Difference between revisions of "Part:BBa K2201241"

 
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===Functional Parameters===
 
===Functional Parameters===
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To investigate the influence of <html><a href="http://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/photoswitching">photoswitching</a> </html> on the lycopene production, we cultivated three biological replicates of the three variants and each with one of the AzoF conformations for 24&nbsp;hours in a 6-wellplate at 37&nbsp;°C and 400&nbsp;rpm. The media was supplemented with 1&nbsp;mM of AzoF and then split in to charges. Both were irradiated for 40&nbsp;minutes and 100&nbsp;% brightness, one with 367 nm and the other with 465 nm to photoswitch the amino acids. After the cultivation, we measured the OD<sub>600</sub> of each sample (Figure 1). The growth was not influenced in a noticeable way by the different AzoF variants, since the error bars overlap each other.
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/f/f0/T--Bielefeld-CeBiTec--YKE_OD_after_irradiation.png">
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<p class="figure subtitle"><b>Figure 1:</b> OD<sub>600</sub> of three biological and three technical replicated of the <i>crtI</i> variants after cultivation.<p>
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We then extracted the lycopene from the cell pellet and measured the lycopene amount (Figure 2). It can be seen that the TAG353 variant with the <i>trans</i>-AzoF has the highest lycopene production, followed by the TAG353 with the <i>cis</i>-AzoF and TAG318 with the <i>trans</i>-AzoF nearly equal. The TAG318 variant with the <i>cis</i>-AzoF has the lowest lycopene amount.
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<div class="figure small">
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/9/95/T--Bielefeld-CeBiTec--YKE_lycopene8.png">
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<p class="figure subtitle"><b>Figure 2:</b> Absorption spectrum of the four samples of the <i>crtI</i> variants, cultivated with AzoF supplemented to the media photoswitched to <i>cis</i>- or <i>trans</i>-conformation.<p>
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The absorption at 476 nm was measured and normed to the OD<sub>600</sub> of the samples. The relative lycopene production of each <i>crtI</i> and AzoF variant is shown in Figure 3 compared to the unmodified lycopene producer.
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<div class="figure small">
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/f/f0/T--Bielefeld-CeBiTec--YKE_switch_proof.png">
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<p class="figure subtitle"><b>Figure 3:</b> Absorption at 476&nbsp;nm (indicator for lycopene) normalized to the OD<sub>600</sub> (indication for the cell density) to calculate the relative lycopene production of each <i>crtI</i> variant cultivated with AzoF in <i>cis</i>- and <i>trans</i>-conformation.
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Figure 3 shows the effect on the lycopene production based on the incorporation of photoswitched AzoF. The <i>trans</i>-conformation seems to favor the binding activity of the active site, while the <i>cis</i>-conformation seems to reduce the binding activity. The highest difference in the lycopene production is present at the TAG353 variant. Here the cotransformant shows a lycopene production similar to the unmodified lycopene producer when cultivated with <i>trans</i>-AzoF while the productivity is reduced to nearly a third when cultivated with <i>cis</i>-AzoF. The AzoF-variants do not seem to influence the lycopene production when no amber-codon is present in <i>crtI</i>. Concluding, we provided strong evidence that that the observed difference in lycopene production in the three variants is caused by the incorporation and <html><a href="http://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/photoswitching">photoswitching</a> </html>of AzoF.
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<partinfo>BBa_K2201241 parameters</partinfo>
 
<partinfo>BBa_K2201241 parameters</partinfo>
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Revision as of 19:00, 30 October 2017


CrtEBI under constitutive promoter with an amber codon at amino acid position 318 of crtI

This parts contains the part BBa_K274110 with an amber codon at position 318 of the crtI enzyme. With the help of an photoinducable amino acid crtI can be regulated by irradiation with light.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 2037
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1573
    Illegal NgoMIV site found at 1703
    Illegal AgeI site found at 788
  • 1000
    COMPATIBLE WITH RFC[1000]


Functional Parameters

To investigate the influence of photoswitching on the lycopene production, we cultivated three biological replicates of the three variants and each with one of the AzoF conformations for 24 hours in a 6-wellplate at 37 °C and 400 rpm. The media was supplemented with 1 mM of AzoF and then split in to charges. Both were irradiated for 40 minutes and 100 % brightness, one with 367 nm and the other with 465 nm to photoswitch the amino acids. After the cultivation, we measured the OD600 of each sample (Figure 1). The growth was not influenced in a noticeable way by the different AzoF variants, since the error bars overlap each other.

<img class="figure image" src="T--Bielefeld-CeBiTec--YKE_OD_after_irradiation.png">

Figure 1: OD600 of three biological and three technical replicated of the crtI variants after cultivation.<p> </div> We then extracted the lycopene from the cell pellet and measured the lycopene amount (Figure 2). It can be seen that the TAG353 variant with the trans-AzoF has the highest lycopene production, followed by the TAG353 with the cis-AzoF and TAG318 with the trans-AzoF nearly equal. The TAG318 variant with the cis-AzoF has the lowest lycopene amount.

<img class="figure image" src="T--Bielefeld-CeBiTec--YKE_lycopene8.png"> <p class="figure subtitle">Figure 2: Absorption spectrum of the four samples of the crtI variants, cultivated with AzoF supplemented to the media photoswitched to cis- or trans-conformation.<p>

The absorption at 476 nm was measured and normed to the OD600 of the samples. The relative lycopene production of each crtI and AzoF variant is shown in Figure 3 compared to the unmodified lycopene producer.

<img class="figure image" src="T--Bielefeld-CeBiTec--YKE_switch_proof.png"> <p class="figure subtitle">Figure 3: Absorption at 476 nm (indicator for lycopene) normalized to the OD600 (indication for the cell density) to calculate the relative lycopene production of each crtI variant cultivated with AzoF in cis- and trans-conformation.

Figure 3 shows the effect on the lycopene production based on the incorporation of photoswitched AzoF. The trans-conformation seems to favor the binding activity of the active site, while the cis-conformation seems to reduce the binding activity. The highest difference in the lycopene production is present at the TAG353 variant. Here the cotransformant shows a lycopene production similar to the unmodified lycopene producer when cultivated with trans-AzoF while the productivity is reduced to nearly a third when cultivated with cis-AzoF. The AzoF-variants do not seem to influence the lycopene production when no amber-codon is present in crtI. Concluding, we provided strong evidence that that the observed difference in lycopene production in the three variants is caused by the incorporation and photoswitching of AzoF.

</div>

</div>