Difference between revisions of "Part:BBa K5058000"

Latest revision as of 01:33, 21 September 2024

ADH2 promoter (Saccharomyces cerevisiae)

The ADH2 promoter is a well-characterized and widely used regulatory element in Saccharomyces cerevisiae that controls the expression of the alcohol dehydrogenase 2 gene. Its activity is tightly regulated by glucose levels, exhibiting repression during high glucose conditions and strong induction during glucose depletion, making it a key component in metabolic and synthetic biology applications. Due to its glucose-repressible nature, the ADH2 promoter is frequently employed in metabolic engineering strategies to drive gene expression in the later stages of fermentation, allowing for a temporal control.

• LacZ test

The lacZ gene, which encodes the enzyme β-galactosidase, is extensively used as a reporter in synthetic biology for evaluating promoter strength and gene expression across various organisms, including S. cerevisiae. When fused to a promoter of interest, lacZ enables quantitative assessment of promoter activity through straightforward enzymatic assays. β-galactosidase catalyzes the hydrolysis of substrates such as ortho-Nitrophenyl-β-galactoside (ONPG), resulting in a quantifiable yellow product. So we used the lacZ assay technique and builded a construct of the ADH2 promoter and the LacZ gene (BBa_K5058009)

An initial inoculum of BY4741 cells transformed with pESC-URA-ADH2p-LACZ was cultured for 16 hours in YNB medium containing 2% glucose, supplemented with leucine, methionine, and histidine. Following this, a secondary inoculum was prepared in YPD medium with 2% glucose, using the previous culture at an OD of 0.2 (600 nm). Samples were collected for both β-galactosidase activity and glucose level assays. β-galactosidase activity was measured using a liquid ONPG assay, and the resulting values were normalized to protein concentration determined by the Bradford assay.

Specific promoter activity calculation:

Measure the absorbance (OD420) of the solution containing o-nitrophenol at 420 nm after the reaction.

Calculate the concentration of o-nitrophenol formed during the assay using the conversion factor: o-nitrophenol concentration (nmol/mL) = OD420/0.0045

Multiply the amount of o-nitrophenol by the total volume of the sample (in mL) to obtain the total nmol produced: Total nmol = (OD420/0.0045) × Volume of extract (mL)

Divide the total nmol by the reaction time (minutes) to calculate the enzymatic activity: Enzymatic activity (nmol/min)=Total nmol / Time (min)

Calculate the specific activity by dividing the enzymatic activity by the protein concentration (mg/mL): Specific activity (nmol/min/mg) = Enzymatic activity (nmol/min) / Protein concentration (mg/mL)

This formula provides the specific activity of β-galactosidase, expressed as nmol of o-nitrophenol formed per minute per mg of protein.

The results indicate that as glucose levels decrease, ADH2p activity increases, which is consistent with our initial expectations, with peak activity observed at the 24-hour mark. The β-galactosidase activity detected at 7 hours may be attributed to residual lacZ expression from the initial inoculum or a minor leakage in promoter regulation.

Sequence and Features