Difference between revisions of "Part:BBa K5047036"

Latest revision as of 11:34, 2 October 2024

Sequence encodes for constitutive promoter pTEF1 from S. cerevisiae.

pTEF1 is a constitutive promoter for translation elongation factor 1alpha in Saccharomyces cerevisiae (Mumberg D et al Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds Gene vol 1561 1995 119-22 doi:10.1016/0378-1119(95)00037-7). This promoter is known for its robust and stable expression making it a preferred choice for applications that require consistent gene expression in yeast. To evaluate the strength of the pTEF1 promoter in comparison to other commonly used promoters we constructed a series of plasmids encoding the red fluorescent protein mCherry under the control of different promoters pTEF1 (BBa_K5047036) pADH1 (BBa_J63005) and pCYC1 (BBa_K12400). These plasmids were used to determine relative expression levels in yeast.

Plasmid pDA95 Origins of replication in E coli AmpR URA3 pTEF1 mCherry

Plasmid pDA120 Origins of replication in E coli AmpR URA3 pCYC1 mCherry

Plasmid pDA122 Origins of replication E coli AmpR URA3 pADH1 mCherry

Experimental Design

The plasmids were linearized using the restriction enzyme BstBI for targeted integration into the URA3 locus of the Saccharomyces cerevisiae strain W303 This method ensures that each colony would have a single defined integration site (Wosika Victoria et al New families of single integration vectors and gene tagging plasmids for genetic manipulations in budding yeast Molecular genetics and genomics MGG vol 2916 2016 2231-2240 doi101007s00438-016-1249-1) enabling direct comparison of promoter activity.

Following transformation the yeast cultures were incubated for 2 days at 30C After sufficient growth 12 colonies were selected from each transformation plate for further analysis. These colonies were cultured in SD full medium a non-fluorescent medium to avoid background fluorescence from affecting measurements. The fluorescence intensity of mCherry expression in the samples was measured using a plate reader. Fluorescence was normalised by the optical density (OD) of each sample to account for differences in cell density. This normalisation enabled accurate comparison of the expression levels driven by the three different promoters.

Fluorescence Measurement and Flow Cytometry Analysis

The samples with sufficient levels of fluorescence expression were further analysed using flow cytometry which allows for a detailed quantification of fluorescence intensity at the single-cell level.

Results

The data obtained from these experiments are consistent with each other and demonstrate that pTEF1 drives significantly higher expression of mCherry compared to both pADH1 and pCYC1. This confirms that pTEF1 is a stronger promoter making it ideal for experiments where high and constitutive expression of a target gene is required.

Conclusion

The pTEF1 promoter shows higher performance in driving gene expression in Saccharomyces cerevisiae when compared to pADH1 and pCYC1 This makes pTEF1 a valuable tool for experiments requiring strong and consistent gene expression in yeast.

Sequence and Features