Tag Archives: Publications

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.

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

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!!! ūüôā 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. ūüė¶ 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! ūüė¶ 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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Submission predicament!

I’m¬†finally¬†starting to write the last big (relative definition please) paper of my PhD!! ūüôā I’ve run into a bit of a predicament though. ūüė¶ So, for all of the other papers that I’ve been a part of over the last ~10 years there has been a clear first, second and third (yes, that’s right,¬†third…this is what happens when you are crazy) choice for where to submit. My boss/adviser¬†were part of the¬†same research community that typically populate the kind of journals that my work was appropriate for. This was great; we could submit to the journals that 1) we were most familiar with, b) our friends were most familiar with¬†and¬†c) our type of work was most typically found. Three birds, one stone. Perfect.

Unfortunately, it is not quite that simple this time around. My adviser is a theorist whose work has spanned from inorganic polymer physics to biopolymers over the last 15 years, and is now moving down the even darker and scarier road of experimental¬†synthetic and applied biology. Enter my project. We are very much at the interface of (micro)biology, food science and engineering — three fields that, at first glance, seem like quite the stretch to connect in one fell swoop. Hence the current question: where do we submit?

Do we submit to the journal that’s most appropriate for our subject matter? ¬†If we go here, no one will know us. People who have these publications included in their¬†monthly journal email updates¬†will not recognize our names or our department as they scroll through their announcement for the most recent issue. This unfortunately means that our chances of being read, much less cited, decrease dramatically. (The sad truth is that if people don’t know you, odds are ¬†stacked against them reading your work.) And at the same time, if we are to take this route, our friends will continue¬†indulging¬†in the periodicals that we all browse/read regularly, and they will miss our publication as well. Booohooohooo ūüė¶

So then, do we submit to the journal that all of our friends are familiar with and regularly publishing in? If all of our friends jumped off of a cliff would we go jump too? (Obviously the answer to this question depends. Are the beautiful blue waters of Serifos below us? Are we wearing a parachute/hang-glider? Is there a rabid tiger chasing us?) Now if we do this (submit the paper among friends, not go cliff jumping…although really, how cool would that be?!?! Sans the tiger…) ¬†our friends will recognize our name and our department as they scroll though their email updates. They will probably click on our article with the expectation that they are opening a computational paper that integrates multiscale¬†molecular dynamic simulations with stochastic modeling approaches. However, they will be greeted by a solution (based on experimental research) for curbing the emergence of antibiotic resistance. WTF?!?! Now they will probably still read the paper (we have nice friends AND curtailing this world health issue is pretty damn cool whether you’re in the field or not…in my humble opinion) but will they cite it? Why would they? It is SO outside of their field! And of course, the people who would be interested in this project are above in paragraph 3, unaware of anything that we have done.

So what do we do? Do we stick with our friends that we already know and love? Do we stick our necks and our work out there into the unknown? Regardless, this is a pretty clear wake up call that we do need to start making new friends (….but keep the old…one is silver and the other’s gold… Sorry.) as we move into different fields. Fine.

But, what do we do now?

Help please!

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