Difference between revisions of "Part:BBa K3930014"
 
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<h2>Introduction</h2>
 
<h2>Introduction</h2>
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<p>The promoter TetO7 is an doxycycline-inducible promoter. It must be used with the part coding for the activator protein rtTA advanced (BBa_K3930019). The sequence comes from the publication Garí et al. (1997).</p>
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<p>The promoter TetO7 is a doxycycline-inducible promoter. It must be used with the part coding for the activator protein rtTA advanced <a href="https://parts.igem.org/Part:BBa_K3930019" class="pr-0" target="_blank">(BBa K3930019)</a>. The sequence comes from the publication Garí et al. (1997).</p>
 
<h2>Results</h2>
 
<h2>Results</h2>
  
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<h3>Production of &beta;-carotene (BBa_K3930003)</h3>
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<h3>Production of &beta;-carotene </h3>
  
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<p> All the experiments that characterized this part are related to the final construct <a href="https://parts.igem.org/Part:BBa_K3930003" class="pr-0" target="_blank">(BBa K3930003)</a>, which was cloned into the <i>S. cerevisiae</i> LycoYeast strain. For more information on the experimental background, please refer to this part.</p>
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<p> All the experiments that characterized this part are related to the final construct pFRAMBOISE-notfused <a href="https://parts.igem.org/Part:BBa_K3930002" class="pr-0" target="_blank">(BBa K3930002)</a>, which was cloned into the <i>S. cerevisiae</i> LycoYeast strain. For more information on the experimental background, please refer to this part.</p>
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<p>The promoter controls in the pFRAMBOISE-notfused construct the expression of the CrtY gene, which converts lycopene to &beta;-carotene. The production of &beta;-carotene is therefore a control of the functionality when the expression is induced with doxycycline. The carotenoids, produced by the LcyE part of our enzymatic fusion, are contained in the cells. They were extracted using the method described by López et al. (2020). Yeast cells were lysed in acetone using glass beads and the supernatant obtained after lysis was analyzed by RP-HPLC on a C18 column. In the LycoYeast-pFRAMBOISE-notfused strains (which express the LcyE-ofCCD1 fusion), lycopene is converted into a new product with a higher retention time (Figure 1). Considering the &beta;-ionone production results, we concluded this new peak most likely corresponds to &beta;-carotene. Nonetheless, the peak is also observed in the condition with no induction. It seems like there is no negative regulation of the promoter TetO7.</p>
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<p>The promoter controls in the pFRAMBOISE-notfused construct the expression of the <i>CrtY</i> gene, which converts lycopene to &beta;-carotene. The production of &beta;-carotene is therefore a control of the functionality when the expression is induced with doxycycline. The carotenoids are contained in the cells, they were so extracted using the method described by López et al. (2020). Yeast cells were lysed in acetone using glass beads and the supernatant obtained after lysis was analyzed by RP-HPLC on a C18 column. In the LycoYeast-pFRAMBOISE-notfused strains (which express the CrtY), lycopene is converted into a new product with a higher retention time (Figure 1). Considering the &beta;-ionone production results, we concluded this new peak most likely corresponds to &beta;-carotene. Nonetheless, the peak is also observed in the condition with no induction. It seems like there is no negative regulation of the promoter TetO7.</p>
 
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Latest revision as of 08:55, 17 October 2021


Doxycycline inducible promoter 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 282
    Illegal XhoI site found at 239
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Introduction

The promoter TetO7 is a doxycycline-inducible promoter. It must be used with the part coding for the activator protein rtTA advanced (BBa K3930019). The sequence comes from the publication Garí et al. (1997).

Results

Production of β-carotene

All the experiments that characterized this part are related to the final construct pFRAMBOISE-notfused (BBa K3930002), which was cloned into the S. cerevisiae LycoYeast strain. For more information on the experimental background, please refer to this part.

The promoter controls in the pFRAMBOISE-notfused construct the expression of the CrtY gene, which converts lycopene to β-carotene. The production of β-carotene is therefore a control of the functionality when the expression is induced with doxycycline. The carotenoids are contained in the cells, they were so extracted using the method described by López et al. (2020). Yeast cells were lysed in acetone using glass beads and the supernatant obtained after lysis was analyzed by RP-HPLC on a C18 column. In the LycoYeast-pFRAMBOISE-notfused strains (which express the CrtY), lycopene is converted into a new product with a higher retention time (Figure 1). Considering the β-ionone production results, we concluded this new peak most likely corresponds to β-carotene. Nonetheless, the peak is also observed in the condition with no induction. It seems like there is no negative regulation of the promoter TetO7.



Figure 1: Production of β-carotene upon doxycycline activation

β-carotene is produced in vivo by our strain. On the right are presented the mass spectra matching between the standard and the observed peak.


We concluded this TetO7 promoter is not negatively regulating our gene expression and seems always active under those lab conditions.


References

  1. Garí E, Piedrafita L, Aldea M, Herrero E. 1997. A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in Saccharomyces cerevisiae. Yeast. 13(9):837–848. doi:10.1002/(SICI)1097-0061(199707)13:9<837::AID-YEA145>3.0.CO;2-T.