Difference between revisions of "Part:BBa C0056"
 
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_C0056 parameters</partinfo>
 
<partinfo>BBa_C0056 parameters</partinfo>
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<h2>Improvement </h2>
 
<h2>Improvement </h2>
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<p>    In 2019, UCAS-China made several improvements to CI434, BBa_C0056 by constructing a new part, CI434-TEVsite, BBa_K2969020. Firstly, we inserted the TEV protease recognition sequence between N-terminal and C-terminal of CI434 so that it can be cleaved by TEV and then lose activity, which can regulate gene expression at both transcriptional and proteolytic levels. As shown in Figure 1 and Figure 2, we can see that CI434-TEVsite and original TEV have little difference. However, when there exists TEV protease, CI434-TEVsite can lose its activity so that the genes expression under CI434-TEVsite can be activated significantly.  
 
<p>    In 2019, UCAS-China made several improvements to CI434, BBa_C0056 by constructing a new part, CI434-TEVsite, BBa_K2969020. Firstly, we inserted the TEV protease recognition sequence between N-terminal and C-terminal of CI434 so that it can be cleaved by TEV and then lose activity, which can regulate gene expression at both transcriptional and proteolytic levels. As shown in Figure 1 and Figure 2, we can see that CI434-TEVsite and original TEV have little difference. However, when there exists TEV protease, CI434-TEVsite can lose its activity so that the genes expression under CI434-TEVsite can be activated significantly.  
 
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<div>[[File:T--UCAS-China--CI434.png |700px|thumb|center|<b>Figure 1:</b>Relationship between the wavelength of light exposed on liquid medium and the intensity of BFP, GFP and RFP E. coli expressed from left figure to right figure respectively]]</div>
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<div>[[File:T--UCAS-China--LALA.png|700px|thumb|center|<b>Figure 1:</b>The effect of CI434 and CI434-TEVsite at different temperatures]]</div>
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[[File:T--UCAS-China--PTac-TEV.png|700px|thumb|center|<b>Figure 2:</b>The cleavage efficiency of TEV to CI434-TEVsite induced by a series of concentrations of IPTG
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<div>[[File:T--UCAS-China--PTac-TEV.png|700px|thumb|center|<b>Figure 2:</b>The cleavage efficiency of TEV to CI434-TEVsite induced by a series of concentrations of IPTG]]</div>
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<p>  Besides, we also developed CI434ts-TEVsite through an evolutionary strategy which use temperature as a screening condition. CI434ts is a heat-inducible ON-transcription factor which is a thermo-sensitive mutation type of CI434 transcription factor, which can regulate gene expression at different temperatures. As shown in Figure 3, when the temperature is high, CI434ts will function and repress its promoter to inhibit the process of under-stream genes transcription more tightly than original CI434.  
 
<p>  Besides, we also developed CI434ts-TEVsite through an evolutionary strategy which use temperature as a screening condition. CI434ts is a heat-inducible ON-transcription factor which is a thermo-sensitive mutation type of CI434 transcription factor, which can regulate gene expression at different temperatures. As shown in Figure 3, when the temperature is high, CI434ts will function and repress its promoter to inhibit the process of under-stream genes transcription more tightly than original CI434.  
 
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https://static.igem.org/mediawiki/parts/3/39/T--UCAS-China--CI434ts.png
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<div>[[File:T--UCAS-China--CI434ts.png|700px|thumb|center|<b>Figure 3:</b>The effect of CI434 and CI434ts at different temperatures]]</div>
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<h5> Figure 3. The effect of CI434 and CI434ts at different temperatures
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<p>Therefore, our part, CI434ts-TEVsite have both functions and can be applied in many ways.
 
<p>Therefore, our part, CI434ts-TEVsite have both functions and can be applied in many ways.
 
