Latest revision as of 22:17, 11 October 2023

pprm1 Ultra

Multiple copies of putative Ste12 binding sites are prevalent in pheromone-responsive promoters of Saccharomyces cerevisiae, which are also known as pheromone response elements (PREs). pprm1 contains 3 copies of PRE which is in the opposite direction of the promoter. pprm1 Ultra is a modified pheromone-responsive promoter. It contains 6 copies of PRE in the opposite direction of the promoter.

Characterization

When assembled with promotor BBa_K3384314, GFP BBa_K3112009, and CYC1 terminator BBa_K3384311 in pRS415, this construct expressed GFP. Then the activity of these promoters can be quantitatively measured through flow cytometer. As is shown in figure 1, the fluorescence intensity of the pprm1 is higher than that of the pprm1 Ultra under high concentration treatment conditions of pheromones. The pheromone concentration has no significant effect on the GFP expression intensity under the control of pprm1 Ultra, indicating that this engineered pprm1 remains a stable expression level when induced by pheromone. As pprm1 Ultra can provide a stable expression level regardless of the fluctuations in external conditions, it is expected to be applied to gene expression regulation in cell factories.

Nanjing-BioXstem

Switch

The fluorescence expression intensity of BY4741 pRS415-prm1 Ultra-GFP-CYC1 BBa_K3384314 was characterized using flow cytometry under of 0uM-5uM α-pheromone treatment. The data were analyzed as follows: under 0uM-5uM α-pheromone treatment, the fluorescence intensity is basically unchanged as α-pheromone concentration increases. It can be designed as a switch.

Figure 1. Characterization of the fluorescence intensity of BY4741 pRS415-prm1 Ultra-GFP-CYC1 (BBa_K3384314) by using flowjo software.

Figure 2. Characterization of the fluorescence intensity of BY4741 pRS415-prm1 Ultra-GFP-CYC1 (BBa_K3384314) under 0μM-1.5μM α-pheromone treatment by using prism software.

information learned from literatures

Sengupta and colleagues have discovered that an increase in the number of PRE copies enhances promoter activity in MATa cells. Furthermore, compared to single-copy PRE, PRE trimers strongly drive mating factor induction in cells. Meanwhile, Sprague’s group assembled a synthetic PRE oligonucleotide chain with different copy numbers upstream of the CYC1-lacZ vector. The results indicate that multiple copies of PRE confer a higher pheromone-inducible activity to the promoter in a non-additive, cooperative manner.

Reference

1. LSengupta, P., and Cochran, B. H. (1990) The Pre and Pq Box Are Functionally Distinct Yeast Pheromone Response Elements, Molecular and Cellular Biology 10, 6809-6812.
2:Hagen, D. C.; McCaffrey, G.; Sprague, G. F., Jr., Pheromone response elements are necessary and sufficient for basal and pheromone-induced transcription of the FUS1 gene of Saccharomyces cerevisiae. Mol Cell Biol 1991, 11 (6), 2952-61.
3:Liu, Y.; Huang, Y.; Lu, R.; Xin, F.; Liu, G., Synthetic biology applications of the yeast mating signal pathway. Trends Biotechnol 2022, 40 (5), 620-631.

Sequence and Features