Difference between revisions of "Part:BBa K2656020"
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Part BBa_K2656020 is the Yellow fluorescent protein with LVA degradation tag sequence compatible with both Biobrick and [http://2018.igem.org/Team:Valencia_UPV/GB3 GoldenBraid 3.0] assemply methods. It can be combined with other compatible parts from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Valencia UPV IGEM 2018 Printeria Collection] to assemble transcriptional units with the [http://2018.igem.org/Team:Valencia_UPV/Protocols Golden Gate assembly protocol].
 
Part BBa_K2656020 is the Yellow fluorescent protein with LVA degradation tag sequence compatible with both Biobrick and [http://2018.igem.org/Team:Valencia_UPV/GB3 GoldenBraid 3.0] assemply methods. It can be combined with other compatible parts from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Valencia UPV IGEM 2018 Printeria Collection] to assemble transcriptional units with the [http://2018.igem.org/Team:Valencia_UPV/Protocols Golden Gate assembly protocol].
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<html>
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<p>
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First, we adapted the CDS BBa_K592101 to be used to assemble composite parts using the Golden Gate method, creating <a href="https://parts.igem.org/Part:BBa_K2656021">BBa_K2656021</a> and we added the LVA degradation tag, creating <a href="https://parts.igem.org/Part:BBa_K2656020">BBa_K2656020</a>, our improved part. Next, we performed an <a href="http://2018.igem.org/Team:Valencia_UPV/Experiments#spectra">experiment</a> to obtain the excitation and emission spectra. To do this, we created the transcriptional unit <a href="https://parts.igem.org/Part:BBa_K2656112"> BBa_K2656112</a> and we used the parameters of the Table 1:
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</p>
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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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<h6> Table 1. Parameters used to obtain the spectra </h6>
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</html>
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{|class='wikitable'
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|'''Parameter'''
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|'''Value'''
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|-
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|Number of samples
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|6
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|-
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|Excitation Wavelength measurement range (nm)
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|[450-550]
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|-
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|Emission wavelenght (nm)
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|580
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|-
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|Emission Wavelength measurement range (nm)
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|[500-580]
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|-
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|Excitation wavelenght (nm)
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|470
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|-
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|Gain (G)
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|50
 +
|-
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|}
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 +
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[[File:T--Valencia_UPV--YFP_spectrum.png|900px|thumb|none|alt=YFP spectra.|Figure 1. YFP emission and excitation spectra]]
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<html>
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<p>
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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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<p>
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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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<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 2 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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</html>
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[[File:T--Valencia_UPV--YFP-YFP_LVA.png|900px|thumb|none|alt=sfGFP spectra.|Figure 2.  Experimental results of the fluorescence comparison experiment between the transcriptional unit with BBa_K2656021 and the one with BBa_K2656020.]]
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<html>
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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 2 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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<h6>Table 2. Optimized values of translation rate, degradation rate and dilution rate from experimental data
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</h6>
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<table class="wikitable">
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  <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>
 +
 +
  </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>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 15:52, 9 October 2018


YFP_LVA Coding Sequence

Part BBa_K2656020 is the Yellow fluorescent protein with LVA degradation tag sequence compatible with both Biobrick and [http://2018.igem.org/Team:Valencia_UPV/GB3 GoldenBraid 3.0] assemply methods. It can be combined with other compatible parts from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Valencia UPV IGEM 2018 Printeria Collection] to assemble transcriptional units with the [http://2018.igem.org/Team:Valencia_UPV/Protocols Golden Gate assembly protocol].

First, we adapted the CDS BBa_K592101 to be used to assemble composite parts using the Golden Gate method, creating BBa_K2656021 and we added the LVA degradation tag, creating BBa_K2656020, our improved part. Next, we performed an experiment to obtain the excitation and emission spectra. To do this, we created the transcriptional unit BBa_K2656112 and we used the parameters of the Table 1:

Table 1. Parameters used to obtain the spectra

Parameter Value
Number of samples 6
Excitation Wavelength measurement range (nm) [450-550]
Emission wavelenght (nm) 580
Emission Wavelength measurement range (nm) [500-580]
Excitation wavelenght (nm) 470
Gain (G) 50


YFP spectra.
Figure 1. YFP emission and excitation spectra

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 2 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.


sfGFP spectra.
Figure 2. 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 2 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 2. 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


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]