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<h3>Reference
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<p>Zheng, Y., Meng, F., Zhu, Z., Wei, W., Sun, Z., Chen, J., . . . Chen, G.-Q. (2019). A tight cold-inducible switch built by coupling thermosensitive transcriptional and proteolytic regulatory parts. Nucleic Acids Research. doi:10.1093/nar/gkz785
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==Functional Parameters: Austin_UTexas==
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<h3><center>Burden Imposed by this Part:</center></h3>
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<center><img src = "https://static.igem.org/mediawiki/parts/7/7a/T--Austin_Utexas--high_significant_burden.png" style = "width:250px;height:120px"></center>
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<figcaption><center><b>Burden Value: 28.2 ± 3.9% </b></center></figcaption>
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<p> Burden is the percent reduction in the growth rate of <i>E. coli</i> cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This part exhibited a significant burden. Users should be aware that BioBricks with a burden of >20-30% may be susceptible to mutating to become less functional or nonfunctional as an evolutionary consequence of this fitness cost. This risk increases as they used for more bacterial cell divisions or in larger cultures. Users should be especially careful when combining multiple burdensome parts, as plasmids with a total burden of >40% are expected to mutate so quickly that they become unclonable. Refer to any one of the
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<a href="https://parts.igem.org/Part:BBa_K3174002">BBa_K3174002</a> - <a href="https://parts.igem.org/Part:BBa_K3174007">BBa_K3174007</a> pages for more information on the methods and other conclusions from a large-scale measurement project conducted by the <a href="https://2019.igem.org/Team:Austin_UTexas">2019 Austin_UTexas team.</a></p>
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<p>This functional parameter was added by the <a href="https://2020.igem.org/Team:Austin_UTexas/Contribution">2020 Austin_UTexas team.</a></p>
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Latest revision as of 00:53, 4 September 2020


cI repressor from phage 434 (no LVA)

-- No description --

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]


Functional Parameters

directionForward
functionRep
ligandsRecA?
proteinCI 434
swissproP16117
tagNone


Improvement

Group: UCAS-China

Summary: We improved the function of CI434, BBa_C0056, by constructing CI434ts-TEVsite, BBa_K2969020 and characterized their function. Compared with original CI434, firstly, CI434ts-TEVsite can be recognized by TEV protease and then lose its activity, which can regulate gene expression at transcriptional and proteolytic levels. What’s more, it becomes a cold-inactivated transcriptional factor, which can regulate gene expression at different temperatures.

Characterization

In 2019, UCAS-China made several improvements to CI434, BBa_C0056 by constructing a new part, CI434-TEVsite, BBa_K2969020. Firstly, we inserted the TEV protease recognition sequence between N-terminal and C-terminal of CI434 so that it can be cleaved by TEV and then lose activity, which can regulate gene expression at both transcriptional and proteolytic levels. As shown in Figure 1 and Figure 2, we can see that CI434-TEVsite and original TEV have little difference. However, when there exists TEV protease, CI434-TEVsite can lose its activity so that the genes expression under CI434-TEVsite can be activated significantly.

Figure 1:The effect of CI434 and CI434-TEVsite at different temperatures


Figure 2:The cleavage efficiency of TEV to CI434-TEVsite induced by a series of concentrations of IPTG

Besides, we also developed CI434ts-TEVsite through an evolutionary strategy which use temperature as a screening condition. CI434ts is a heat-inducible ON-transcription factor which is a thermo-sensitive mutation type of CI434 transcription factor, which can regulate gene expression at different temperatures. As shown in Figure 3, when the temperature is high, CI434ts will function and repress its promoter to inhibit the process of under-stream genes transcription more tightly than original CI434.

Figure 3:The effect of CI434 and CI434ts at different temperatures


Therefore, our part, CI434ts-TEVsite have both functions and can be applied in many ways.

Reference

Zheng, Y., Meng, F., Zhu, Z., Wei, W., Sun, Z., Chen, J., . . . Chen, G.-Q. (2019). A tight cold-inducible switch built by coupling thermosensitive transcriptional and proteolytic regulatory parts. Nucleic Acids Research. doi:10.1093/nar/gkz785



Functional Parameters: Austin_UTexas

Burden Imposed by this Part:

Burden Value: 28.2 ± 3.9%

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This part exhibited a significant burden. Users should be aware that BioBricks with a burden of >20-30% may be susceptible to mutating to become less functional or nonfunctional as an evolutionary consequence of this fitness cost. This risk increases as they used for more bacterial cell divisions or in larger cultures. Users should be especially careful when combining multiple burdensome parts, as plasmids with a total burden of >40% are expected to mutate so quickly that they become unclonable. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.