Team:Tianjin/Contribution

Introduction

We added some information of basic parts to the library. Below we will make a detailed introduction to our contribution.

Characterization of TetR

In our project, we want to use TetR operon to control the expression of mcherry gene. Thus, we want to test whether TetR operon can strictly regulate the expression of gene or not.
We constructed a carotene plasmid with TetR operon, and transformed it into Saccharomyces cerevisiae. If the carotene gene controlled by TetR operon leaks, the yeasts will turn orange. then cultured the yeasts without Atc inducer. By observing the color of the colony, the leakage of tetR promoter can be roughly determined.

Result

We cultured the yeasts after transforming the carotene plasmid. We were surprised to find that, before using Atc inducer, a large proportion of yeasts was orange. Our experimental results show that the tetR promoter is very easy to leak, and the expression amount of the circuit is different and difficult to control. Therefore, the other iGEMers should aware that TetR operon is not a good choice if you want to strictly control the expression of structure gene.

Fig.1 carotene plasmid with tetR operon

Fig 2. Saccharomyces cerevisiae with carotene plasmids shows that TetR operon is easy to leak

Characterization of TDH3

GFP is an important reporter gene used in our project. We integrated GFP gene into yeast’s chromosomes, and used it to represent the degradation of chromosomes. In order to verify whether the TDH3 promoter can express the GFP signal intensity required in experiment, we characterized the TDH3 promoter’s efficiency. We constructed a GFP fragment that was controlled by the TDH3 promoter and bound it to the chromosome genome of 4742 yeast. We measured the GFP expression levels of yeast at 15h, 20h, 25h and 30h using microplate analyzer. Meanwhile, we used 4742 yeast (no GFP fragment) as control group.

Fig 1. Gene part using GFP as reporter gene to characterize TDH3 promoter strength

Result

We use GFP as reporter gene to test the intensity of TDH3 promoter, and draw a bar chart with fluorescence intensity /OD600nm as the ordinate and time as the abscissa. The excitation/emission wavelength of GFP in the microplate detector was set as 488/535nm.

Fig2. Characterization of TDH3 promoter’s strength

Characterization of ADH1

SY14 is a special kind of Artificially modified yeasts which contains only one chromosome fused by 16 chromosomes. SY14 yeast differs from normal yeast in many properties: growth rate, metabolic activity etc. We use GFP as reporter gene to test the ADH1 promoter’s strength in SY14. We constructed a GFP fragment that was controlled by the ADH1 promoter and bound it to the chromosome genome of SY14 yeast. We measured the GFP expression levels of SY14 yeast at 15h, 20h,25h and 30h by using microplate analyzer. Meanwhile, we used SY14 yeast (no GFP fragment) as control group.

Result

We use GFP as reporter gene to test the intensity of ADH1 promoter, and draw a bar chart with fluorescence intensity /OD600nm as the ordinate and time as the abscissa. The excitation/emission wavelength of GFP in the microplate detector was set as 488/535nm. The strength of ADH1 promoter reaches its peak at 15h.

Fig1. Characterization of ADH1 promoter’s strength