Difference between revisions of "Part:BBa K3580003"

 
 
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<partinfo>BBa_K3580003 short</partinfo>
 
<partinfo>BBa_K3580003 short</partinfo>
  
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This part express GFP with ssrA tag, which activated by luxR protein and suppress by tetR protein.
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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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We construct this parts by improving BBa_K934025 to test the effect of degradation tag under the luxR regulation.
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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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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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To compare the
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 16:46, 27 October 2020


Plux/tet-GFPssrA(LVA)

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 745