Difference between revisions of "Part:BBa J24813"

(Yeast strain construction)
 
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The constitutive promoter  <i>pURA3</i> controls the activity of the <i>URA3</i> gene. This gene encodes for orotidine 5-phosphate decarboxylase (ODCase), an enzyme that plays a vital role in biosynthesis of pyrimidine ribonucleotides (Umezu et al., 1971).
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The <i>pURA3</i> promoter controls the <i>URA3</i> gene. This gene is responsible for producing the enzyme orotidine 5-phosphate decarboxylase (ODCase), which is crucial for the synthesis of pyrimidine ribonucleotides (Umezu <i>et al</i>., 1971).
  
 
===Plasmid formation===
 
===Plasmid formation===
  
The promoters were PCR-amplified from the yeast genome using primers that contained <i>SacI</i> (forward primer) and <i>BamHI</i> (reverse primer) restriction sites in their 5’-overhangs. After PCR and restriction digestion, the DNA fragments containing the promoters were ligated into <i>SacI/BamHI</i>-restricted pRS304-based vector carrying sfGFP coding sequence and <i>tCYC1</i> terminator.  
+
The promoter was PCR-amplified from the yeast genome using primers that contained <i>SacI</i> (forward primer) and <i>BamHI</i> (reverse primer) restriction sites in its 5’-overhangs. After PCR and restriction digestion, the DNA fragment containing the promoter was ligated into <i>SacI/BamHI</i>-restricted pRS304-based vector carrying EGFP coding sequence and <i>tCYC1</i> terminator.
 
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         <td style = "border: 1px solid black"><i>pURA3</i></td><td style = "border: 1px solid black">sfGFP</td><td style = "border: 1px solid black">Restriction-ligation</td>
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         <td style = "border: 1px solid black"><i>pURA3</i></td><td style = "border: 1px solid black">EGFP</td><td style = "border: 1px solid black">Restriction-ligation</td>
 
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</table>
  
 
===Yeast strain construction===
 
===Yeast strain construction===
Prior to yeast transformation, the integration plasmids were restricted with <i>HindIII</i> to linearise the plasmids for homologous recombination into the yeast genome <i>TRP1</i> locus. The restricted plasmids were used to transform the <i>S. cerevisiae</i> DOM90 strain. Transformants were selected for Trp+ phenotype on tryptophan-dropout synthetic media (CSM-TRP) agar plates containing 2% glucose. All yeast strains generated and used for promoter characterization are listed in table:
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Prior to yeast transformation, the integration plasmid was restricted with <i>HindIII</i> to linearise the plasmid for homologous recombination into the <i>TRP1</i> locus in the yeast genome. The restricted plasmid was used to transform the <i>S. cerevisiae</i> DOM90 strain. Transformants were selected for Trp<sup>+ </sup>phenotype on tryptophan-dropout synthetic media (CSM-TRP) agar plates containing 2% glucose. All yeast strains generated and used for promoter characterization are listed in the table:
  
 
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         <td style = "border: 1px solid black"><i>DOM90</i></td><td style = "border: 1px solid black">MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+] &nbsp;&nbsp;</td><td style = "border: 1px solid black">Background strain used for transformation and as a negative control</td>
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         <td style = "border: 1px solid black"><i>DOM90</i></td><td style = "border: 1px solid black"><i>MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+] </i> &nbsp;&nbsp;</td><td style = "border: 1px solid black">Background strain used for transformation and as a negative control</td>
 
     </tr>
 
     </tr>
 
<tr>
 
<tr>
         <td style = "border: 1px solid black"><i>I86</i></td><td style = "border: 1px solid black">DOM90 trp1::pRS304-pURA3-sfGFP-tCYC1&nbsp;&nbsp;</td><td style = "border: 1px solid black">Strain with sfGFP under <i>pURA3</i> promoter, integrated into Trp1-1 locus</td>
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         <td style = "border: 1px solid black"><i>I86</i></td><td style = "border: 1px solid black"><i>DOM90 trp1::pRS304-pURA3-EGFP-tCYC1</i>&nbsp;&nbsp;</td><td style = "border: 1px solid black">Strain with <i>EGFP</i> under <i>pURA3</i> promoter, integrated into trp1-1 locus</td>
 
