Characterization of yeast promoters

Driven by the central importance of promoters in regulating gene expression, this year, we continue the characterization of yeast promoters. After careful examination of the iGEM part registry, we chose two constitutive promoters pREV1 and pPAB1, and one inducible promoter pFUS1 (Table 1). Constitutive pREV1 and pPAB1 promoters were characterized using Venus fluorescent protein as a reporter, while EGFP was used in the experiments with the inducible pFUS1. Integration vectors carrying the coding sequences of these fluorescent proteins under the control of target promoters were transformed into yeast cells. The resultant yeast strains were analyzed by quantitative fluorescence microscopy, and the fluorescence intensity of the reporter protein was used as a measure of promoter activity.

Table 1. Characterized promoters

pFUS1 promoter

pFUS1 controls the expression of the FUS1gene that encodes a membrane protein from the yeast mating pathway. Fus1 protein localizes to the tip of the shmoo (a protrusion formed during yeast mating) (Lim et al., 2014). FUS1 protein coordinates the signaling, cell fusion, and polarization events required for the fusion of yeast cells. Transcription of Fus1 is mediated in haploid yeast by four pheromone response elements (PRE) of the pFUS1 promoter (Hagen et al., 1991). To induce the pFUS1, we used the α-factor to activate the mating pathway in MATa yeast strain.

pREV1 promoter

pREV1 is a constitutive promoter responsible for the expression of REV1, a gene encoding bi-functional DNA-directed DNA polymerase/deoxycytidyl transferase (Lee et al., 2015). This enzyme is involved in error-prone translesion synthesis, one of the pathways for DNA repair (Lawrence, 2004).

pPAB1 promoter

Constitutive pPAB1 regulates the expression of an essential gene encoding for poly(A) binding protein, which is involved in the regulation of poly(A) tail length. Pab1 is localized to the nucleus and cytoplasm. It was shown that modulation of Pab1 levels could improve cell fitness under stress conditions (Martani et al., 2015).

Plasmid construction

To characterize the promoters of interest, we inserted them into plasmids where the fluorescent proteins were placed under the control of target promoters. Three different plasmids were constructed (Table 2). Vectors p182 and p183 with constitutive pPAB1 and pREV1, respectively, were created using the Golden Gate assembly procedure and type IIs restriction enzyme BsaI from MoClo yeast toolkit (Lee et al., 2015). The MoClo toolkit parts used for assemblies are listed in Table 2.

To create p166 plasmid, pFUS1 promoter was amplified from the yeast genome with primers containing NotI (forward primer) and SacI (reverse primer) restriction sites in their 5’-overhangs. After PCR and digestion, the DNA fragment containing the promoter was ligated into NotI/SacI-restricted vector carrying Venus coding sequence.

Table 2. Features of constructed plasmids

Yeast strain construction

Prior to yeast transformation, p182 and p183 integration plasmids were restricted with NotI, and p166 was cut with Apa1. Restricted plasmids were used to transform the S. cerevisiae DOM90 strain. Transformants were selected for URA+ phenotype on uracil-dropout complete synthetic media (CSM-URA) plates containing 2% glucose. All yeast strains generated and used for promoter characterization are listed in Table 3.

Table 3. Constructed yeast strains.

Time-lapse microscopy

Prior to time-lapse microscopy, the yeast cells were grown in complete synthetic media (CSM) or CSM-URA, depending on the needs of the experiment. The cultures were grown to OD600 0.2-1, the cells were pipetted onto a 0.08 mm cover glass slip and were covered with 1.5% agar-CSM with 2% glucose (in the case of pREV1 and pPAB1) or with 1.5% agar-CSM with 2% glucose and 1 μg/mL α-factor (for pFUS1 promoter induction).

Zeiss Observer Z1 microscope with 63C/1.4NA oil immersion objective and Axiocam 506 mono camera were used for imaging. The focus was maintained using Definite Focus, and the experiment was conducted at 30 °C using PeCon TempControl 37-2 digital. EGFP and Venus fluorescent proteins were excited by the 470 LED Colibri module at 25% intensity. The exposure time was set at 15 ms for 470 LED, and the filter set 61 HE (Zeiss) was used. Images (fluorescence and phase contrast) were taken every 3 minutes. Image segmentation and quantification of fluorescent signals was performed in Matlab as described in (Doncic et al., 2013).

Results

We quantified the expression driven by pREV1, pPAB1, and pFUS1 promoters using fluorescent proteins as reporter genes. The expression cassettes were integrated into the yeast genome, and the fluorescence of the reporter proteins was monitored by quantitative time-lapse microscopy. The fluorescence levels were compared to the cell background fluorescence of the control DOM90 strain, which does not express any fluorescent proteins.

pFUS1 promoter activity was induced with 1 μg/mL of α-factor. We observed gradual pheromone-dependent activation of the pFUS1 promoter (Fig. 1) throughout the experiment. When α-factor was not present (uninduced strain), the level of GFP fluorescence was similar to those observed in the control DOM090 strain (data not shown). Fus1 protein is associated with the tip of the shmoo involved in a mating process. Since the α-factor is not removed from the environment and mating did not happen, it leads to the constant activation of pFUS1 promoter and, as a result, an increase in GFP fluorescence throughout the experiment (Fig. 1).

Figure 1. pFUS1-EGFP fluorescence intensity in time during α-factor exposure . Plot shows the mean fluorescence levels of a population of cells from cultures without α-factor (uninduced) or with α-factor (induced).

Figure 2. The expression levels of Venus controlled by pPAB1 and pREV1. Bars indicate the mean fluorescence intensity (expressed in arbitrary units, AU) of pPAB1-Venus, pREV1-Venus, and DOM090 control from analyzed cell population. Error bars show standard deviation.

pPAB1 and pREV1 promoters showed constitutive levels of Venus fluorescence during the experiment, confirming that they are constitutive promoters. Compared to the background fluorescence of the DOM90 strain, pPAB1 showed a 19-fold higher expression level, and pREV1 demonstrated a five-fold higher level of fluorescence intensity, respectively (Fig. 2). Our data are in agreement with the data from Michael E. Lee’s article from 2015, where the pREV1 was characterized as the weakest promoter present in the kit, while the pPAB1was a promoter of medium strength (Lee et al., 2015).

Overall, we characterized three yeast promoters from the iGEM part registry. Analyzed constitutive promoters can be recommended when constant moderate (pPAB1 promoter) or weak (pREV1 promoter) expression of a target gene is required. Inducible pheromone-dependent pFUS1 can be used to ensure a gradual increase in gene expression throughout the experiment.