Here is some real-world data describing expected yields we may expect from some of these routine lab procedures or services.
Oh, and this is a good one:
How well my determination of flask “confluency” actually correlated with cell counts. I mean, sure, there must be some error being imparted by the actual measurement of the cells when counting, but I think we all know it’s mostly that my estimate really isn’t precisely informative.
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…
iCasp9 as a negative selection cassette is amazing. Targeted protein dimerization with AP1903 / Rimiducid is super clean and potent, and the speed of its effect is a cell culturist’s dream (cells floating off the plate in 2 hrs!). It really works.
But when there are enough datapoints, sometimes it doesn’t. I have three recorded instances of email discussions with people that have mentioned it not working in their cells. First was Jeff in Nov 2020 with MEFs. Then Ben in June 2021 with K562s. And Vahid in July 2021 with different MEFs. Very well possible there’s one or two more in there I missed with my search terms.
Reading those emails, it’s clear that I had already put some thought into this (even if I can’t remember doing so), so I may as well copy-paste what some them were:
1) Could iCasp9 not work in murine cells, due to the potential species-based sequence differences in downstream targets? Answer seems to be no, as a quick google search yields a paper that says “Moreover, recent studies demonstrated that iPSCs of various origin including murine, non-human primate and human cells, effectively undergo apoptosis upon the induction of iCasp9 by [Rimiducid]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7177583/
Separately, after the K562s (human-derived cells) came into the picture:
This is actually the second time this subject has come up for me this week; earlier, I had a collaborator working in MEF cells note that they were seeing slightly increased but still quite incomplete cell death. That really made me start thinking about the mechanism of iCasp9-based killing, which is chemical dimerization and activation of caspase 9, which then presumably cleaves caspases 3 and 7 to then start cleaving the actual targets actually causing apoptosis. So this is really starting to make me think / realize that perhaps those downstream switches aren’t always available to be turned on, depending on the cellular context. In their case, I wondered whether the human caspase 9 may not recognize the binding / substrate motif in murine caspase 3 or 7. In yours, perhaps K562’s are deficient in one (or both?) of those downstream caspases?
Now for the most recent time, which happened in the lab rather than by email: It was recently brought up that there is a particular landing pad line (HEK293T G417A1) which we sometimes use, that apparently has poor negative selection. John and another student each separately noticed it. Just so I could see it in a controlled, side-by-side experiment, I asked John if he’d be willing to do that experiment, and the effect was convincing.
So after enough attempts and inadvertently collecting datapoints, we see the cases where things did not go the way we expected. Perhaps all of these cases share a common underlying mechanism, or perhaps they all have unique ones; we probably won’t ever know. But there are also some potentially interesting perspective shifts (eg. a tool existing only for a singular practical purpose morphing into a potential biological readout), along with the practical implications (ie. if you are having issues with negative selection, you are not alone).
This is the post I will refer people to when they ask about this phenomenon (or what cell types they may wish to avoid if they want to use this feature).
In this previous post, I showed how many reads we’ve gotten from our Plasmidsaurus and AMP-EZ submissions. Well, now’s also time to see whether the amount of DNA that we gave correlated with the number of reads we got back.
As you can see above, since this is miniprepped DNA, it’s usually quite easy to reach the 300 ng needed for submission. One time, when we submitted closer to 200ng, it worked perfectly fine. One other time, when we submitted ~ 100ng, it did not, albeit this was not plasmid DNA and instead was a PCR product, so it’s an outlier for that reason as well.
This is the more important graph though, since all of our AMP-EZ submissions are from gel extracted PCR amplifications, and it can be quite difficult to do it in such a way that we have the 500 ng of total qubitted DNA available for submission. Well, turns out that it’s probably not all that important for us to hit 500 ng of DNA, since it’s worked perfectly fine in our attempts between 200 and 500 ng. I imagine people in my lab will simultaneously be happy (knowing they don’t have to hit 500 ng) and sad (knowing they had spent a bunch of extra effort in the past unnecessarily trying to reach that number) seeing the above data, but hey, it’s good to finally know this and better late than never!
1/2/2025 edit: FYI, we do all of our lentivector transformations in NEB stable cells now (NEB equivalent of STBL2 or STBL3 like cells); we still grow in TB, although perhaps we should systematically test this at some point too (or maybe I told someone to do this and we already have, and I’ve simply forgotten the results…)
At some point, I was chatting with Melissa Chiasson about plasmid DNA yields, and she mentioned that her current boss had suggested using terrific broth instead of Luria broth for growing transformed bacteria. I think both of us were skeptical at first, but she later shared data with me showing that DNA from e.coli grown in TB had actually given her better yield. I thus decided it was worth trying myself to see if I could reproduce it in my lab.
There are two general types of plasmids we tend to propagate a lot in my lab. attB recombination vectors, for expressing transgenes within the landing pad, and also lentiviral vectors of the “pLenti” variety, which play a number of different roles including new landing pad cell line generation and pseudovirus reporter assays.
I first did side-by-side preps of the same attB plasmids grown in TB or LB, and TB-grown cultures yielded attB plasmid DNA concentrations that were slightly, albeit consistently worse. But I eventually I tested some lentiviral vector plasmids and finally saw the increase in yield from TB that I had been hoping for. Relaying this to Melissa, she noted she had been doing transformations with (presumably unrelated sets of) lentiviral vectors herself, so these observations had been consistent after all.
Thus, if you get any attB or pLenti plasmids from me, you should probably grow them in LB (attB plasmids) and TB (pLenti plasmids), respectively, to maximize the amount of DNA yields you get back for your efforts.
