Difference between revisions of "Part:BBa I766556"

(Yeast strain construction)
(Yeast strain construction)
 
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The promoter labeled as <i>pADH1</i> regulates the expression of the <i>ADH1</i> gene. The enzyme encoded by the <i>ADH1</i> gene is called alcohol dehydrogenase and it is crucial in converting acetaldehyde into ethanol in fermentation. Furthermore, Adh1 has alternative functions, as it possesses methylglyoxal reductase activity, it is involved in oxidation of NADH, and in the synthesis of fusel alcohol through the breakdown of amino acids (Bennetzen & Hall, 1982). <i>pADH1</i> promoter is widely used in yeast research and biotechnology to drive exogenous protein expression.
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The <i>pADH1</i> promoter governs the <i>ADH1</i> gene's function. This gene encodes for the enzyme alcohol dehydrogenase, essential for converting acetaldehyde to ethanol in fermentation. Besides, Adh1 has additional functions such as methylglyoxal reduction, assisting in NADH oxidation, and producing fusel alcohol by decomposing amino acids (Bennetzen & Hall, 1982). Researchers and biotechnologists frequently use the <i>pADH1</i> promoter in yeast to promote the expression of foreign proteins.
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===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.  
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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 their 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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===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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===EGFP fluorescence measurements===
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.
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 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.
 
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===
 
===Results===
  
In this study, we assessed the level of gene expression driven by the promoters <i>pADH1</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.
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In this study, we assessed the level of gene expression driven by the <i>pADH1</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 strain with sfGFP under the control of <i>pADH1</i> promoter  displayed a 2-fold increase in sfGFP fluorescence intensity.
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Compared to the background fluorescence of DOM90, yeast strains with EGFP under the control of <i>pADH1</i> displayed a 4.42-fold increase in EGFP fluorescence when grown with glucose, a 2.62-fold increase with both raffinose and galactose, and  a 1.3-fold with raffinose or glycerol. The results from <i>pADH1</i>-induced EGFP expression in glycerol are in compliance with prior findings which noted the suppression of <i>ADH1</i> transcription during non-fermentative sugar consumption (Denis <i>et al</i>., 1983).
  
  
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<img alt=""
 
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style="width: 800px"
 
style="width: 800px"
src="https://static.igem.wiki/teams/4917/wiki/contribution/adh1.png">
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src="https://static.igem.wiki/teams/4917/wiki/contribution/padh1.png">
<figcaption>Bars indicate the mean fluorescence intensity (expressed in arbitrary units, AU) measured in pADH1-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 the <i> pADH1-EGFP</i> strain 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 found that constitutive promoter like <i>pADH1</i> is a 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. For the constructs harboring the <i>pADH1</i> promoter, strong gene expression was observed when glucose was used as the carbon source, moderate expression was detected in the presence of both raffinose and galactose, and weak expression was noted with either raffinose or glycerol alone.
 
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===References:===
 
===References:===

Latest revision as of 03:24, 11 October 2023


pAdh (Strong) Promoter

Strong Expression level Constitutive promoter in yeast

Team Estonia_TUIT 2023 characterization of BBa_I766556 (pADH1)

The pADH1 promoter governs the ADH1 gene's function. This gene encodes for the enzyme alcohol dehydrogenase, essential for converting acetaldehyde to ethanol in fermentation. Besides, Adh1 has additional functions such as methylglyoxal reduction, assisting in NADH oxidation, and producing fusel alcohol by decomposing amino acids (Bennetzen & Hall, 1982). Researchers and biotechnologists frequently use the pADH1 promoter in yeast to promote the expression of foreign proteins.


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 their 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  
pADH1EGFPRestriction-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
I84DOM90 trp1::pRS304-pADH1-EGFP-tCYC1  Strain with EGFP under pADH1 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 pADH1 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 under the control of pADH1 displayed a 4.42-fold increase in EGFP fluorescence when grown with glucose, a 2.62-fold increase with both raffinose and galactose, and a 1.3-fold with raffinose or glycerol. The results from pADH1-induced EGFP expression in glycerol are in compliance with prior findings which noted the suppression of ADH1 transcription during non-fermentative sugar consumption (Denis et al., 1983).


Bars indicate the mean fluorescence intensity in arbitrary units (AU) measured in the pADH1-EGFP strain 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. For the constructs harboring the pADH1 promoter, strong gene expression was observed when glucose was used as the carbon source, moderate expression was detected in the presence of both raffinose and galactose, and weak expression was noted with either raffinose or glycerol alone.

References:

Bennetzen, J. L., & Hall, B. D. (1982). The primary structure of the Saccharomyces cerevisiae gene for alcohol dehydrogenase. The Journal of Biological Chemistry, 257(6), 3018–3025.


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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 224
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 874