Revision as of 13:21, 25 October 2017

Introduction

P_fus(2 Ste12 binding sites)

General

The original P_fus (BBa_K1154001) is 201bp long with three Ste12 binding sites, and the Ste12 is P_fus’s transcriptional activator in the endogenous GPCR pathways of yeast.

Fig.1 The sequence of original P_fus

In order to decrease the transcription initiation activity of the promoter, we used the method of one-step mutation to remove a binding sites. (from tgaaaca to ggacac)

Fig.2 Sequencing result of original P_fus and mutant P_fus

After the modified promoter assembling into the [http://2017.igem.org/Team:BIT-China/Project/#Detection detection circuit], we added 5umol/L α pheromone to detect it, and the result is shown in Fig.3.

Fig.3 The mRFP intensity of two kinds of promoters

The intensity of modified promoter was about 35% of the wild type, which did match with our expectation. We got the new Pfus with different transcription initiation activity. With different promoters, we can apply them in the regulation subsystem according to various situations or wide-ranging needs.

But from another point of view, still, we got the new P_fus with different transcription initiation activity. With different promoters, we can apply them in the regulation subsystem according to various situations or wide-ranging needs.

This part is an improvement of BBa_K1154001 of 2013 RHIT team.

[1] Su, T.-C., Tamarkina, E. and Sadowski, I. (2010), Organizational constraints on Ste12 cis-elements for a pheromone response in Saccharomyces cerevisiae. FEBS Journal, 277: 3235–3248. doi:10.1111/j.1742-4658.2010.07728.x

Sequence and Features