Yeast vectors Flashcards

1
Q

What are vectors?

A

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

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2
Q

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

A

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

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3
Q

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

A

Shuttle vectors

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4
Q

What 4 features will be common to most eukaryotic vectors?

A

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

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5
Q

What is a multiple cloning site?

A

Small region rich in restriction enzyme cut sites

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6
Q

What is a yeast origin of replication called?

A

ARS: autonomously replicating sequences

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7
Q

What are the 4 types of yeast vectors?

A

YIPs, YEPs, YRPs, and YCPs

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8
Q

What are YIPs?

A

Yeast integrative plasmids. Designed to integrate into the genome

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9
Q

What features are present on a YIP vector?

A

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

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10
Q

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

A

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

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11
Q

What is the transformation efficiency of a YIP?

A

10^2 ug/DNA, so rather low

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12
Q

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

A

«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

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13
Q

How do YIPs persist in cells?

A

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

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14
Q

What are the pros of using YIPs?

A

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

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15
Q

What are the cons of using YIPs?

A

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

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16
Q

What are YEPs?

A

Yeast episomal plasmids

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17
Q

What features are present on a YEP?

A

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

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18
Q

What are 2 um plasmids?

A

Endogenous yeast plasmids present in some strains

19
Q

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?

A

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

20
Q

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

A

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

21
Q

How many copies of a YEP are in a cell?

22
Q

What is the transformation efficiency of a YEP?

A

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

23
Q

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

24
Q

What type of studies are YEPs really useful for?

A

Overexpression

25
What are the pros of using YEPs?
Easy to recover with a miniprep, high copy number, great for overexpression studies, high transformation efficiency
26
What are the cons of using YEPs?
Can recombine with the endogenous 2 um plasmids, can recombine into the genome, has no centromere and it can be lost after many generations
27
What are YRPs?
Yeast replicative plasmids
28
What features are present on a YRP?
Bacterial Ori, bacterial selectable marker, MCS, yeast selectable marker, yeast ARS
29
Where does the ARS on a YRP come from?
Nuclear genome, so it isn't reliant on endogenous 2 um plasmids to be maintained
30
How many copies of a YRP are maintained in a cell?
1-20
31
What is the transformation efficiency of a YRP?
10^4 cells/ug DNA
32
What percentage of cells per generation will lose a YRP on non-selective media?
>>1%/gen. Very substantial loss
33
What types of studies are YRPs good for?
Short term overexpresssion studies where too much gene product is toxic
34
What are the pros of using YRPs?
Can recover with a miniprep, high copy number, useful in overexpression studies, high transformation efficiency
35
What are the cons of using YRPs?
May recombine into the genome, and very substantial loss in non-selective media
36
What is a YCP?
Yeast centromeric plasmids
37
What features are present on a YCP?
Bacterial ori, bacterial selectable marker, MCS, yeast selectable marker, yeast ARS, yeast centromeric sequences
38
Why does a YCP behave like a normal chromosome in terms of inheritance?
They have centromeric sequences
39
How many copies of a YCP will be in a cell?
1-2 copies, because of the normal chromosome behaviour
40
What is the transformation efficiency of a YCP?
10^4 cells/ug DNA. Pretty high
41
What percentage of cells per generation will lose a YCP on non-selective media?
<1%/gen
42
What type of experiment uses YCPs?
Plasmid rescue and for toxic gene products
43
What are the pros of using YCPs?
Copy number is beneficial for toxic products, high transformation efficiency, typically shows Mendelian inheritance and segregation, useful for complementation studies
44
What are the cons of using YCPs?
Low miniprep yields, may recombine into the genome