Difference between revisions of "Part:BBa K3930015"
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<h2>Results</h2>
 
<h2>Results</h2>
  
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<h3>Production of &beta;-ionone (BBa_K3930003)</h3>
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<h3>Production of &beta;-ionone</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;-ionone was measured by GC-MS. Figure 1 shows the mass spectrum of a CrtY expressing strain.</p>
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<p>All the experiments that characterized this part are related to the final construct pFLEUR <a href="https://parts.igem.org/Part:BBa_K3930002" class="pr-0" target="_blank">(BBa_K3930002)</a> which was cloned into the S. cerevisiae LycoYeast strain. For more information on the experimental background, please refer to this part</p>
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<p>In the part (BBa_K3930002), the TEF1 promoter control the expression of CrtY, an enzyme that produce &beta;-ionone from &beta;-carotene. The production of &beta;-ionone can be so considered as a contol of functionality of the promoter TEF1. pFLEUR was transformed into the <i>S.cerevisiae</i> LycoYeast strain. The &beta;-ionone is very volatile. 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). The production of &beta;-ionone, was successfully achieved with this construction. This mean that the TEF1 promoter is functional to express gene in S. cerevisiae</p>
 
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Revision as of 15:10, 16 October 2021


Constitutive promoter TEF1 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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 168

Introduction

The promoter TEF1 is a constitutive promoter that comes from the plasmid p405TEF1 from Nicolas Buchler & Fred Cross (unpublished). The TEF1 promoter coming from the yeast Y. lipolitica is already described and used in part (BBa_K2117000), but ours comes from the S. cerevisiae genome.

Results

Production of β-ionone

All the experiments that characterized this part are related to the final construct pFLEUR (BBa_K3930002) which was cloned into the S. cerevisiae LycoYeast strain. For more information on the experimental background, please refer to this part

In the part (BBa_K3930002), the TEF1 promoter control the expression of CrtY, an enzyme that produce β-ionone from β-carotene. The production of β-ionone can be so considered as a contol of functionality of the promoter TEF1. pFLEUR was transformed into the S.cerevisiae LycoYeast strain. The β-ionone is very volatile. 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 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). The production of β-ionone, was successfully achieved with this construction. This mean that the TEF1 promoter is functional to express gene in S. cerevisiae



Figure 1: Production of β-ionone upon galctose activation

β-ionone is produced in vivo by our strain when it is induced by galactose. On the right are presented the mass spectra that correspond between the standard and the observed peak


This promoter TEF1 works under those lab conditions.