Difference between revisions of "Part:BBa K3930017"
 
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<h2>Introduction</h2>
 
<h2>Introduction</h2>
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<p>This sequence codes for a phCCD1 enzyme, that transforms beta-carotene into beta-ionone, fused with a fyn anchor, which allows relocation of phCCD1 to the membrane. This sequence is codon optimized for its expression into <i>S. cerevisiae</i>. The phCCD1 sequence comes from <i>Petunia hybrida</i> and is described in the publication of López et al. (2020).</p>
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<p>This sequence codes for a fyn-phCCD1 enzyme, that transforms beta-carotene into beta-ionone, fused with a fyn anchor, which allows relocation of phCCD1 to the membrane. This sequence is codon optimized for its expression into <i>S. cerevisiae</i>. The phCCD1 sequence comes from <i>Petunia hybrida</i> and is described in the publication of López et al. (2020).</p>
 
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<h2>Characterization</h2>
 
<h2>Characterization</h2>
 
<h3>Production of &beta;-ionone</h3>
 
<h3>Production of &beta;-ionone</h3>
 
<p>All the experiments that characterized this part are related to the final construct pFRAMBOISE-notfused <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>
 
<p>All the experiments that characterized this part are related to the final construct pFRAMBOISE-notfused <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>&beta;-ionone, which is produced by fyn-phCCD1, is a very volatile molecule. A common strategy to avoid losing these molecules during the culture is to grow the engineered microorganisms in a culture medium supplemented with an organic phase to trap the molecules of interest. The most common organic solvent used is dodecane for ionones (Chen et al. 2019; López et al. 2020). Figure 1 shows the GC-MS spectrum for the LycoYeast-FRAMBOISE-notfused strain. A peak can be observed at the same retention time as the &beta;-ionone standard for the induced LycoYeast-FRAMBOISE-notfused strain. The mass spectra associated with this peak matched with the one obtained with the analytical standard. The &beta;-ionone attribution was further confirmed by the NIST mass spectral library (National Institute of Standards and Technology). Thus, production of &beta;-ionone, the main molecule of the violet odor, was successfully achieved with this construction.</p>
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<p>&beta;-ionone, which is produced by fyn-phCCD1, is a very volatile molecule. A common strategy to avoid losing these molecules during the culture is to grow the engineered microorganisms in a culture medium supplemented with an organic phase to trap the molecules of interest. The most common organic solvent used is dodecane for ionones (Chen et al. 2019; López et al. 2020). Figure 1 shows the GC-MS spectrum for the LycoYeast pFRAMBOISE notfused strain. A peak can be observed at the same retention time as the &beta;-ionone standard for the induced LycoYeast-FRAMBOISE-notfused strain. The mass spectra associated with this peak matched with the one obtained with the analytical standard. The &beta;-ionone attribution was further confirmed by the NIST mass spectral library (National Institute of Standards and Technology). Thus, production of &beta;-ionone, the main molecule of the violet odor, was successfully achieved with this construction.</p>
 
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                     <b>Figure 1: </b> <b>GC-MS analysis of the dodecane layer of the LycoYeast-pFRAMBOISE-notfused</b>  
 
                     <b>Figure 1: </b> <b>GC-MS analysis of the dodecane layer of the LycoYeast-pFRAMBOISE-notfused</b>  
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                     <p>&beta;-ionone is produced <i>in vivo</i> by our strain LycoYeast-pFRAMBOISE-notfused. On the right are presented the matching mass spectra between the standard and the observed peak.</p>
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                     <p>&beta;-ionone is produced <i>in vivo</i> by our strain LycoYeast-pFRAMBOISE-notfused. On the right are presented the matching mass spectra between the standard and the observed peak. First panel is the β-ionone standard. Second panel is the LycoYeast-pFRAMBOISE-notfused induced strain. Third panel is the LycoYeast-pFRAMBOISE-notfused non-induced strain. Forth panel is the LycoYeast WT strain.</p>
 
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<p><b> We concluded the fyn-phCCD1 part works under those lab conditions </b></p>
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<p><b> We concluded the fyn-phCCD1 part works under those lab conditions. </b></p>
 
<h2>References</h2>
 
<h2>References</h2>
 
<ol>
 
<ol>

Latest revision as of 06:15, 17 October 2021


Fusion between phCCD1 and the fyn anchor with linker to produce β-ionone 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 864
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Introduction

This sequence codes for a fyn-phCCD1 enzyme, that transforms beta-carotene into beta-ionone, fused with a fyn anchor, which allows relocation of phCCD1 to the membrane. This sequence is codon optimized for its expression into S. cerevisiae. The phCCD1 sequence comes from Petunia hybrida and is described in the publication of López et al. (2020).


Characterization

Production of β-ionone

All the experiments that characterized this part are related to the final construct pFRAMBOISE-notfused (BBa_K3930003) which was cloned into the S. cerevisiae LycoYeast strain. For more information on the experimental background, please refer to this part.

β-ionone, which is produced by fyn-phCCD1, is a very volatile molecule. A common strategy to avoid losing these molecules during the culture is to grow the engineered microorganisms in a culture medium supplemented with an organic phase to trap the molecules of interest. The most common organic solvent used is dodecane for ionones (Chen et al. 2019; López et al. 2020). Figure 1 shows the GC-MS spectrum for the LycoYeast pFRAMBOISE notfused strain. A peak can be observed at the same retention time as the β-ionone standard for the induced LycoYeast-FRAMBOISE-notfused strain. The mass spectra associated with this peak matched with the one obtained with the analytical standard. The β-ionone attribution was further confirmed by the NIST mass spectral library (National Institute of Standards and Technology). Thus, production of β-ionone, the main molecule of the violet odor, was successfully achieved with this construction.


Figure 1: GC-MS analysis of the dodecane layer of the LycoYeast-pFRAMBOISE-notfused

β-ionone is produced in vivo by our strain LycoYeast-pFRAMBOISE-notfused. On the right are presented the matching mass spectra between the standard and the observed peak. First panel is the β-ionone standard. Second panel is the LycoYeast-pFRAMBOISE-notfused induced strain. Third panel is the LycoYeast-pFRAMBOISE-notfused non-induced strain. Forth panel is the LycoYeast WT strain.


We concluded the fyn-phCCD1 part works under those lab conditions.

References

  1. López J, Bustos D, Camilo C, Arenas N, Saa PA, Agosin E. 2020. Engineering Saccharomyces cerevisiae for the Overproduction of β-Ionone and Its Precursor β-Carotene. Front Bioeng Biotechnol. 8:578793. doi:10.3389/fbioe.2020.578793.
    2.