Part:BBa_K3852001

Modified Gal promoter

Introduction

By looking through the literature, we found that this promoter can be induced by estradiol to promote the expression of subsequent genes.

Experiment

Initially, when we build a receptor expression system, the promoters we use are Pfba1, but if the plasmids that express the umami receptor, the plasmids that express the sweet receptor, and the plasmids that express the bitter receptor are all imported into the Saccharomyces cerevisiae at the same time, it may cause the growth difficulties of the Saccharomyces cerevisiae and aggravate it metabolic burden. After discussion and literature reading, we decided to add different induced promoters to the plasmids that express the taste receptor, so that when different inducers are added, the Saccharomyces cerevisiae can express different taste receptor proteins to detect different tastes. We selected three induced promoters: Modified Gal promoter, PADH7, and PCUP1. Modified Gal promoter is induced by estradiol, PADH7 is induced by balsamic, and PCUP1 is induced by copper ions. The experiments on Modified Gal promoter are described below.

Figure1. Genetic circuit design

Promoter strength determination

We design the following plasmids, import them into the Saccharomyces cerevisiae, and prepare to measure the fluorescence intensity with an enzyme meter to characterize the promoter strength with fluorescent intensity/OD600.

Figure2. Plasmid design diagram

The measured data are drawn as the figure below. According to the figure, in a certain range, increasing the dosage of estradiol can enhance the strength of Modified Gal promoter and facilitate the expression of subsequent genes.

Figure3. Promoter strength diagram

Reference

[1] McIsaac, R. Scott & Oakes, Benjamin & Wang, Xin & Dummit, Krysta & Botstein, David & Noyes, Marcus. (2012). Synthetic gene expression perturbation systems with rapid, tunable, single-gene specificity in yeast. Nucleic acids research. 41. 10.1093/nar/gks1313.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 300
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 215
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 300
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 300
  Illegal AgeI site found at 324
  
• 1000
  COMPATIBLE WITH RFC[1000]