Difference between revisions of "Part:BBa K592101"
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To test the effect of the degradation tag, we designed an <a href="http://2018.igem.org/Team:Valencia_UPV/Model#tag_exp">experiment</a> with which we measured the increase in protein degradation due to this tag. To perform this experiment, we assembled two composite parts with the same promoter, RBS and terminator:
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</p>
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<ul>
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<li>
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<a href="https://parts.igem.org/Part:BBa_K2656112"> BBa_K2656112</a>:
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</p>
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</li>
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<ul style="
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padding-left: 3em !important;">
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<li><p><a href="https://parts.igem.org/Part:BBa_K2656004">BBa_K2656004</a>: the <a href="https://parts.igem.org/Part:BBa_J23106">J23106</a> promoter in its GoldenBraid compatible version from our <a href="http://2018.igem.org/Team:Valencia_UPV/Part_Collection">Part Collection</a></p></li>
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<li><p><a href="https://parts.igem.org/Part:BBa_K2656009">BBa_K2656009</a>: the <a href="https://parts.igem.org/Part:BBa_B0030">B0030</a> ribosome biding site in its GoldenBraid compatible version from our <a href="http://2018.igem.org/Team:Valencia_UPV/Part_Collection">Part Collection</a></p></li>
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<li><p><a href="https://parts.igem.org/Part:BBa_K2656021">BBa_K2656021</a>: The original part  <a href="https://parts.igem.org/Part:BBa_K592101"> BBa_K592101</a> compatible with the GoldenBraid assembly method</p></li>
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<li><p><a href="https://parts.igem.org/Part:BBa_K2656026">BBa_K2656026</a>: the <a href="https://parts.igem.org/Part:BBa_B0015">B0015</a> transcriptional terminator in its GoldenBraid compatible version from our <a href="http://2018.igem.org/Team:Valencia_UPV/Part_Collection">Part Collection</a></p></li>
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</ul>
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<li>
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<p>
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<a href="https://parts.igem.org/Part:BBa_K2656111"> BBa_K2656111</a>:
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</p>
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</li>
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<ul style=" padding-left: 3em !important;">
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<li><p><a href="https://parts.igem.org/Part:BBa_K2656004">BBa_K2656004</a></p></li>
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<li><p><a href="https://parts.igem.org/Part:BBa_K2656009">BBa_K2656009</a></p></li>
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<li><p><a href="https://parts.igem.org/Part:BBa_K2656020">BBa_K2656020</a>: The original part with the added LVA ssRA degradation tag compatible with the GoldenBraid assembly method</p></li>
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<li><p><a href="https://parts.igem.org/Part:BBa_K2656026">BBa_K2656026</a></p></li>
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</ul>
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            </ul></div>
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<p> Once the experiment was carried out, the results were plotted and Figure 1 was obtained, in which we can observe that the growth of the bacteria with both constructions was very similar, while the fluorescence had a clear variation.</p>
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<img src="https://static.igem.org/mediawiki/2018/7/72/T--Valencia_UPV--optimization_exp1_YFP_graphUPV2018.png" />
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<h6 style="
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    text-align: left !important;
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"> Figure 1. Experimental results of the fluorescence comparison experiment between the transcriptional unit with BBa_K2656021 and the one with BBa_K2656020. </h6>
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<p>These data were optimized with our <a href="http://2018.igem.org/Team:Valencia_UPV/Model#const_models"> model</a> and the parameters from Table 1 were obtained. With these parameters it is possible to obtain that <b>the protein degradation of the protein with the degradation tag is around twice as much as without the tag</b>.
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<table>
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<h6> Table 1. Optimized values of translation rate, degradation rate and dilution rate from experimental data </h6>   <tbody><tr>
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    <th><p>Optimized parameters</p></th>
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    <th><p>Values</p></th>
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  </tr>
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  <tr>
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  <td><p>Translation rate <b>p</b></p></td>
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  <td>
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  <ul>
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  <li><p>
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  For YFP with LVA tag <a href="https://parts.igem.org/Part:BBa_K2656020" target="_blank" style="padding-right: 0">BBa_K2656020</a>: p = 0.4622 min<sup>-1</sup>
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  </p></li>
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  <li><p>
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  For YFP without LVA tag  <a href="https://parts.igem.org/Part:BBa_K2656021" target="_blank" style="padding-right: 0">BBa_K2656021</a>: p = 1.053 min<sup>-1</sup>
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  </p></li>
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  </ul>
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  </td></tr>
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  <tr><td><p>PoI degradation rate <b>d<sub>p</sub></b></p></td>
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  <td><p>
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  </p><ul>
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  <li><p>
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  For YFP with LVA tag <a href="https://parts.igem.org/Part:BBa_K2656020" target="_blank" style="padding-right: 0">BBa_K2656020</a>: d<sub>p</sub> = 0.01492 min<sup>-1</sup>
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  </p></li>
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  <li><p>
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  For YFP without LVA tag  <a href="https://parts.igem.org/Part:BBa_K2656021" target="_blank" style="padding-right: 0">BBa_K2656021</a>: d<sub>p</sub> = 0.0080 min<sup>-1</sup>
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  </p></li>
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  </ul> 
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  <p></p></td>
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  </tr>
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  <tr>
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  <td><p>Dilution rate <b><meta charset="utf-8">μ</b></p></td>
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  <td><p>
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  </p><ul>
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  <li><p>
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  For YFP with LVA tag <a href="https://parts.igem.org/Part:BBa_K2656020" target="_blank" style="padding-right: 0">BBa_K2656020</a>: <meta charset="utf-8">μ = 0.01166 min<sup>-1</sup>
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  </p></li>
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  <li><p>
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  For YFP without LVA tag  <a href="https://parts.igem.org/Part:BBa_K2656021" target="_blank" style="padding-right: 0">BBa_K2656021</a>: <meta charset="utf-8">μ = 0.01307 min<sup>-1</sup>
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  </p></li>
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  </ul>
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  <p></p></td>
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</tr></tbody></table>
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        </div></html>
  
