Yeast vectors Flashcards
What are vectors?
DNA molecules able to have fragments of DNA of interest inserted that are capable of replication in a host organism
Why are eukaryotic vectors based off well established E. coli vectors?
Much easier to put a vector into bacteria and make a ton of copies
What is a vector called when it can propagate in 2 hosts?
Shuttle vectors
What 4 features will be common to most eukaryotic vectors?
Bacterial Ori
Bacterial selectable marker
Multiple cloning site
Backbone features and selectable marker for eukaryotic system of choice
What is a multiple cloning site?
Small region rich in restriction enzyme cut sites
What is a yeast origin of replication called?
ARS: autonomously replicating sequences
What are the 4 types of yeast vectors?
YIPs, YEPs, YRPs, and YCPs
What are YIPs?
Yeast integrative plasmids. Designed to integrate into the genome
What features are present on a YIP vector?
Bacterial Ori, bacterial selectable marker, MCS, yeast selectable marker
How many copies of a YIP will be in a cell?
Usually only 1 or 2, since they are supposed to integrate into the genome
What is the transformation efficiency of a YIP?
10^2 ug/DNA, so rather low
What percentage of cells per generation will lose a YIP when plated on non-selective media?
«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
How do YIPs persist in cells?
They need to integrate into the genome to stick around, since they have no sequences to allow for their own maintenance and replication
What are the pros of using YIPs?
Most stable maintenance out of all the plasmids, and behaves like nuclear DNA in terms of inheritance
What are the cons of using YIPs?
Low transformation efficiency and hard to recover the plasmid since you can’t miniprep it out of the cell
What are YEPs?
Yeast episomal plasmids
What features are present on a YEP?
Bacterial Ori, bacterial selectable marker, MCS, yeast selectable marker, 2 um sequences
What are 2 um plasmids?
Endogenous yeast plasmids present in some strains
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?
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
What happens if we put a YEP into a yeast cell that has no endogenous 2 um plasmids?
There are no transformants because the YEP can’t be maintained without the 2 um plasmids
How many copies of a YEP are in a cell?
25-200
What is the transformation efficiency of a YEP?
10^3-10^5 cells/ug DNA. Which is high
What percentage of cells per generation will lose a YEP on non-selective media?
~1%
What type of studies are YEPs really useful for?
Overexpression
What are the pros of using YEPs?
Easy to recover with a miniprep, high copy number, great for overexpression studies, high transformation efficiency
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
What are YRPs?
Yeast replicative plasmids
What features are present on a YRP?
Bacterial Ori, bacterial selectable marker, MCS, yeast selectable marker, yeast ARS
Where does the ARS on a YRP come from?
Nuclear genome, so it isn’t reliant on endogenous 2 um plasmids to be maintained
How many copies of a YRP are maintained in a cell?
1-20
What is the transformation efficiency of a YRP?
10^4 cells/ug DNA
What percentage of cells per generation will lose a YRP on non-selective media?
> > 1%/gen. Very substantial loss
What types of studies are YRPs good for?
Short term overexpresssion studies where too much gene product is toxic
What are the pros of using YRPs?
Can recover with a miniprep, high copy number, useful in overexpression studies, high transformation efficiency
What are the cons of using YRPs?
May recombine into the genome, and very substantial loss in non-selective media
What is a YCP?
Yeast centromeric plasmids
What features are present on a YCP?
Bacterial ori, bacterial selectable marker, MCS, yeast selectable marker, yeast ARS, yeast centromeric sequences
Why does a YCP behave like a normal chromosome in terms of inheritance?
They have centromeric sequences
How many copies of a YCP will be in a cell?
1-2 copies, because of the normal chromosome behaviour
What is the transformation efficiency of a YCP?
10^4 cells/ug DNA. Pretty high
What percentage of cells per generation will lose a YCP on non-selective media?
<1%/gen
What type of experiment uses YCPs?
Plasmid rescue and for toxic gene products
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
What are the cons of using YCPs?
Low miniprep yields, may recombine into the genome
