Revision as of 15:32, 25 October 2017

Introduction

P_fus(4 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 enhance the transcription initiation activity of the promoter, we add additional one binding site(gatgaaacaa) in the front of the promoter.

File:T BIT-China 2017part K2368026.22.png

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 1/3 of the wild type, which didn’t match with our expectation. According to Ting-Cheng Su’s[1] result, there were serious limitations in Ste12 multimers identifying Ste12 binding sites. So when we changed the structure of P_fus, the steric hindrance of Ste12 may increases and Ste12 couldn’t combine the promoter as smoothly as before.

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