How to disrupt the Yap 1801 and 1802 gene in Genetic lab? (what media will you n

Question

How to disrupt the Yap 1801 and 1802 gene in Genetic lab? (what media will you need to grow and how to grow your yeast?) And Come up with some tests to proved that we have successfully done it?

This is a method that i found online which is related to the question but i don really understand the process.

METHODS
Yeast strains and media
Yeast strains (listed in supplementary material Table S1) were transformed with plasmids carrying the different chimeric constructs under the GAL10 promoter and were grown overnight on dextrose medium (0.67% yeast nitrogen base, 2% dextrose) supplemented with 0.67% casamino acids or with some of the following amino acids: 20 mg/l histidine (H), 20 mg/l lysine (K) and 60 mg/l leucine (L). Upon reaching exponential phase (OD600?1), the cells were placed in galactose medium supplemented with either 0.67% casamino acids or HKL to induce the expression of chimeric proteins. After 6 hours, cells were either collected to obtain total cell extracts or were observed under epifluorescence.

The ?yap1801/1802 strain was constructed by mating the two simple deleted strains ?yap1801 and ?yap1802. The mating type of the ?yap1801 strain was changed by transforming the strain with a plasmid carrying the HO gene. The two strains were then crossed by patching them mixed on a selective medium. After verifying mating by microscopy, the diploid strain was patched onto a sporulation medium. After 3 days of growth, tetrads were dissected and the resistance of the spores to G418 was tested. The G418- resistant spores from the double recombined tetrads were kept as ?yap1801/1802 strains. To lose the plasmid carrying the HO gene, ?yap1801/1802 was then plated onto rich medium containing 5FOA.

Oligonucleotides are listed in supplementary material Table S2. The A?1-42 sequence was amplified by PCR from pSG5-APP (a kind gift from Agne?s He?mar) using oligonucleotide 792, which introduces a BamHI restriction site at the 5 end of the fragment and an ATG codon at the beginning of the A?1-42 sequence, and oligonucleotide 794. The PCR fragment was then inserted into the plasmid pYecHetsYGFP (Couthouis et al., 2009), which had been previously linearized by BamHI using a gap repair method (Orr-Weaver and Szostak, 1983). The pYe?A?YGFP and pYe?A?ARCYGFP plasmids were constructed by cloning a synthetic sequence in a BamHI-BstXI-digested pYeA?YGFP plasmid. These synthetic sequences, made by GeneScript, were composed of BamHI restriction site followed by ?-factor prepro sequence, A? wild-type or arctic mutant coding sequence, the 5 end of the GFP sequence, and a BstXI restriction site. The pYeA?ARCYGFP plasmid was created by overlapping PCR using pYe?A?ARCYGFP as a template (with oligonucleotides 705, 706, 859 and 860). This allowed the amplification of a PGAL-A?ARC-GFP fragment, which was introduced by a gap repair method into a BamHI-BstXI- digested pYe?A?ARCYGFP plasmid. Similarly, PGAL-?-factor prepro-GFP and PGAL-GFP sequences were created by overlapping PCR using pYe?A?YGFP and pYeA?YGFP as templates (with oligonucleotides 705, 706, 856, 857 and 858). The fragments were respectively inserted into pYe?A?YGFP and pYeA?GFP BamHI- BstXI-digested plasmids. Each of these plasmids is a multicopy yeast-expression plasmid with the URA3 selectable marker and a GAL10 promoter in a pYeHFN2U backbone (Cullin and Minvielle- Sebastia, 1994). CALM long splice variant (CALM-L; GenBank ID BC011470) and short splice variant (CALM-S; GenBank ID

BC021491) from the American Tissue Culture Collection (ATCC) were amplified by PCR using oligonucleotide 951, which introduced a BamHI site at the 5 end of the cDNA and oligonucleotide 952 which introduced a NotI site at the 3 end of the cDNA. After digestion by BamHI and NotI, the PCR fragment was inserted into the pYeHFN2L plasmid. This plasmid is a multicopy yeast- expression plasmid with an LEU2 selectable marker and a GAL10 promoter (Cullin and Minvielle-Sebastia, 1994).

Explanation / Answer

Yeast genes can be disrupted by integrative transformation i.e integrating a heterologus gene segment into the gene of interest.Use of restriction enzymes can yield DNA fragment that contains the disruption and flanking homology.The DNA fragment has a selection marker like URA3 selection marker,, wherein yeasts that have stably integrated the DNA fragment can be selected for Ura+.

Gene disruption can also be tested by PCR.

Gene disrution helps in ascertaining gene function and phenotype analysis.

Several mediums can be used to grow yeast, common one being YPD medium ,dextrose medium and others.These media can be used as is, or can be supplemented with more nutrients, for example phosphate, or even be depleted for a particular nutrient.The choice of medium or supplementation or specficic character like a nutrient depletion often depends upon the nature and objective of experiment.

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