Part:BBa_I766556

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
pADH1	EGFP	Restriction-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
DOM90	MATa {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
I84	DOM90 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