Difference between revisions of "Part:BBa J24813"

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.

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:

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.

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