 
===References===
 
===References===

Revision as of 12:10, 9 October 2018

Yellow Fluorescent Protein (YFP)

Yellow Fluorescent Protein derived from Aequorea victoria GFP. This sequence is cloned from the pZE12-YFP plasmid used by Elowitz (see reference). The original gene was made by the The Yeast Resource Center (YRC) based at the University of Washington in Seattle, Washington.


iGEM12_Uppsala_University: If you are looking for a bright yellow fluorescent protein, the improved gene SYFP2 BBa_K864100 is a better choice than this part.


Usage and Biology

This part is useful as a reporter.

A15 UU.JPG A16 UU.jpg

The images above show E coli constitutively expressing YFP BBa_K592101 (yellow) and mTagBFP BBa_K592100 (blue) illuminated on a UV table.

[http://2017.igem.org/Team:AFCM-Egypt# Egypt-AFCM Team] tried to improve YFP gel characterization and function at BBa_K592101 regarding its expression by the lac promoter (BBa_K2217017) as a weak constitutive promoter in one composite part to help enhancing YFP expression to characterize non-coding RNA regulatory activity. Part characterization and usage can be found at BBa_K2217023_Experience.

Contribution

Group: Valencia_UPV iGEM 2018
Author: Adrián Requena Gutiérrez
Summary:We have adapted the part to be able to assemble transcriptional units with the Golden Gate method and we have added the degradation tag ssRA LVA. After that, we have characterized the protein degradation variation due to this tag.
Documentation:

To test the effect of the degradation tag, we designed an experiment with which we measured the increase in protein degradation due to this tag. To perform this experiment, we assembled two composite parts with the same promoter, RBS and terminator:

Once the experiment was carried out, the results were plotted and Figure 1 was obtained, in which we can observe that the growth of the bacteria with both constructions was very similar, while the fluorescence had a clear variation.

Figure 1. Experimental results of the fluorescence comparison experiment between the transcriptional unit with BBa_K2656021 and the one with BBa_K2656020.

These data were optimized with our model and the parameters from Table 1 were obtained. With these parameters it is possible to obtain that the protein degradation of the protein with the degradation tag is around twice as much as without the tag.

Table 1. Optimized values of translation rate, degradation rate and dilution rate from experimental data

Optimized parameters

Values

Translation rate p

PoI degradation rate dp

Dilution rate μ

References

[http://www.ncbi.nlm.nih.gov/pubmed/12183631] Elowitz, M. B., A. J. Levine, et al. (2002). "Stochastic gene expression in a single cell." Science 297(5584): 1183-6.

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 644