Difference between revisions of "Part:BBa K1346004"

Revision as of 06:29, 27 October 2020

pPRM1

Promoter endogenous to s. cerevisiae that is induced by alpha factor. Characterized on flow cytometer by measuring its mean GFP fluorescence over 8 concentrations of alpha factor.

Compared to the other alpha factor responsive promoters, pPRM1 has a low basal expression level, a broad range of expression, and the third highest maximum output, less than pAGA1 and pPCL2.

Alpha factor responsive promoters were selected from the following paper: Roberts, Christopher J., et al (2000 Feb 4). Signaling and Circuitry of Multiple MAPK Pathways Revealed by a Matrix of Global Gene Expression Profiles. Science, 287(5454):873-80. http://www.ncbi.nlm.nih.gov/pubmed/10657304.

iGEM2020_NJTech_China Improvement:

pprm1 Ultra BBa_K3384314

An improved part has been constructed. Based on the characterization results, we found that although pprm1 possesses three PREs, it has a low induced expression level which indicates a good potential for transformation. And the background expression level of this promoter is low which means it can be used to express some proteins that require lower concentrations in specific stage. Based on the literature, we learned the general effect of PREs on promoter strength. For example, an increase in the copy number of PREs can enhance the induced expression level of the promoter. We doubled the 3 × PREs sequence in natural pprm1 to futher evaluate the influence of copy number of PREs on the activity of promoters. The modified pprm1 is referred to as pprm1 Ultra which contains 6 × PREs.

When assembled with GFP BBa_K3112009, the activity of pprm1 can be reflected through the intensity of GFP. As is shown in figure1, we quantitatively characterized the activity of pprm1 at different pheromone concentrations.

When assembled with pprm1 Ultra promotor BBa_K3384314, CYC1 terminator 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 2, 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 factory.

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.

Sequence and Features