Difference between revisions of "Part:BBa K2572000"
 
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https://parts.igem.org/Part:BBa_K2447012
 
https://parts.igem.org/Part:BBa_K2447012
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<h2>Improvement</h2>
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<p>Group：UCAS-China</p>
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<h3>Characterizetion</h3>
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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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</p>
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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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<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.
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</p>
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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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Latest revision as of 15:09, 19 October 2019


TlpA39 thermal regulator

TlpA39 is from Salmonella typhimurium,quite similar to TlpA36 (BBa_K2447012).It is a temperature sensitive protein which is capable of binding to promoter pTlpA. Under temperature of above 39 degrees Celsius, TlpA39 protein would stay in monomeric form and hence it loses the ability to bind to the promoter pTlpA39. If the temperature drops below 39 degree Celsius, TlpA39 proteins would form dimers; which are capable of repressing the pTlpA promoter and prevent the recruitment of RNA polymerase to repress downstream gene expression. <a href="https://parts.igem.org/Part:BBa_K2447012">BBa_K2447012**</a>

In our work, we characterized its threshold temperature. We found that expression is not detected on the microplate spectrophotometre at 37 degrees Celsius(but the plate showed a little florescence), and the downstream gene is fully expressed at 39 degrees Celsius.

We characterized a set of thermal sensitive regulators by culturing them under different temperature, and read them on a petri dish or plate reader. 800px-T--RDFZ-China--TSRquali.jpeg

We can see that at 35 degree Celsius, TlpA36-K2447012 starts to derepress. At 37 degree Celsius, TlpA39-K2572000 starts to derepress. At 39.5 degree Celsius, Tcl42-K2572001 starts to express. So they do initiate at different temperature. T--RDFZ-China--TSR.png Also, we can see there are differences of expression for same regulators are different under different temperature.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 508
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 793
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1096
  • 1000
    COMPATIBLE WITH RFC[1000]



https://parts.igem.org/Part:BBa_K2447012

Improvement

Group：UCAS-China

Characterizetion

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)