I’m making a bunch of promoter reporter constructs by Gateway for MosSCI targeting and fluorescent microscopy.
We’re using the 3-fragment Gateway kit, and initially I’m making promoter reporters (with the gene proximal primer within the first exon, in the interests of decent primer design and capturing all 5’ regulatory sequence, in frame with GFP/mCherry::unc-54 3’UTR).
But later I also want to be able to swap in gene coding region (into site 2) and/or native 3’ UTR (into site 3), nice and modular.
My question is this … does anyone have an opinion on whether it would be detrimental to include a ~100bp ‘stuffer’ coding fragment as a placeholder within the final Gateway reporter construct? I’ve codon optimised it and there’s not much in the way of predicted secondary structure (I’ve literally taken a sequence from a random DNA generator and added att sites) or homology to anything in the genome. 100bp is the smallest size I’ve seen reference to for use with Gateway cloning.
I suspect the answer will be ‘suck it and see’, but I thought I’d see if anyone’s tried anything similar.
Thanks
Maybe I don’t quite understand what you are trying to do (for example, what slot is the placeholder DNA going into?) but would it work if you used three different plasmids:
[1-2] GFP
[2-3] unc-54 3’UTR
[2-3] GFP:unc-54 3’UTR
([1-2] = slot 2, [2-3] = slot 3)
Then you can swap in your gene of interest in the [1-2] slot or the native 3’UTR in the [2-3] slot and just change the fluorophore/3’UTR plasmids. It will not solve your problem if you want to tag the native gene with a fluorophore and the native 3’ UTR but I suspect you’d have the same problem with a placeholder. If you can live with using the tbb-2 3’ UTR instead of unc-54 then we have the above plasmids - send me an email directly if you think you’ll use them.
If you decide to use placeholder DNA I would probably not use random DNA sequence (even if you codon optimize it!). You’re probably better off with some kind of flexible linker sequence. We use flexible linkers in the lab for attaching fluorophores (5’ linker: STSGGSGGTGGSS , 3’ linker: GGTGGTGGSGGTG ). If you combine the two you’ll be close to the 100bp.
Thanks for the reply and sorry if that wasn’t clear. But I think you got it.
The idea for phase 1 is …
[1] 5’ regulatory sequence + start of 1st exon
[2] placeholder
[3] in frame GFP::unc-54 3’UTR (already fused by PCR)
… final form in the unc-19 pDEST ready for injection.
Then for phase 2 I can reuse [1]5’ and [3]GFP::3’ DONR vectors, but add in [2]gene cds to site instead of the placeholder.
For phase 3 reuse [1]5’ and [2]cds DONR vectors, but swap in [3]native GFP::3’UTR (again, a single DONR PCR fusion - the MosSCI kit not being compatible with the 4-site Gateway kit sadly).
Yes, your 3-plasmid method would work fine, I guess I’m trying to optimise the workflow (i.e. being impatient and cheap). For the moment I’m trialling a few. I’ll take your linker suggestion on board and see how it goes, but your plasmids could definitely come in handy, thanks. tbb-2 3’UTR looks nice and small too, which is a factor in all of this.