Part:BBa_I766556

pAdh (Strong) Promoter

Strong Expression level Constitutive promoter in yeast

Team Estonia_TUIT 2023 characterization of BBa_I766556 (pADH1)

The promoter labeled as pADH1 regulates the expression of the ADH1 gene. The enzyme encoded by the ADH1 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). pADH1 promoter is widely used in yeast research and biotechnology to drive exogenous protein expression.

Plasmid formation

The promoters were 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 fragments containing the promoters were ligated into SacI/BamHI-restricted pRS304-based vector carrying sfGFP coding sequence and tCYC1 terminator.

Promoter	Reporter	Assembly methods
pADH1	EGFP	Restriction-ligation

Yeast strain construction

Prior to yeast transformation, the integration plasmids were restricted with HindIII to linearise the plasmids for homologous recombination into the yeast genome TRP1 locus. The restricted plasmids were 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 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 promoters pADH1 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. Compared to the background fluorescence of DOM90, yeast strain with EGFP under the control of pADH1 promoter displayed a 2-fold increase in sfGFP fluorescence intensity.

Bars indicate the mean fluorescence intensity (expressed in arbitrary units, AU) measured in pADH1-EGFP strain or in DOM90 negative control strain. Error bars show standard deviation.

In our study, we found that constitutive promoter like pADH1 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.

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