Difference between revisions of "Part:BBa K2969021"
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<h2> Characterization
 
<h2> Characterization
 
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<h3><center>Heat-inducible switches
 
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<p>In 2019, UCAS-China developed a collection of thermosensitive parts with high-performance, versatility and robustness. Based on TCI transcription factor family and TlpA family, we collected five TCI and TlpA mutants and used sfGFP as reporter to build some heat-inducible ON-switches, which can open gene expression under high temperature. To characterize them quantitatively, we first characterized the performance of them by flow cytometer in Top10 strain.
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<img class="img-responsive img-center" width="500px;" src="https://2019.igem.org/wiki/images/5/58/T--UCAS-China--zlc4.png">
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<h5>    Figure 8. The heat-inducible ON-switch is encoded on one plasmid. The circuit is shown with the genetic parts and relationships among them.
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<p>   In this circuit we just make full use of the cold-inducible transcription repressors. We found TCI transcription factor family and TlpA family whose activity will be lost under high temperature. The structure of the corresponding transcription factor changes with the temperature’s change.
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<p>As shown in Figure 1, most of the transcription repressors show sharp thermal transitions, especially TCI and TCI42, with more than 100-fold induction within 10 degrees Celsius. Their impressive performances make them candidate parts for our further circuit design.
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</p>
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<p>  When it is above threshold temperature, TF will be allosteric, losing its activity. Then the inhibition of target gene is relieved while the target gene is expressed. We characterized the performance of this series of switch combinations, and the results are as follows.
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<p>   We first characterized the performance of 6 members from the two family in Top10 strain, the result shows that most of the transcription repressors show sharp thermal transitions, especially TCI and TCI42, with more than 100-fold induction within 10 degrees Celsius. Their impressive performances make them candidate parts for our further circuit design.
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<div>[[File:T--UCAS-China--TOP10_Heat-I.png|700px|thumb|center|<b>Figure 1:</b>The induction curves of the heat-inducible switches (TOP10)]]</div>
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<p>What’s more, we also tested these heat-inducible ON-switch in the chassis E.coli Nissle 1917, a probiotic with more than 100 years of medical application, their robustness give us more confidence in the stability and preciseness of our ark. The result is shown in Figure 2.
 
</p>
 
</p>
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<img class="img-responsive img-center" width="700px;" src="https://2019.igem.org/wiki/images/3/34/T--UCAS-China--TOP10_Heat-I.png">
 
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<h5>  Figure 9. The induction curve of the heat-inducible ON-switches (TOP10)
 
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</h5>
 
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<p>  Also, we tested these heat-inducible ON-switch in the chassis E.coli Nissle 1917, a probiotic with more than 100 years of medical application, their robustness give us more confidence in the stability and preciseness of our ark.
 
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</p>
 
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<img class="img-responsive img-center" width="700px;" src="https://2019.igem.org/wiki/images/8/8d/T--UCAS-China--Nissle_Heat-I.png">
 
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<h5>  Figure 10. The induction curve of the heat-inducible ON-switches (Nissle 1917)
 
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</h5>
 
  
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<div>[[File:T--UCAS-China--Nissle_Heat-I.png|700px|thumb|center|<b>Figure 2:</b>The induction curves of the heat-inducible switches (Nissle 1917)]]</div>
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<h2>Reference
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</h2>
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<p>Piraner, D. I., Abedi, M. H., Moser, B. A., Lee-Gosselin, A., and Shapiro, M. G., Tunable thermal bioswitch for in vivo control of microbial therapeutics. DOI: 10.1038/NCHEMBIO.2233
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</p>
  
  

Revision as of 18:09, 19 October 2019


TCI

TCI is a temperature-sensitive variant of the bacteriophage λ repressor cI. It consists of N-terminal, C-terminal and the fragment between N-terminal and C-terminal whose function is the linkage. It is a cold-inducible transcription factor. When the temperature is below 35℃, the activity of TCI will gradually rising. When the temperature is above 35℃, the activity of TCI will remain at a low level.

Characterization

In 2019, UCAS-China developed a collection of thermosensitive parts with high-performance, versatility and robustness. Based on TCI transcription factor family and TlpA family, we collected five TCI and TlpA mutants and used sfGFP as reporter to build some heat-inducible ON-switches, which can open gene expression under high temperature. To characterize them quantitatively, we first characterized the performance of them by flow cytometer in Top10 strain.

As shown in Figure 1, most of the transcription repressors show sharp thermal transitions, especially TCI and TCI42, with more than 100-fold induction within 10 degrees Celsius. Their impressive performances make them candidate parts for our further circuit design.

Figure 1:The induction curves of the heat-inducible switches (TOP10)

What’s more, we also tested these heat-inducible ON-switch in the chassis E.coli Nissle 1917, a probiotic with more than 100 years of medical application, their robustness give us more confidence in the stability and preciseness of our ark. The result is shown in Figure 2.

Figure 2:The induction curves of the heat-inducible switches (Nissle 1917)


Reference

Piraner, D. I., Abedi, M. H., Moser, B. A., Lee-Gosselin, A., and Shapiro, M. G., Tunable thermal bioswitch for in vivo control of microbial therapeutics. DOI: 10.1038/NCHEMBIO.2233


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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]