How far can the promoter be from the fluorophore?

Model Organism: Worms Methods & Reagents
1

Hello everyone,

I am trying to generate a reporter strain by cloning my promoter into a vector that has mcherry in it. Unfortunately,
I don’t have many options for restriction sites, even taking enzyme compatibility into account. My only option results in my promoter being
168bp upstream of my mcherry. Is this too much? Does anyone know how much wiggle room there is for generating reporter strains?

Any help or advice would be greatly appreciated!!

2

I don’t have a direct answer to your question, but I can tell you that I haven’t done restriction cloning in >5 years. Check this out: https://www.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/gibson-assembly. You can fuse your promoter directly to mCherry even if there’s no convenient restriction site.

3

Two things:

  1. I recommend studying the documentation for the Fire vector kits and looking at the annotated plasmid sequences in the documentation and on Addgene’s site. You can learn a lot about how those widely-used plasmids were assembled - including that in some of the standard gfp reporters there’s about 165 bp between the start of the multiple cloning site (SphI) and the start of the reporter cassette (AgeI). I believe this stretch includes synthetic introns that contribute to expression. It’s not necessarily the case that you should try to make your construct by fusing the start of your gene’s open reading frame directly to the start codon of mCherry.

  2. In addition to Dan’s suggestion that you can splice your gene’s promoter to the reporter using Gibson cloning, you can still use restriction enzyme cloning if you want to. All you have to do is amplify your promoter by PCR using oligonucleotides that add compatible restriction sites to the ends of the products.

4

Yeah, both Hillel and Dan’s suggestions are great. In addition, if you’re looking to just generate a quick and dirty reporter fusion you could make one through PCR and just inject the product.

5

I am a big fan of overlap PCR, as Snug suggested. However, I tended to be overly cautious, so I would use a high fidelity enzyme (my fave is Takara PrimeStar) and drop it into a TOPO-Blunt vector. I sequence-verified and then injected. But in all honesty, if there was a deleterious SNP in either the promoter or the fluorophore, you wouldn’t get expression, so if you see mCh fluorescence it must have worked! :slight_smile:

There’s also another great cloning method I like - USER cloning. It’s a bit heavy on the cost of reagents but is very effective. PM me if you want more details.

If you have limited resources, overlap PCR and injection is definitely an inexpensive option.

Steve

6

We do almost everything now by Gibson, per Dan Dickinson’s recommendation to me a few years back. And PCR fusion (or stitching, or splicing, or overlap: whatever name you prefer) works well, too.

And we now Gibson any gBlocks into a vector: we have a tube full of EcoRV-opened pBluescript in the freezer, and order all gBlocks with overlaps so we can drop then straight into a plasmid. That way we have a permanent copy of that particular sequence. Example: we ordered germline codon-optimized mKate2::AID (Auxin Inducible Degron) as a gBlock and dropped it into pBS by Gibson, and sequence verified. Now we have that fusion as a template for ever, and can amplify the whole thing for CRISPR, or each piece separately for other applications. So we don’t need to go back to more gBlocks. It can be difficult to amplify straight from a gBlock if it is over 1 kb.