Operation damage control

What do you do when one stupid ugly Western blot informs you that all of your efforts of the last year and a half may amount to squat? It’s like someone snuck into the lab and took a giant crap on my bench-top.


And this time, I can’t even pin the blame on PETA! Not cool. 😦

The question now, is whether or not I can salvage what I do have. If anyone has an opinion on the following — long story (very) short, I’d love love love to hear it —

So I’m trying to coax my cells to make molecule M that has function F. I’ve given the cells the blueprints and all of the resources that they should need to make M and send them to work.  Unfortunately, M is really small and sneaky; it’s super difficult to catch and see. But, when I bring my cells to chill with their cell friends (well, cell enemies actually :-/) I see function F! Yeah!!! 🙂 Right?!?! Well, this is good of course, but is it good enough? There is F, but not as much as we were hoping for. And we still can’t see M. 😦 So, if we show F across many different groups of cell enemies, is this good enough? If yes, how many is “many”? If not, is anyone looking to hire a damn good molecular biologist with 6 years of chemical engineering training but no PhD? I can make a mean cheesecake too…

For the Sheldons out there – here is a translation of the above that might suit your fancy —

I’m trying to express and secrete a heterologous protein (P) on a plasmid expression vector from Gram-positive bacteria. I’ve included a robust secretion peptide on the protein’s N-terminus that should promote functional protein secretion from the cell. Now, I see significant upregulation of P at the transcript level by qPCR and intracellular protein by Western blot (via a C-termial 6xHIS tag) but I can’t detect my protein in the culture media! 😦 Now the point of this protein is to kill and/or inhibit growth of other bacteria strains. When I culture these other strains in the sterilized (supernatant) media that should contain P, I do see a decrease in viability (relative to viability in supernatant media from just an empty vector control). I have run this kind of viability experiment a handful of times, collecting the supernatant media (hopefully with the peptide), sterilizing it, and then trying to grow the other strains in it, and it seems to be relatively reproducible. (You know, unless the damn stars aren’t aligned properly, but never mind…)

So, either I’m a) making the protein and not secreting it (and the reduction in viability is a nasty coincidence) or b) secreting it (probably in low concentrations) and I’m not effectively concentrating (via ammonium sulfate precipitation or freeze drying) and/or detecting it via Western. Obviously I’m hoping to god that the answer is b. Assuming this is the case (please go with me on this for now):

  1. May it be good enough to show functional reproducibility across a number of strains? If so, how many is a good target (3, 5, 600)?
  2. Will I still probably have to back out the protein either by Western or mass spec regardless of the functional results? — If I will have to prove specific protein production, are there any suggestions or tricks on how to do this aside from what I’m already trying?

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