Latest revision as of 22:19, 11 October 2023

pprm1 Pro

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 × PREs which is in the opposite direction of the promoter. pprm1 Pro is a modified pheromone-responsive promoter. It contains 3 × PREs in the same direction of the promoter.

Characterization

When assembled with promotor BBa_K3384314, BBa_K3112009, and 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 pheromone concentration has no significant effect on the GFP expression intensity under the control of pprm1 Pro, indicating that this engineered pprm1 remains a stable expression level when induced by pheromone. With a low and stable induced expression level, this part assembled with reporter genes, for example, chromoproteins, can be used in co-expression scenarios with some colored products, and its weak expression intensity will not affect the observation of its colored products.

Nanjing-BioXstem

Varistor

The fluorescence expression intensity of BY4741 pRS415-prm1 Pro-GFP-CYC1 BBa_K3384313 under 0uM-1.5uM α-pheromone treatment was characterized using flow cytometry. The data were analyzed as follows: under 0uM-1.5uM α-pheromone treatment, the fluorescence intensity increases as α-pheromone concentration increases, and they are linearly related. It can be designed as a varistor.

Figure 1. Characterization of the fluorescence intensity of BY4741 pRS415-prm1 Pro-GFP-CYC1 (BBa_K3384313) by using flowjo software.

Figure 2. Characterization of the fluorescence intensity of BY4741 pRS415-prm1 Pro-GFP-CYC1 (BBa_K3384313) under 0μM-1.5μM α-pheromone treatment by using prism software.

information learned from literatures

Sengupta and colleagues have demonstrated that while PRE is active and effective in both directions, its activity is significantly stronger when oriented in the same direction as the promoter, as opposed to the opposite direction. This is consistent with the experimental results of Hagen and others. When three copies of the PRE sequence from pfus1 are inverted, meaning there are more copies in the opposite direction to the promoter. Although the synthetic promoter still exhibits pheromone-inducible activity, but at a lower level, approximately 2-3 times lower.

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