Difference between revisions of "Part:BBa K3930019"
 
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<h2>Introduction</h2>
 
<h2>Introduction</h2>
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<p>The rtTA advanced cassette is composed of a promoter HXT7, the gene <i>rtTA</i> which allows the induction of the promoter Teto7 upon binding of doxycycline, and the terminator GYP7. This part is related to the already used part (BBa_K2748001), but we added to it the terminator and the promoter. It must be used with the sequence of Teto7 promoter (BBa_K3930014). The sequence comes from the publication of (Garí et al. 1997)</p>
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<p>The rtTA advanced cassette is composed of the promoter Hxt7, the gene <i>rtTA</i> which allows the induction of the promoter TetO7 upon binding of doxycycline, and the terminator Gyp7. This part is related to the previous part <a href="https://parts.igem.org/Part:BBa_K2748001" class="pr-0" target="_blank">(BBa_K2748001)</a>, but we added to it the terminator and the promoter. It must be used with the sequence of TetO7 promoter <a href="https://parts.igem.org/Part:BBa_K3930014" class="pr-0" target="_blank">(BBa_K3930014)</a>. The sequence comes from the publication of Garí et al. (1997).</p>
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<h2>Results</h2>
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<h2>Characterization</h2>
  
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<h3>Production of &beta;-caotene (BBa_K3930003)</h3>
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<h3>Production of &beta;-carotene </h3>
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<p> The part (BBa_K3930002) was linearized and transformed into the <i>S. cerevisiae</i> LycoYeast strain. The production of &beta;-carotene was measured by HPLC. Figure 1 shows the chromatogram of an induced strain with 10 &mu;g.ml-1 of doxycycline.</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 rtTA activator allows the positive control of the TetO7 promoter controls, upon binding with doxycycline. In the pFRAMBOISE-notfused construct the expression of the CrtY gene, which converts lycopene to &beta;-carotene, is controlled by this promoter. 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 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. So, while there is clearly some TetO7 promoter activity, the regulatory part rtTA does not appear as functional.</p>
 
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                 <b>Figure 1: </b> <b> Production of &beta;-carotene upon doxycycline activation</b>
 
                 <b>Figure 1: </b> <b> Production of &beta;-carotene upon doxycycline activation</b>
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                 <p>&beta;-carotenes produced in vivo by our strain. On the right are presented the mass spectra that correspond between the standard and the observed peak</p>
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                 <p>&beta;-carotenes produced in vivo by our strain. On the right are presented the mass spectra of the the standard and the observed peak, showing their similarity.</p>
 
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<p><b>This promoter Teto7 is leaky under these lab conditions.</b><p>
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<p><b>We concluded that the promoter TetO7 is leaky under these lab conditions and rtTA functionality was not proved here.</b><p>
 
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<h2>References</h2>
 
<h2>References</h2>

Latest revision as of 07:47, 17 October 2021


Inducer rtTA for the TetO7 promoter 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]

Introduction

The rtTA advanced cassette is composed of the promoter Hxt7, the gene rtTA which allows the induction of the promoter TetO7 upon binding of doxycycline, and the terminator Gyp7. This part is related to the previous part (BBa_K2748001), but we added to it the terminator and the promoter. It must be used with the sequence of TetO7 promoter (BBa_K3930014). The sequence comes from the publication of Garí et al. (1997).

Characterization

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 rtTA activator allows the positive control of the TetO7 promoter controls, upon binding with doxycycline. In the pFRAMBOISE-notfused construct the expression of the CrtY gene, which converts lycopene to β-carotene, is controlled by this promoter. 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 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. So, while there is clearly some TetO7 promoter activity, the regulatory part rtTA does not appear as functional.



Figure 1: Production of β-carotene upon doxycycline activation

β-carotenes produced in vivo by our strain. On the right are presented the mass spectra of the the standard and the observed peak, showing their similarity.


We concluded that the promoter TetO7 is leaky under these lab conditions and rtTA functionality was not proved here.


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.