm created by CsgA-Spytag. Therefore, there will not be significant improvement in enzyme activity. However, while applying with CsgA-Spytag, the enzyme activity dramatically increased, as shown in the graph below.

The more *, the larger difference between each group

Some experiments showed that, while using singly, the existence of SpyCatcher actually result in a decrease in enzyme activity compared to CotA wild type, as shown in the graph below.

The problem might be:
1. The inserted gene Spycatcher affected expression of laccase.
2. The extra structure of CotA laccase result in a decrease in enzyme activity

For more experimental details, please visit http://2018.igem.org/Team:SHSBNU_China/Experiments