     </tr>
 
     </tr>
 
</table>
 
</table>
  
 
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===EGFP fluorescence measurements===
 
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Before conducting fluorescence measurements, yeast seed cultures were cultivated in complete synthetic media (CSM) containing 2% (m/v ratio) raffinose until the cultures reached an optical density (OD<sup>600</sup>) ranging from 1 to 2. Subsequently, the yeast cultures were diluted to an OD<sup>600</sup> of 0.3, and various carbon sources, including glucose, raffinose, galactose, or glycerol, were added into the cultures to achieve a 2% (m/v) concentration of the respective carbon source. After 6 hours of growth, 200 μl of the cell suspension was carefully transferred into designated wells on 96-well plates for subsequent fluorescence measurements.
sfGFP fluorescence measurements
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To measure EGFP fluorescence, a BioTek Synergy Mx Microplate Reader equipped with a 458 nm wavelength LED for GFP excitation was utilized. The emitted fluorescence was measured at a wavelength of 528 nm.
Prior to fluorescence measurements, yeast cells were cultivated in complete synthetic media (CSM) with 2% glucose until the cultures reached an optical density (OD600) in the range of 0.6 to 1. Subsequently, 200 μl of the cell suspension was transferred into the designated wells on 96-well plates.
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To measure sfGFP fluorescence, a BioTek Synergy Mx Microplate Reader equipped with a 458 nm wavelength LED for GFP excitation was utilized. The emitted fluorescence was measured at a wavelength of 528 nm.
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===Results===
 
===Results===
  
In this study, we assessed the level of gene expression driven by the promoter <i>pCYC1</i> by employing a fluorescent protein as a reporter. The promoter-containing constructs were integrated into the yeast genome, and the resulting reporter protein fluorescence was quantified in a 96-well plate. To establish a baseline of background fluorescence in the culture, we measured the fluorescence in a control strain, DOM90, which does not express any fluorescent proteins.
+
In this study, we assessed the level of gene expression driven by the <i>pURA3</i> promoter in different growth conditions by employing a fluorescent protein as a reporter. The promoter-containing constructs were integrated into the trp1-1 locus in the yeast genome, and the EGFP reporter protein fluorescence was quantified in a 96-well plate. To establish a baseline of background fluorescence in the culture, we measured the fluorescence in a control strain, DOM90, which does not express any fluorescent proteins.
Compared to the background fluorescence of DOM90, yeast strains with sfGFP under the control of <i>pURA3</i> promoter  displayed a 3.5-fold increase in sfGFP fluorescence intensity.
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Compared to the background fluorescence of DOM90, yeast strains with EGFP regulated by the <i>pURA3</i> promoter, a 1.17-fold increase was observed with glucose as the carbon source, and a 2.6-fold increase in EGFP fluorescence was noted with raffinose and galactose. In contrast, using either raffinose or glycerol alone as carbon sources resulted in fluorescence levels comparable to those of the DOM90 background.
  
 
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<figcaption>Bars indicate the mean fluorescence intensity (expressed in arbitrary units, AU) measured in pURA3-sfGFP strain or in DOM90 negative control strain. Error bars show standard deviation.</figcaption>
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<figcaption>Bars indicate the mean fluorescence intensity in arbitrary units (AU) measured in <i>pURA3-EGFP</i> strains or in DOM90 negative control strain measured in a plate reader. Error bars show standard deviation. </figcaption>
 
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In our study, we examined three yeast promoters sourced from the iGEM part registry. We found that constitutive promoter <i>pURA3</i> is suitable choice for achieving consistent moderate gene expression. Expanding the quantitative information on yeast promoter activities facilitates the engineering of fine-tuned synthetic biology applications.
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We evaluated the expression levels in the presence of different carbon sources. Our results show that constructs with the <i>pURA3</i> promoter, moderate gene expression was evident when using raffinose and galactose together, while weak expression was observed when glucose was the sole carbon source.
  
