Team:BNU-China/Experiments
Build a periodic expression module of Cas9
To verify that our design enables Cas9 to be expressed periodically, we built a module for the periodic expression of Cas9, and the experiments were carried out as follows.
1. Construct the recombinant plasmid pRH003-URA3-CLB2 promoter-Cas9 by in-fusion cloning. Then transform it into E. coli DH5α. Extract plasmids from transformants. The recombinant plasmids were confirmed by colony PCR and sequencing, then transformed into S. cerevisiae BY4741. Screen for transformants by SD-ura culture medium.
2.Synchronize the cell cycle of S. cerevisiae population to G1 phase.
3.Release yeast cells from synchronization. Remove a time-zero fraction. Collect fractions of culture every 10 min for 150 min. Extract total protein for Western Blot. Strain without being transformed was used as a negative control. GAPDH was chosen as an internal reference.
4.Obtain and analyze data. Draw the image of Cas9 protein levels over time.
Degradation of Cas9 at a proper rate
In order to verify the function of Clb2 (N124aa) as a degradation tag, we constructed the plasmids for an experimental and a control group. We hoped to characterize the protein degradation by determining the fluorescence intensity curve.
1.The two plasmids were constructed with pYES2, containing GAL1 galactose inducible promoter and URA3 marker gene. The vector in the experimental group contains CLB2 (N124aa)- Linker-GFP, while the control group only contains GFP. The successfully constructed vectors were transformed into E.coli DH5α strain.
2.After the plasmids were extracted and linearized, they were transformed into Saccharomyces cerevisiae BY4741. We used the fluorescence microscope and differential interference microscope to verify the transformants.
3.Both experimental and control groups were cultured in SC medium at 30 ℃ until OD600 value reached about 3.000, and then the expression was induced by galactose with a final concentration of 2%. After induction, the fluorescence intensity was measured by flow cytometry at half an hour interval.
4.Cells were then inoculated into the medium containing glucose instead of galactose. In addition to being a carbon source, glucose also inhibits the expression of GFP by inhibiting the GAL1 promoter. Within 7 hours after inoculation, the fluorescence intensity was measured by flow cytometry at the same interval of half an hour.
5.We used SPSS26.0 to process the data and determined the relationship between fluorescence intensity and time, as well as the protein concentration and time. Finally, we calculated the degradation rate of protein modified by Clb2 N124aa through modeling.
Obtaining the lineage information out of the transcriptomic information
In order to read the lineage information out of the transcriptomic information, we design two double promoter modules. And we conducted the experiment to test the function of the cassette. To make results visible, we use gRNA that targets ADE2 gene. Cryptococcus neoformans will turn red if gRNA works. The experiments were carried as follows.
1.Construct recombinant plasmids. Get pGAP from the genome of Cryptococcus neoformans. Insert it upstream of pU6 on PRH003 plasmid. Use the plasmid without pGAP as the control group.
2.Transform the product (2.5μL) into DH5α competent cells (50μL), grow cells on agar plates (containing Ampicillin). Incubate plates at 37°C overnight. Colonies were screened by colony PCR and then grown at 37℃, 200rpm. Plasmids were extracted and sent for sequencing.
3.Use Kpn1 to linearize the plasmids and transform them into Cryptococcus neoformans by electroporation.
4.The C. neoformans was spread on YNBA selection medium, and transformants grew after being cultured in an incubator kept at 30℃ for 4 days. Then the culture was transferred to a refrigerator at 4℃.
5.Red colonies were selected and inoculated into YPD medium, then placed in an incubator kept at 30℃ for 4 days. Finally, it was kept in at 4℃ refrigerator.
6.For both the experimental and the control groups, we first extracted the total mRNA of these red colonies by Trizol. Then the mRNA was reverse transcribed using oligo dT as the primer.
7.To test whether gRNA can be transcribed, we performed PCR on the reverse transcription products by two specific primers. Then sequence the PCR product.
We exchanged pGAL7 with pGAP and repeated steps 1-5. Then we selected red colonies and induced the expression by galactose. After expression for 30mins. We repeated steps 6-7.
Postcode:100875
Email：igem2020bnu_china@163.com
© 2020. All Right Reserved. Designed by BNU-China