Difference between revisions of "Part:BBa K3506011"

Revision as of 16:43, 27 October 2020

GAL1 promoter

It is the promoter of GAL1 gene, which codes galactokinase. For our project，we use it to express GFP fused with the first 124 amino acid of Clb2，then release the induction，if the fluorescence disappears，it can be proved that the first 124 amino acid of Clb2 have the ability to help proteins degrade.

Biology and Usage

It can be generally induced for gene expression by galactose and inhibited by glucose [1][2], making it a highly efficient inducible promoter for expression of foreign protein.

Sequence and Features

Expenrimental approach

1.Clb2 N124aa was amplified by PCR from the Saccharomyces cerevisiae genome using specific primers.

2.The pYES2 plasmid (containing GAL1 promoter), Clb2 N124aa, and GFP fragment were connected by in-fusion cloning as experimental group.

3.Both experimental group and control group were transformed into E. coli DH5α.

4.The transformed E. coli were grown in 20 mL of LB-amphenicol (100 mg/ml) in an incubator at 37°C, 180 rpm for 12 hours.

5.1 ml of bacterial solution was inoculated into LB-amphenicillin (100 mg/ml) medium and incubated at 37°C for 12 hours in the incubator.

6.Screen for single colonies in LB-amphenicol (100 mg/l) medium and perform colony PCR to verify the successful transformation.

7.Select the single colony successfully transformed and incubate overnight in a shaker.

8.Extract the plasmids from E.coli and linearize by single enzyme.

9.Transform the plasmids into competent yeast cell and grow it in SC-ura(glucose) medium at a constant temperature of 30°C for 2 days.

10.Screen single colonies by colony PCR to verify successful transformation.

11.Selected colonies were inoculated into the SC-ura(glucose) medium and grown at 30°C for 14-16h in the incubator.

12.Transfer the yeast cell to the SC-ura(galactose) medium.

13.After 10 hours, transfer the yeast cell to the SC-ura(glucose) medium. Then measure the fluorescence intensity every one and a half hours by flow cytometry for 5 hours.

Reference

[1] Johnston M. (1987). A model fungal gene regulatory mechanism: the GAL genes of Saccharomyces cerevisiae. Microbiological reviews, 51(4), 458–476. [2] Weinhandl, K., Winkler, M., Glieder, A., & Camattari, A. (2014). Carbon source dependent promoters in yeasts. Microbial cell factories, 13, 5.