Yeast transformation

Question 1

What are 4 types of selectable markers we use in yeast?

Answer 1

Nutritional markers, carbon source, drug resistance, colour selection

Question 2

How do we use nutritional markers as a selectable marker in yeast?

Answer 2

Vector contains WT sequence, and transformed cells are auxotrophs. Only cells that picked up a vector will grow on media that is missing the nutrient they are auxotrophs for

Question 3

How do we use carbon source markers as a selectable marker in yeast?

Answer 3

We can select for cells capable of only using glucose vs one capable of using both glucose and galactose, if we have galactose metabolism genes on the vector

Question 4

How do we use drug resistance markers as a selectable marker in yeast?

Answer 4

The yeast die unless they took up a vector, since the vector contains a resistance gene

Question 5

How do we use colour selection markers as a selectable marker in yeast?

Answer 5

We have have a vector with a WT adenine sequence and transform adenine mutants. Untransformed cells are red and transformed cells are white

Question 6

Why is URA3 a very common selectable marker?

Answer 6

We have the means to select against it as well, which is very valuable

Question 7

What are 2 ways to transform yeast cells?

Answer 7

Alteration of membrane permeability with salts or electroporation

Question 8

How do you transform yeast cells using salts?

Answer 8

Put the cells into a lithium or calcium salt solution, which alters their membrane permeability. Then add the vectors, incubate the cells for a while then plate them and select for the transformed cells

Question 9

How do you transform yeast cells with electroporation?

Answer 9

Uses electricity instead of salts to permeabilize the membrane and the DNA gets in through the short-lived pores. Need to find the right amount of electricity to not damage the cells or DNA

Question 10

When using a plain old YIP, how many transformants will have had integration into the genome by homologous recombination, non-homologous recombination, or no integration?

Answer 10

70% will have homologous, 10% will have non-homologous, 20% will have had no integration

Question 11

What would be the procedure to determine if homologous or non-homologous recombination occurred when transforming a YIP?

Answer 11

Cross the transformants to a wild-type strain to generate a diploid. Then sporulate the diploid and look at the resulting haploids

Question 12

What would be the results of sporulating the diploid if homologous recombination occurred?

Answer 12

Only parental ditype tetrads would be formed and they would all be URA+. Each tetrad would have two genotypes that are exactly the same as the parents (AB or ab)

Question 13

What would be the results of sporulating the diploid if non-homologous recombination occurred?

Answer 13

Get tetrads that are parental ditype, non-parental ditype, and tetratype. Most of the progeny will be URA+ but some will be URA-. Some tetrads would have 2 genotypes that look like the parents, some have 2 genotypes that don’t look like the parents, and some have 4 genotypes with 2 that look like the parents and 2 that don’t

Question 14

How do we design YIPs that undergo homologous recombination 100% of the time?

Answer 14

Linearized vectors. We make a double strand break in a vector sequence that is homologous to where we want it to integrate, and it will integrate homologously 100% of the time since double stranded ends are highly recombinogenic