Part:BBa_K2368026

Introduction

P_fus(4 Ste12 binding sites)

General

The original Pfus (BBa_K1154001) is 201bp long with three STE12 binding sites, and the STE12 is Pfus’s transcriptional activator in the endogenous GPCR pathways of yeast.

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fig.1 The sequence of original Pfus

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

And we add 5umol/L α pheromone to detect the modified promoter, and the result is shown in fig.2.

fig.2 Sequencing result of original Pfus and mutant Pfus

After the modified promoter assembling into the detection circuit(加个链接), we added 5umol/L α pheromone to detect it, and the result is shown in fig.3.

fig.3 The intensity of two kinds of promoters

The intensity of modified promoter is about 1/3 of the wild type, which doesn’t match with our expectation. According to Ting-Cheng Su’s[1] result, there are serious limitations in STE12 multimers identifying STE12 binding sites. So when we change the structure of Pfus, the steric hindrance of STE12 may increases and STE12 can't combine the promoter as smoothly as before.

But from another point of view, still, we get the new Pfus with different transcription initiation activity.

Reference

[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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]