Difference between revisions of "Part:BBa K934025"
 
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We constructed this part by ligating Plux/tet hybrid promoter ([https://parts.igem.org/Part:BBa_K934024 BBa_K934024]) to the upstream of promoterless GFP generator ([https://parts.igem.org/Part:BBa_K934025 BBa_I13504]).
 
We constructed this part by ligating Plux/tet hybrid promoter ([https://parts.igem.org/Part:BBa_K934024 BBa_K934024]) to the upstream of promoterless GFP generator ([https://parts.igem.org/Part:BBa_K934025 BBa_I13504]).
  
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[[Image:Pluxtet_assay.png|thumb|center|500px|]]
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[[Image:Pluxtet_assay.png|thumb|center|500px|This work was done by M.M, X.T and S.L.]]
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To characterize the function of the Lux/Tet hybrid promoter(BBa_934024), we constructed this part Plux/tet-GFP (BBa_ K934025) by inserting Plux/tet promoter in front of a GFP coding sequence. By using the reporter cell that contains Plux/tet-GFP and constitutive LuxR and TetR generator (PlacIq-LuxR-Ptrc-TetR), we measured the fluorescence intensity of the reporter cell dependent on the four different combinations of two inducers, 3OC6HSL and aTc (anhydrous tetracycline).
  
 
In the presence of both inducers, the culture showed about 500-fold higher fluorescence intensity than that in the absence of both inducers.
 
In the presence of both inducers, the culture showed about 500-fold higher fluorescence intensity than that in the absence of both inducers.
 +
 +
We improved previous Plux/tet hybrid promoter ([https://parts.igem.org/Part:BBa_K176000:Experience BBa_K176000]).
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For more information, see [http://2012.igem.org/Team:Tokyo_Tech/Project our work in Tokyo_Tech 2012 wiki].
 +
 +
===Contribution and improvement: Waseda 2020 ===
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By addition of LVA degradation tag, Waseda2020 improved this part in terms of quick response of genetic circuit<br>
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See [https://parts.igem.org/Part:BBa_K3580003 BBa_K3580003]
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To see the improvement in degradation effect by ssrA tagging, we construct a improved part (BBa_K3580003) by modification of an existing part: Plux/tet-GFP([https://parts.igem.org/Part:BBa_K934025 BBa_K934025]).
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[[File:T--Waseda--Plux-tet-GFP-improve-ppt.png|500px|thumb|center|<b>Fig1</b> LVA degradation tag improve part(BBa_K358003)]]<br>
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In the behavior of the gene circuit, it is important to reduce the concentration of proteins by degradation and dilution as well as production. Although cells can decrease their concentration by dilution according to growth, cell-free systems can’t because it doesn’t growth. Therefore, an improvement is necessary to incorporate degradation into the cell-free system. In order to compare these parts(BBa_K3580003 and [https://parts.igem.org/Part:BBa_K934025 BBa_K934025]), we first measured the fluorescence of GFP in vivo for 240 minutes. The fluorescence of tagged GFP(BBa_K3580003) was lower than that of normal GFP([https://parts.igem.org/Part:BBa_K934025 BBa_K934025]) at 240 min point (Fig 2). Although GFP is a stable protein with a β-barrel and much difficult to be degraded, this result shows that tagged GFP was successfully degraded as we desired.
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 +
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Then, we compared the fluorescence of GFP in a cell-free system which was extracted from the E.coli containing luxR protein. Fig 3. is the result of the experiment. The fluorescence of tagged GFP(BBa_K3580003) couldn’t be measured because the value was too low. Those results show that ssrA tagged protein can be degraded much both in vivo and in vitro. Based on this data, we modified the degradation terms in the model.
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[[File:T--Waseda--Plux-tet-GFPssrA-vivo-comment_revised.png|600px|thumb|center|<b>Fig2</b> ssrA degradation tag assay <i>in vivo</i>]]<br>
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[[File:T--Waseda--Plux-tet-GFPssrA-vitro-comment.png|600px|thumb|center|<b>Fig3</b> ssrA degradation tag assay <i>in vitro</i>]]<br>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 19:06, 27 October 2020

Plux/tet-GFP

We constructed this part by ligating Plux/tet hybrid promoter (BBa_K934024) to the upstream of promoterless GFP generator (BBa_I13504).

This work was done by M.M, X.T and S.L.

To characterize the function of the Lux/Tet hybrid promoter(BBa_934024), we constructed this part Plux/tet-GFP (BBa_ K934025) by inserting Plux/tet promoter in front of a GFP coding sequence. By using the reporter cell that contains Plux/tet-GFP and constitutive LuxR and TetR generator (PlacIq-LuxR-Ptrc-TetR), we measured the fluorescence intensity of the reporter cell dependent on the four different combinations of two inducers, 3OC6HSL and aTc (anhydrous tetracycline).

In the presence of both inducers, the culture showed about 500-fold higher fluorescence intensity than that in the absence of both inducers.

We improved previous Plux/tet hybrid promoter (BBa_K176000).


For more information, see [http://2012.igem.org/Team:Tokyo_Tech/Project our work in Tokyo_Tech 2012 wiki].

Contribution and improvement: Waseda 2020

By addition of LVA degradation tag, Waseda2020 improved this part in terms of quick response of genetic circuit
See BBa_K3580003

To see the improvement in degradation effect by ssrA tagging, we construct a improved part (BBa_K3580003) by modification of an existing part: Plux/tet-GFP(BBa_K934025).

Fig1 LVA degradation tag improve part(BBa_K358003)

In the behavior of the gene circuit, it is important to reduce the concentration of proteins by degradation and dilution as well as production. Although cells can decrease their concentration by dilution according to growth, cell-free systems can’t because it doesn’t growth. Therefore, an improvement is necessary to incorporate degradation into the cell-free system. In order to compare these parts(BBa_K3580003 and BBa_K934025), we first measured the fluorescence of GFP in vivo for 240 minutes. The fluorescence of tagged GFP(BBa_K3580003) was lower than that of normal GFP(BBa_K934025) at 240 min point (Fig 2). Although GFP is a stable protein with a β-barrel and much difficult to be degraded, this result shows that tagged GFP was successfully degraded as we desired.


Then, we compared the fluorescence of GFP in a cell-free system which was extracted from the E.coli containing luxR protein. Fig 3. is the result of the experiment. The fluorescence of tagged GFP(BBa_K3580003) couldn’t be measured because the value was too low. Those results show that ssrA tagged protein can be degraded much both in vivo and in vitro. Based on this data, we modified the degradation terms in the model.

Fig2 ssrA degradation tag assay in vivo

Fig3 ssrA degradation tag assay in vitro


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 751