I was trying to streamline our existing attB vector. I was prompted to do this for a few reasons: 1) I recently identified a previously unappreciated T7 (bacteriophage) promoter and potential cryptic bacterial promoter in our standard plasmid, 2) There are presumably some weak cryptic eukaryotic promoters hidden somewhere in the plasmid too, and 3) I was trying to “domesticate” the plasmid to get rid of some Type II and Type IIS restriction enzyme sites.

As part of the most recent plan, I decided to delete two different sections of the plasmid; one was the segment of DNA between the attB site and the Amp promoter driving the AmpR gene, and the second was the segment between the origin of replication and the SV40 PolyA signal. First one worked fine (which I knew, since I eventually remembered that I had previously done this back in like….2015, but never used it again for some reason). The second one proved very problematic. Here’s the section in question:

So I had seen those annotations for the lac promoter and lac operon, but assuming the directionality of the map was true, it seemed like they weren’t really pointed at enough bacterial sequence to matter so I just assumed they were vestiges of something. Well, this is what happened in terms of my plasmid yields in this lineage of plasmid.

I’m not going to read into the slightly higher concentration of L036 too much, but man, what really smacks you in the face is just how bad yields became with L048 (a derivative of L036). Well, so I tested the panel for their ability to recombine into landing pad cells, and the phenotype there was obvious as well; all plasmids up to and including L036 recombined at high rates, whereas L048 and one of its sibling plasmids with the same deletion had nonzero but *severely* diminished recombination. So not only is the DNA yield bad, but the “quality” in some sense seems to be much worse in that the DNA that is there is not resulting in good recombination.

I’ve learned my lesson, and I’m now just trying to take out that last BspQI/SapI site with a nucleotide substitution.

But still, this begets the question: so what in the world is in that DNA section, and why is it so important for plasmid propagation? I’m sure some bacteriologists and perhaps some old-school molecular biologists should know, but I’ve always lamented how much of a black box the bacterial portions of plasmids are (my expertise is in eukaryotic / mammalian cell biology). Will I ever figure this out?

Well, I will first try with pLannotate. That’s what told me there was a T7 primer site in this plasmid after all.

Well, so that didn’t really uncover anything new. Hmmm…