Difference between revisions of "Part:BBa K3506010"

Latest revision as of 22:41, 27 October 2020

Clb2 N124aa

Of the four mitotic cyclins, Clb2 has the most important role in Saccharomyces cerevisiae. It alone is sufficient to trigger all essential functions of cyclin-dependent kinases in mitosis. Research shows that the first 124 amino acids of Clb2 can promote the degradation of it because it contains the D-Box and KEN-Box, which are the recognition site by E3 ubiquitin ligase APC (Anaphase-promoting complex)[1].

Biology and Usage

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 phases. In our project, it is used to degrade Cas9 at a proper speed, thus achieve the goal to switch off our system. This part can be fused to another protein to increase the degradation rate of it.

Properties

This part was fused to GFP at the N-terminal and transformed into Saccharomyces cerevisiae BY4741. 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 mintues by flow cytometry for 5 hours. After data collection, the protein degradation curve characterized by fluorescence intensity was processed and plotted. As shown in the figure below, compared with the control group (only GFP), the Clb2 N124aa and GFP fusion protein degrade much faster.

Figure 1. Fluorescence intensity by time

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. The control group is the pYES2 plasmid only with GFP.

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 30 mintues by flow cytometry for 5 hours.

References

[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.