Difference between revisions of "Part:BBa C0071"
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Author: Yoshio Takata  
 
Author: Yoshio Takata  
  
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<span style="margin-left: 10px;">We simulated our final genetic circuits and found that the circuits did not work, because Prhl activity was too weak compared to Plux. (see the Modeling page and the Only Assay page). We therefore considered using the improved Prhl (BBa_K1529310, BBa_K1529300) established by Tokyo_Tech 2014, but we noticed that they were inappropriate for two reasons (see the Discussion). Then, we decided to improve Prhl Promoter and obtain our original improved Prhl (Noticeable Mutant) (BBa_K1949060), hereafter referred to as Prhl(NM), that suited our goal.
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summary of characterization: I. Improved Prhl by iGEM 2014 Tokyo_Tech team and characterize RhlR assay 
  
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During improving Prhl(R0071), we characterize this part. So, we describe that characterization.
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<span style="margin-left: 10px;">We Tokyo Tech 2016 simulated our final genetic circuits and found that the circuits did not work, because Prhl strength was too weak. (see [http://2016.igem.org/Team:Tokyo_Tech  our work in Tokyo_Tech 2016 wiki]). We therefore considered using the improved Prhl (<partinfo>BBa_K1529310</partinfo>,<partinfo>BBa_K1529310</partinfo>) established by Tokyo_Tech 2014, but we noticed that they were inappropriate for two reasons (see [http://2016.igem.org/Team:Tokyo_Tech  our work in Tokyo_Tech 2016 wiki]). Then, we decided to improve Prhl Promoter and obtain our original improved Prhl (included in <partinfo>BBa_K1949060</partinfo>) that suited our goal.
  
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<span style="margin-left: 10px;">We found that Prhl(RL) (<partinfo>BBa_K1529300</partinfo>) activity was weak and the expression level depended on LVA tag (Fig.1); LVA-tagged proteins are prone to be degraded by cellular proteases. Prhl(LR) (<partinfo>BBa_K1529310</partinfo>) activity was strong and unexpectedly reacted with C12 (crosstalk) (Fig.3).
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<span style="margin-left: 10px;">Our purpose is to create strong Prhl for our final genetic circuits.
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I. Improved Prhl by iGEM 2014 Tokyo_Tech team and characterize rhlR assay 
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====I. Improved Prhl by iGEM 2014 Tokyo_Tech item and characterize RhlR assay==== 
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<span style="margin-left: 10px;">We found that Prhl(RL) (<partinfo>BBa_K1529300</partinfo>) activity was weak and the expression level depended on LVA tag (Fig.1); LVA-tagged proteins are prone to be degraded by cellular proteases. Prhl(LR) (<partinfo>BBa_K1529310</partinfo>) activity was strong and unexpectedly reacted with C12 (crosstalk).
  
 
[[Image:prhl1.png|thumb|center|400px|Fig.1 Comparison of the Past improved Prhl  and RhlR ]]<br>
 
[[Image:prhl1.png|thumb|center|400px|Fig.1 Comparison of the Past improved Prhl  and RhlR ]]<br>
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[[Image:prhl2.png|thumb|center|400px|Fig.2 The colonies of transformants with a rhlR (left) or a rhlR-LVA (right)]]<br>
 
[[Image:prhl2.png|thumb|center|400px|Fig.2 The colonies of transformants with a rhlR (left) or a rhlR-LVA (right)]]<br>
  
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If you want more information, you see [http://2016.igem.org/Team:Tokyo_Tech  our work in Tokyo_Tech 2016 wiki] and <partinfo>BBa_K1949060</partinfo>!
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If you want more information, you see [http://2016.igem.org/Team:Tokyo_Tech  our work in Tokyo_Tech 2016 wiki]!
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 20:10, 19 October 2016


rhlR repressor/activator from P. aeruginosa PA3477 (+LVA)

Transcriptional regulator, in complex with N-butyryl-HSL, RhlR binds to the Rhl promoter

Usage and Biology

Transcriptional regulator, binds with N-butyryl-HSL

Characterization

Group: Tokyo Tech 2016

Author: Yoshio Takata

summary of characterization: I. Improved Prhl by iGEM 2014 Tokyo_Tech team and characterize RhlR assay

We Tokyo Tech 2016 simulated our final genetic circuits and found that the circuits did not work, because Prhl strength was too weak. (see [http://2016.igem.org/Team:Tokyo_Tech our work in Tokyo_Tech 2016 wiki]). We therefore considered using the improved Prhl (BBa_K1529310,BBa_K1529310) established by Tokyo_Tech 2014, but we noticed that they were inappropriate for two reasons (see [http://2016.igem.org/Team:Tokyo_Tech our work in Tokyo_Tech 2016 wiki]). Then, we decided to improve Prhl Promoter and obtain our original improved Prhl (included in BBa_K1949060) that suited our goal.

Our purpose is to create strong Prhl for our final genetic circuits.

I. Improved Prhl by iGEM 2014 Tokyo_Tech team and characterize rhlR assay

I. Improved Prhl by iGEM 2014 Tokyo_Tech item and characterize RhlR assay

We found that Prhl(RL) (BBa_K1529300) activity was weak and the expression level depended on LVA tag (Fig.1); LVA-tagged proteins are prone to be degraded by cellular proteases. Prhl(LR) (BBa_K1529310) activity was strong and unexpectedly reacted with C12 (crosstalk).

Fig.1 Comparison of the Past improved Prhl and RhlR

The colonies of transformants with a rhlR (BBa_C0171) plasmid looked rough and the growth rate was low(Fig.2-left), while the colonies of transformants with a rhlR-LVA (BBa_C0071) plasmid looked smooth and the growth rate was normal(Fig.2-right). However, the reason for this result is unclear.

Fig.2 The colonies of transformants with a rhlR (left) or a rhlR-LVA (right)

If you want more information, you see [http://2016.igem.org/Team:Tokyo_Tech our work in Tokyo_Tech 2016 wiki]!


Sequence and Features


Barcodes are discontinued, but one was appended to the sequence of this part. Composite parts using this part will include the barcode. More ...

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 240
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 715