Team:EPF Lausanne/Notebook/Yeast
Express dCas9-VP64Integrate pTPGI_dCas9_VP64
We received plasmid pTPGI_dCas9_VP64 from Addgene. The plasmid was found from the article "Tunable and multifunctional eukaryotic transcription factors based on Crispr/Cas". After glycerol stocks and miniprep, we performed a restriction analysis to check the identity of our plasmid. We linearised the plasmid, in order to integrate it into yeast genome. For more details about our experiments, see here. Only our fourth trial to integrate the plasmid was successful.
Materials and methods
- Glycerol stocks
- Miniprep
- Restriction analysis
- Polymerase Chain Reaction
- Yeast integration A AJOUTER SUR PROTOCOLS
Results
We used four different sets of enzymes for the restriction analysis. Linearized pTPGI_dCas9_VP64 is expected to be 10'987 bp. We observe that the gel (fig.1) corresponds to the expected one. The plasmid was linearised both with EagI HF and NotI HF prior to integration. We integrated each linearised plasmid to obtain two different yeast strains.
Express dCas9-VP64Western Blot of dCas9-VP64
The Western Blot allows to check the expression of dCas9.
Materials and methods
- Western Blot A AJOUTER SUR PROTOCOLS
Results
Results of Western Blot.
Integrate reporter plasmidLinearise reporter plasmid
>We received the plasmid pCYC1m_yeGFP from Addgene. The plasmid was found from the article "Tunable and multifunctional eukaryotic transcription factors based on Crispr/Cas". After glycerol stocks and minipreps. We linearised the plasmid by PCR according to the following protocol.
Materials and methods
- Polymerase Chain Reaction
Results
Linearized pCYC1m_yeGFP is expected to be 5'485 bp. Running an agarose gel electrophoresis allowed to verify that we had linearised the plasmid.
Integrate reporter plasmidSynthesize promoters
From the article "Tunable and multifunctional eukaryotic transcription factors based on Crispr/Cas", we learnt that different regions on the promoters could lead to activation or inhibition when dCas9-VP64 was bound. We chose to modify the region of strongest activation, named c3, and the two regions that lead to the strongest inhibition, c6 and c7. We synthesized promoters CYC_0, CYC_1, CYC_2 and CYC_3. The only differences between them are the three regions c3, c6 and c7. The promoter CYC_0 is the original promoter, already present in the plasmid pCYC1m_yeGFP.
Materials and methods
- Synthesis ??
Results
Four different promoters according to fig...
Integrate reporter plasmidAdd Gibson overlaps by PCR
We amplified the promoters CYC_0, 1, 2, 3 by PCR adding the Gibson overlaps in order to assemble each fragment in the linearised plasmid pCYC1m_yeGFP.The PCR was performed according to the following protocol.
Materials and methods
- Polymerase Chain Reaction
Results
On the gel electrophoresis that we ran after PCR, we observe the three CYC fragments at the right height. These fragments are then used for the Gibson assembly in the plasmid pCYC_yeGFP.
Integrate and express gRNAsPCR-amplify the gRNA expressing cassettes
The gRNA expressing cassettes c3_0, c3_1, c3_2, c3_3 (activating sequences), c6_0, c6_1, c6_2, c6_3, (inhibiting sequences), c7_0, c7_1, c7_2, c7_3 (inhibiting sequences) were ordered together with the promoters CYC_0, CYC_1, CYC_2, CYC_3 promoters. The c3 gRNA expressing cassettes were synthesized along with their corresponding CYC promoter as individual G-Blocks, all other gRNAs were synthesized as individual G-Blocks. Four primers were used in order to PCR out or amplify the gRNAs and the promoters: f_IDT_tri, f_IDT_squ, r_IDT_dia and r_IDT_cir. For detailed reaction mixes, click here LIEN A INSERER
Materials and methods
- Polymerase Chain Reaction
Results
Each amplicon (gRNA expressing cassette or CYC promoter) is 250 bp long (the exact length can vary by max two bp depending on the primers used). Several PCRs were required for amplification, we only show the gels corresponding to the PCR products we kept. Fig...: c6_0, c6_1, c6_2, c6_3, CYC_0, CYC_1, CYC_2 Fig...: c7_0, c7_1, c7_2 Fig...: c7_3 Fig...: CYC_3, c3_1, c3_2, c3_3 Fig...: c3_0
Integrate and express gRNAsPCR out DsRed2
In order to verify the expression of the gRNAs cassettes, we used fluorescent protein DsRed2 as a reporter gene. It was PCRed out from the plasmid CMVp-dsRed2-Triplex-HHRibo-gRNA1-HDVRibo-pA. A synthetic polyA tail followed by a part of the Hammerhead ribozyme sequence was added on the reverse primer.
Materials and methods
- Miniprep
- Polymerase Chain Reaction
Results
The size of the DsRed2_polyA_HH1 amplicon is 750 bp. The plasmid on the gel is CMVp-dsRed2-Triplex-HHRibo-gRNA1-HDVRibo-pA.
