Team:SCUT/Project/Other Work

Testing and Quantification of Promoter Activities

Overview

This is an additional part of our project, designed to testing and quantify promoter activities for having moderate expression level of target protein, or more detailedly saying, for knowing if TEF2 and TDH3 promoter work and for better control of induction of Gal1 promoter, the only inducible promoter used in our project. We employed GFPmut3b ( BBa_K294055 ), one of the most widely used fluorescence protein, as a reporter. After making a comparison between FCM and ELIASA, we chose the latter one for quantification of fluorescence since it was more convenient to manipulate and was accurate enough for comparing promoter activities. Considering that the fermentation process will span a period of time from several hours to several days, we did not have timed induction of Gal1 promoter included in quantification process.

Design and Result

With employing GFPmut3b (BBa_K294055) as a reporter protein, we designed three similar devices only differing in promoters as shown below.

Promoter Testing
Plasmids carrying the above-mentioned devices were transformed into competent S.cerevisiae cells. Transformed cells were plated on 2% agar in media A (yeast nitrogen base, MET, LYS, HIS, URA), complemented with 2% glucose and cultured at 30℃ for 48h. The colonies were then inoculated in the liquid media A complemented with 2% glucose and were grown at 30℃ overnight. For those contained TEF2 or TDH3 promoter, culture was stopped and samples were taken out for testing. For those contained Gal1 promoter, glucose was removed from media with washing cells by PBS till they reached the exponential phase. Cells were then pelleted and diluted and cultured on liquid medium containing galactose concentration of 2% w/v overnight. Samples were then taken out for testing. The result is shown below.

Figure 1 丨 The results show that all the promoters work.

Galactose Dose Response of Gal1 Promoter
Plasmid carrying the device containing Gal1 promoter was transformed into competent S.cerevisiae cells. Transformed cells were plated on 2% agar in media A (yeast nitrogen base, MET, LYS, HIS, URA), complemented with 2% glucose and cultured at 30℃ for 48h. The colonies were then inoculated in the liquid media A complemented with 2% glucose and were grown at 30℃. Culture was stopped and glucose was removed from media with washing cells by PBS till they reached the exponential phase. Cells were then pelleted and diluted and cultured on liquid medium containing galactose concentrations between 0.25% and 2% w/v overnight. Samples were taken out for quantification.

In quantification process, concentrations of liquid media containing cells were measured at λ600nm and then liquid media were removed and cells were washed twice and refolded to a OD600 = 1.0 concentration by Buffer ( 0.05 M NaH2PO4, 0.1 M NaCl, 0.5 M Imidazole ) for ELIASA analysis. Fluorescence intensity of the refolded cells were then measured by ELIASA (Tecan, Infinite M200 and i-Control)) with an excitation wavelength of 488 nm (the suggested one is 501 nm), and an emission filter of 520 nm (the suggested one is 511nm) to quantify fluorescence intensities. The result is shown below.

Figure 2 丨 Dose responsiveness of the Gal1 promoter to galactose induction

The graph above summarises the ELIASA data, and shows that the intensity of GFP expressing cells induced by low or high dose of galactose are higher then those induced by the middle doses. This is similar to the result given by iGEM 2010 team, Aberdeen_Scotland. We therefore plan to set more experiment groups between these doses to figure out what happened.