 
===References:===
 
===References:===

Latest revision as of 03:18, 11 October 2023

URA3 Promoter from S. cerevisiae

This is the whole regulatory region from the URA3 gene coding for OMP decarboxylase, an essential protein in the uracil synthesis pathway in S. cerevisiae budding yeast.

Team Estonia_TUIT 2023 characterization of BBa_I766556 (pADH1)

The pURA3 promoter controls the URA3 gene. This gene is responsible for producing the enzyme orotidine 5-phosphate decarboxylase (ODCase), which is crucial for the synthesis of pyrimidine ribonucleotides (Umezu et al., 1971).

Plasmid formation

The promoter was PCR-amplified from the yeast genome using primers that contained SacI (forward primer) and BamHI (reverse primer) restriction sites in its 5’-overhangs. After PCR and restriction digestion, the DNA fragment containing the promoter was ligated into SacI/BamHI-restricted pRS304-based vector carrying EGFP coding sequence and tCYC1 terminator.

Promoter  Reporter  Assembly methods  
pURA3EGFPRestriction-ligation

Yeast strain construction

Prior to yeast transformation, the integration plasmid was restricted with HindIII to linearise the plasmid for homologous recombination into the TRP1 locus in the yeast genome. The restricted plasmid was used to transform the S. cerevisiae DOM90 strain. Transformants were selected for Trp+ phenotype on tryptophan-dropout synthetic media (CSM-TRP) agar plates containing 2% glucose. All yeast strains generated and used for promoter characterization are listed in the table:

Strain name  Genotype  Description  
DOM90MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]   Background strain used for transformation and as a negative control
I86DOM90 trp1::pRS304-pURA3-EGFP-tCYC1  Strain with EGFP under pURA3 promoter, integrated into trp1-1 locus

EGFP fluorescence measurements

Before conducting fluorescence measurements, yeast seed cultures were cultivated in complete synthetic media (CSM) containing 2% (m/v ratio) raffinose until the cultures reached an optical density (OD600) ranging from 1 to 2. Subsequently, the yeast cultures were diluted to an OD600 of 0.3, and various carbon sources, including glucose, raffinose, galactose, or glycerol, were added into the cultures to achieve a 2% (m/v) concentration of the respective carbon source. After 6 hours of growth, 200 μl of the cell suspension was carefully transferred into designated wells on 96-well plates for subsequent fluorescence measurements. To measure EGFP fluorescence, a BioTek Synergy Mx Microplate Reader equipped with a 458 nm wavelength LED for GFP excitation was utilized. The emitted fluorescence was measured at a wavelength of 528 nm.

Results

In this study, we assessed the level of gene expression driven by the pURA3 promoter in different growth conditions by employing a fluorescent protein as a reporter. The promoter-containing constructs were integrated into the trp1-1 locus in the yeast genome, and the EGFP reporter protein fluorescence was quantified in a 96-well plate. To establish a baseline of background fluorescence in the culture, we measured the fluorescence in a control strain, DOM90, which does not express any fluorescent proteins.

Compared to the background fluorescence of DOM90, yeast strains with EGFP regulated by the pURA3 promoter, a 1.17-fold increase was observed with glucose as the carbon source, and a 2.6-fold increase in EGFP fluorescence was noted with raffinose and galactose. In contrast, using either raffinose or glycerol alone as carbon sources resulted in fluorescence levels comparable to those of the DOM90 background.

Bars indicate the mean fluorescence intensity in arbitrary units (AU) measured in pURA3-EGFP strains or in DOM90 negative control strain measured in a plate reader. Error bars show standard deviation.


We evaluated the expression levels in the presence of different carbon sources. Our results show that constructs with the pURA3 promoter, moderate gene expression was evident when using raffinose and galactose together, while weak expression was observed when glucose was the sole carbon source.

References:

Umezu, K., Amaya, T., Yoshimoto, A., & Tomita, K. (1971). Purification and properties of orotidine-5’-phosphate pyrophosphorylase and orotidine-5’-phosphate decarboxylase from baker’s yeast. Journal of Biochemistry, 70(2), 249–262. https://doi.org/10.1093/oxfordjournals.jbchem.a129637


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]