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Revision as of 22:23, 26 October 2020

GFP with degradation tag Clb2 N124aa

GFP coupled with Clb2 N124aa is composed of Clb2 N124aa(BBa_K3506010) and GFP(BBa_E0040) through Linker(BBa_K3506013). Clb2 N124aa is the first 124 amino acids of Clb2. In Saccharomyces cerevisiae BY4741, its function is to help the degradation of CLB2 during M and G1 phase. GFP is from 2019 Distribution kit. A linker, “GSGGSG”, was added between Clb2（N124aa）and GFP.

Sequence and Features

Usage and Biology

Green fluorescent protein (GFP) is used as a reporter to provide an easily-observed characterization. Clb2 N124aa is the first 124 amino acids of Clb2. In Saccharomyces cerevisiae BY4741, it promotes the degradation of CLB2 during M and G1 phase.

We fused Clb2 N124aa to GFP at the N-terminal, therefore the green fluorescence will degrade more quickly after expression. Hence, when GFP is used as a reporter to monitor the state of an inducible promoter, it will react more timely and accurately.

Properties

We constructed a plasmid containing GAL1 promoter, Clb2N124aa(BBa_K3506010) and GFP(BBa_E0040), using it as experimental group. After induced by galactose for 10 hours, Saccharomyces cerevisiae BY4741 were transferred to the SC-ura(glucose) medium. Then the fluorescence intensity was measured every 30 mins by flow cytometry for 5 hours. After data collection, the protein degradation curve characterized by fluorescence intensity was processed and plotted. As shown in figure below, compared with control group, the Clb2N124aa and GFP fusion protein degrades much faster.

Figure 1. The expression of GFP in Saccharomyces cerevisiae BY4741

Figure 2. Fuorenscence intensity by time

Experimental 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] Hendrickson, C., Meyn, M. A., 3rd, Morabito, L., & Holloway, S. L. (2001). The KEN box regulates Clb2 proteolysis in G1 and at the metaphase-to-anaphase transition. Current biology : CB, 11(22), 1781–1787.