does size matter - how many genes in 1 vector (Feb/02/2007 )
I was just curious, I am going to try to get 4 genes (+ gal1 promoter) into a yeast. My question is this: has anyone here tried to get more than 2 genes into a single vector and succeeded? if so how big is the final vector, how many genes did you get?
Do you think it would be easier to form 2 gene constructs and do 2 successive transformations?
Thanks,
EJ
I have a vector where I have 4 genes being expressed. 2 Antibiotic resistance genes and 2 other genes. the total size of the vector is bit more than 10kb.
ooo ...
I have a plasmid with 2 prokaryote antibiotic genes, 2 eukaryote antibiotic gene, 1 eukaryote auxotrophic gene, and a yeast autonomous replication site (not a gene but hey it is a piece)
total size 19kb
EDIT: Well that depends. How big is your plasmid? And what do you want to do in your experiment. Do you want integration into the genome? Or simply a floating self replicating plasmid?
As scolix and I have shown, it is possible to make an all singing all dancing plasmid.
I have a plasmid with 2 prokaryote antibiotic genes, 2 eukaryote antibiotic gene, 1 eukaryote auxotrophic gene, and a yeast autonomous replication site (not a gene but hey it is a piece)
total size 19kb
EDIT: Well that depends. How big is your plasmid? And what do you want to do in your experiment. Do you want integration into the genome? Or simply a floating self replicating plasmid?
As scolix and I have shown, it is possible to make an all singing all dancing plasmid.
I'm planning on using pYES2 (~6KB) with URA and amp resistance already there. I want to add ~8 kb worth of genes, all using the same promoter (totalling about 14kb). The point of the experiment is to show multiple steps in a metabolic pathway. No need for integration. Seems feasable now. thanks for your replies. Any other advise is welcome though.
i have a plasmid of 14 kbp which includes 4 gene excluding ori
HI,
I have inserted a casette which has a promoter, two genes, two terminators and an antibiotic gene and the vector is 3 kb. And i was successsful in transforming it. All the best!!!.
Cheers!!!
Just to clarify... your using one promoter driving expression of multiple genes? whats the smallest linker you would trust between the two genes? If my target is 4 genes, would you guys trust more than 2 genes/promoter? (I'm using Gal 1 promoter on pYES2 all for yeast expression). Also if I go for the 2 genes/1 promoter would I need a terminator on both cassettes? Could I put all 4 genes behind a single promoter making a sort of pseudo operon?
Last question...for now, has anyone tried this kind of cloning?
Thanks
EJ
that is a different kettle of fish. In my previous examples, each gene had their own promoter and terminator.
I have never made a real pseudo operon before.
The nearest thing I have done in yeast, was to stick 3 proteins together, spaced by viral 2A self cleaving peptides. I made sure by experimentation, that the self cleaving peptide worked and did not intefere with the 3 individual protein's activity.
Last question...for now, has anyone tried this kind of cloning?
Thanks
EJ
Hi,
Well you could clone two genes after the promoter, provided u have included a proper Kozak or SD btween the genes, that hlps in translation efficiency. Four genes is possible with two promoters in one casette but definitly u need to clone in a terminator on both sides/casettes. I hav done this and it works.
cheers
I'm interested in the 2A peptide, fairly new science but seems robust. Is the best way to insert the 2A peptide to just use synthesized oligo and then ligate it into the vector. Is there any other tips or tricks to 2A peptides? you know of any publicly available vectors that already have usable 2A peptide encoding regions in them (esp. for yeast expression)?
EJ.