Question

2. A homolog of your favorite gene, YFG1, has been identified in S. cerevisiae. You were tasked with: i. generating a null allele of YFG1 in a diploid strain with the genotype: his3/his3, TRP1/trp1, ura3/URA3, YFG/YFG using a one-step gene replacement. [ So after the gene replacement genotype will be his3/his3, TRP1/trp1, ura3/URA3, yfg:marker/YFG] i. Sporulating the knockout strain generated in (i) iii. dissecting the resulting tetrads (asci) iv. testing the resulting spores on a variety of growth media The growth pattern of spores from 6 dissected asci (tetrads) are shown in the figure below. Yeast colonies arising from spores that germinated are illustrated as white spots on a black background. Answer the questions that follow rich medium tetrad: 1 2 3 4 5 6 1 8 3 4 replica plated to defined medium lacking -his -ura 4 (a) Outline in detail (point form is fine) the one step gene replacement method used to generate your null allele in the knockout strain. Be sure to mention all the tools you would have used and their specific properties (as if you were having to do this). Feel free to use well labeled neatly drawn figures to assist you in your response

Answer 1

Null allele generation is possible by one step gene replacement method, by replacing a functional gene with a dominant selectable marker, involving homologous recombination. This involves the standard cloning techniques.

1. The plasmid will be cut with the restriction enzymes to generate a linear DNA that contains the selectable marker and several hundred DNA base pairs on each side of the selectable marker.

2. After the transformation into the yeast, the homologous DNA, flanking the selectable marker induces two recombination events which replace the gene copy of the chromosome with the inactive copy.

3. After the transformation, the dominant marker allows the selection.

Now a days, PCR based methods simplify one step gene disruption because no cloning is needed. In this method,

1. The primers will be designed immediately upstream and downstream of the gene sequence, needed to be deleted. To delete the ORF, the forward 5' PCR primer will contain 40-60 bp of DNA 5' to the ATG, and the reverse primer of 3' end will contain 40-60 bp of DNA, 3' to the stop codon.

2. The marker will be amplified in the way that both the primers contain sequences to recognize the deletion module.

3. After the PCR amplification, the 40-60 bp of gene specific homology will be sufficient to direct gene replacement at the particular locus.

4. Integration into the target locus will be verified by PCR by using primers that amplify part of the integrated marker and the adjacent area of genomic DNA which confirms that the integration site is correct. The deleted part of ORF will also be verified by the PCR.

In the attached image, we can see that PCR of the deletion template with forward and reverse primers results in a PCR product that has short homology regions to the target gene and can direct homologous recombination and further gene replacement. After the transformation and subsequent selection, cassette integration and chromosomal target sequence deletion has been analysed by PCR. After the recovery of the haploids, following the sporulation and tetrad dissection, a single band should be found only in the new deletion strain.