Part:BBa_I766555

pCyc (Medium) Promoter

Mid-expression level constitutive promoter in yeast

Team Estonia_TUIT 2023 characterization of BBa_I766555 (pCYC1)

The pCYC1 promoter is a constitutive promoter responsible for the expression of CYC1, which codes for the iso-1 variation of cytochrome c. Cytochrome c serves as an electron carrier protein and it is an essential component of the mitochondrial respiratory chain. Cyc1, the protein encoded by CYC1, has a heme group, which aids in transferring electrons from respiratory complex III to respiratory complex IV. Additionally, it facilitates the final electron transfer to oxygen, a process catalyzed by complex IV, also known as cytochrome c oxidase. Cyc1 is localized in the inner mitochondrial membrane facing trans-membrane space. Iso-1 variation of cytochrome c constitutes 95% of the total cytochrome c content in cells that are growing aerobically (Sherman, 2005; Sherman et al., 1966). pCYC1 is also widely used in research as a weaker promoter compared to pADH1.

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
pCYC1>	sfGFP	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
I85	DOM90 trp1::pRS304-pCYC1-sfGFP-tCYC1	Strain with sfGFP under pCYC1 promoter, integrated into Trp1-1 locus

sfGFP fluorescence measurements Prior to fluorescence measurements, yeast cells were cultivated in complete synthetic media (CSM) with 2% glucose until the cultures reached an optical density (OD600) in the range of 0.6 to 1. Subsequently, 200 μl of the cell suspension was transferred into the designated wells on 96-well plates. To measure sfGFP 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 sfGFP 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-sfGFP strain or in DOM90 negative control strain. Error bars show standard deviation.

In our study, we examined three yeast promoters sourced from the iGEM part registry. We found that constitutive promoters like pADH1 and pURA3 are suitable choices for achieving consistent moderate gene expression, while the pCYC1 promoter is preferable when a weaker level of gene expression is needed. 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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]