Yeast vectors

Question 1

What are vectors?

Answer 1

DNA molecules able to have fragments of DNA of interest inserted that are capable of replication in a host organism

Question 2

Why are eukaryotic vectors based off well established E. coli vectors?

Answer 2

Much easier to put a vector into bacteria and make a ton of copies

Question 3

What is a vector called when it can propagate in 2 hosts?

Answer 3

Shuttle vectors

Question 4

What 4 features will be common to most eukaryotic vectors?

Answer 4

Bacterial Ori
Bacterial selectable marker
Multiple cloning site
Backbone features and selectable marker for eukaryotic system of choice

Question 5

What is a multiple cloning site?

Answer 5

Small region rich in restriction enzyme cut sites

Question 6

What is a yeast origin of replication called?

Answer 6

ARS: autonomously replicating sequences

Question 7

What are the 4 types of yeast vectors?

Answer 7

YIPs, YEPs, YRPs, and YCPs

Question 8

What are YIPs?

Answer 8

Yeast integrative plasmids. Designed to integrate into the genome

Question 9

What features are present on a YIP vector?

Answer 9

Bacterial Ori, bacterial selectable marker, MCS, yeast selectable marker

Question 10

How many copies of a YIP will be in a cell?

Answer 10

Usually only 1 or 2, since they are supposed to integrate into the genome

Question 11

What is the transformation efficiency of a YIP?

Answer 11

10^2 ug/DNA, so rather low

Question 12

What percentage of cells per generation will lose a YIP when plated on non-selective media?

Answer 12

«1% per generation. Since YIPs are integrated into the genome, they’re very stable since it’s difficult for a sequence to come out of the genome and get lost

Question 13

How do YIPs persist in cells?

Answer 13

They need to integrate into the genome to stick around, since they have no sequences to allow for their own maintenance and replication

Question 14

What are the pros of using YIPs?

Answer 14

Most stable maintenance out of all the plasmids, and behaves like nuclear DNA in terms of inheritance

Question 15

What are the cons of using YIPs?

Answer 15

Low transformation efficiency and hard to recover the plasmid since you can’t miniprep it out of the cell

Question 16

What are YEPs?

Answer 16

Yeast episomal plasmids

Question 17

What features are present on a YEP?

Answer 17

Bacterial Ori, bacterial selectable marker, MCS, yeast selectable marker, 2 um sequences

Question 18

What are 2 um plasmids?

Answer 18

Endogenous yeast plasmids present in some strains

Question 19

What is in the 2 um sequences on a YEP? Why doesn’t the rest of the 2 um plasmid need to be on the vector?

Answer 19

ARS and REP3 sequences that are needed in cis with the rest of the vector. All the other 2 um sequences are needed, but they can be in trans

Question 20

What happens if we put a YEP into a yeast cell that has no endogenous 2 um plasmids?

Answer 20

There are no transformants because the YEP can’t be maintained without the 2 um plasmids

Question 21

How many copies of a YEP are in a cell?

Answer 21

25-200

Question 22

What is the transformation efficiency of a YEP?

Answer 22

10^3-10^5 cells/ug DNA. Which is high

Question 23

What percentage of cells per generation will lose a YEP on non-selective media?

Answer 23

~1%

Question 24

What type of studies are YEPs really useful for?

Answer 24

Overexpression

Question 25

What are the pros of using YEPs?

Answer 25

Easy to recover with a miniprep, high copy number, great for overexpression studies, high transformation efficiency

Question 26

What are the cons of using YEPs?

Answer 26

Can recombine with the endogenous 2 um plasmids, can recombine into the genome, has no centromere and it can be lost after many generations

Question 27

What are YRPs?

Answer 27

Yeast replicative plasmids

Question 28

What features are present on a YRP?

Answer 28

Bacterial Ori, bacterial selectable marker, MCS, yeast selectable marker, yeast ARS

Question 29

Where does the ARS on a YRP come from?

Answer 29

Nuclear genome, so it isn’t reliant on endogenous 2 um plasmids to be maintained

Question 30

How many copies of a YRP are maintained in a cell?

Answer 30

1-20

Question 31

What is the transformation efficiency of a YRP?

Answer 31

10^4 cells/ug DNA

Question 32

What percentage of cells per generation will lose a YRP on non-selective media?

Answer 32

> > 1%/gen. Very substantial loss

Question 33

What types of studies are YRPs good for?

Answer 33

Short term overexpresssion studies where too much gene product is toxic

Question 34

What are the pros of using YRPs?

Answer 34

Can recover with a miniprep, high copy number, useful in overexpression studies, high transformation efficiency

Question 35

What are the cons of using YRPs?

Answer 35

May recombine into the genome, and very substantial loss in non-selective media

Question 36

What is a YCP?

Answer 36

Yeast centromeric plasmids

Question 37

What features are present on a YCP?

Answer 37

Bacterial ori, bacterial selectable marker, MCS, yeast selectable marker, yeast ARS, yeast centromeric sequences

Question 38

Why does a YCP behave like a normal chromosome in terms of inheritance?

Answer 38

They have centromeric sequences

Question 39

How many copies of a YCP will be in a cell?

Answer 39

1-2 copies, because of the normal chromosome behaviour

Question 40

What is the transformation efficiency of a YCP?

Answer 40

10^4 cells/ug DNA. Pretty high

Question 41

What percentage of cells per generation will lose a YCP on non-selective media?

Answer 41

<1%/gen

Question 42

What type of experiment uses YCPs?

Answer 42

Plasmid rescue and for toxic gene products

Question 43

What are the pros of using YCPs?

Answer 43

Copy number is beneficial for toxic products, high transformation efficiency, typically shows Mendelian inheritance and segregation, useful for complementation studies

Question 44

What are the cons of using YCPs?

Answer 44

Low miniprep yields, may recombine into the genome