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 drives the expression of CYC1, which yields the iso-1 variant of cytochrome c. The protein, Cyc1, plays a pivotal role in the mitochondrial respiratory chain by aiding electron transport. With its heme group, it facilitates electron transfer across respiratory complexes, ending with oxygen. Cyc1 is predominantly located in the inner mitochondrial membrane. In oxygen-rich cell environments, the iso-1 type accounts for 95% of total cytochrome c (Sherman, 2005; Sherman et al., 1966). pCYC1 is 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 EGFP coding sequence and tCYC1 terminator.

Promoter	Reporter	Assembly methods
pCYC1	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 the 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

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 pCYC1 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 under the control of pCYC1 displayed a 1.16-fold increase in EGFP fluorescence when using glucose as a carbon source. The pCYC1-driven EGFP fluorescence levels were indistinguishable from the DOM90 background fluorescence in other carbon sources.

Bars indicate the mean fluorescence intensity in arbitrary units (AU) measured in the pCYC1-EGFP strain or in DOM90 negative control strain measured in a plate reader. Error bars show standard deviation.

We evaluated the expression levels in the presence of different carbon sources. Our results show that pCYC1 promoter should be used when a very weak gene expression is desired.

References:

Sherman, F. (2005). The importance of mutation, then and now: studies with yeast cytochrome c. Mutation Research, 589(1), 1–16. https://doi.org/10.1016/j.mrrev.2004.07.001

Sherman, F., Stewart, J. W., Margoliash, E., Parker, J., & Campbell, W. (1966). The structural gene for yeast cytochrome C. Proceedings of the National Academy of Sciences of the United States of America, 55(6), 1498–1504. https://doi.org/10.1073/pnas.55.6.1498

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]