Question

What would be the composition of the canavanine selection plates (see Lab 9) if we did not chose to select for the presence of the gRNA plasmid?
Plate counting Last week you transformed the PMD2 plasmid that contains the ORNA sequence. The yeast cells were selected on a YNB + histidine + methionine plate in order to make sure that only the cells that contain the plasmid grow and give a colony. Simply count the number of colonies that grew and record that number. Take also a photo of your plate and insert it in your online lab notebook Selection of the CRISPR-induced yeast cells Yesterday, Charles has inoculated one colony from your plate into a culture medium that contains galactose. This colony contains two plasmids: • PMD1 that carries the Cas9 gene under a galactose promoter PMD2 that carries the RNA sequence that targets the CAN1 gene in the yeast The presence of galactose in the overnight culture induces the translation of the Cas9 protein directly from the PMD1 plasmid because it is controlled by the galactose promoter. The gRNA being under constitutive primer called SNR52 it is always transcribed within the cell (i.e. it does not actually need to be induced because it is always ON). Therefore since yesterday, the yeast cells in your liquid culture has started editing their genome in the CAN1 gene using both the Cas9 and the GRNA present in the cell: CRISPR editing is underway! You will now select the yeast cells whose genomes have successfully been CRISPR-edited. Because our ORNA targets the CAN1 yeast gene (coding for a permease), we should hopefully get a few cells that have a mutation in that gene (insertion, deletion, substitution). Some mutations might lead to a non-functional CAN1 gene (premature STOP codon, frameshift) which would modify the amino acid sequence and the protein 3D structure. This is what we are hoping for because this permease lets the antibiotic canavanine enter and kill the yeast cell. A successful CRISPR edit would lead to yeast cells that are canavanine resistance and that can grow on a selective petri dish that contains canavanine.

Answer 1

I guess you are talking about selection of yeast cells containing pMD2 plasmid with gRNA sequence.

Canavanine selection plate is for selection of mutated yeast cells (after CRISPR editing), not for selection of yeast cells containing pMD2 plasmid with gRNA sequence.

In the worksheet, such yeast cells were selected on a YNB + Histidine + Methionine plate.

'Yeast Nitrogen Base (YNB) without amino acids' lacks the amino acids histidine, methionine and tryptophane. So YNB mentioned here must be without amino acids  Because two amino acids (Histidine + Methionine) were supplemented seperately for selection of yeast cells.

YNB w/o amino acids is used to select auxotrophic strains of yeasts that require certain supplements. (Auxotrophy is the inability of an organism to synthesize a particular organic compound required for its growth)

Wild Type yeast cells may require all above three amino acids to grow. Yeast cells containing pMD2 plasmid with gRNA sequence may be carrying gene for tryptophan synthesis i.e. yeast cells carrying pMD2 plasmid with gene for both tryptophan (selectable marker) and gRNA (gene of interest). (Note: Here I am assuming that your yeast strain require all three amino acids to grow)

If you donot wish to select for the presence of gRNA plasmid, your plate composition could be YNB + Histidine + Methionine + Tryptophan. Since presence of Tryptophan will grow both gRNA plasmid containing and lacking yeast cells. So there would be no selection.

To answer your questoion, its all depend upon your yeast strain, selectable marker and the method of selecting your cells.

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