Tuesday, July 10, 2012

Follow the biology (pt. 2)...Why not sequence that thing?

I spent a good part of my time as a postdoc figuring out how to sequence microbial genomes cheaply, and learning how to deal with the flood of data that ensued. After my first post on this project, I was asked a very good and relevant question by Olin Silander...specifically, why not just spend a couple of hundred dollars and sequence the interesting brown mutant rather than go through transposon mutagenesis? I'm guessing that if you're reading this and are somewhat up to date on modern microbiology, you probably have the same question. Here's a couple of reasons that I came up with with varying levels of importance:

1) I'm a sucker for old school genetic screens. To be honest, it's fun coming into the lab and checking whether cultures are brown or yellow. There's so much about being a PI that can stress you out, and that feeling of "I got one!" is one little simple pleasure that I love about research. Agar plates, media, and bacterial conjugations are cheap...and I've got a very talented recently post-grad summer student that's helping me out with this experiment to ease the time investment so it's really not a big waste of anything.

2) It's possible that sequencing and transposon mutagenesis will yield different results. Whereas sequencing will likely tell me exactly what the mutation is, it's possible (maybe likely given that my gut is telling me that the brown color is a product of overproduction or disruption of negative regulation) that the exact mutation yielding the brown phenotype is in some regulatory region/protein. However, I'll admit that it is equally as likely at this moment that the brown phenotype is due to disruption of an enzymatic pathway and that what I'm seeing is the buildup of an intermediate product that is getting oxidized. IF the mutation is regulatory though, the transposon screen will likely yield disruptions in the pathways producing the brown product and that act downstream of the initial mutation. With only the sequence of a regulatory mutation I could be left guessing what pathway was being affected and what the next step is.

3) Transposon disruptions give me knockout mutants, and I can directly verify cause and effect of the transposon disruption on knocking out the brown coloration by transforming these loci back into the original brown mutant. If this ever is publishable, I need to be able to demonstrate genetic causation of a phenotype and natural transformation of the brown mutant with an antibiotic resistance locus present within a transposon is slightly easier than the molecular biology gymnastics necessary to recreate the original brown mutant in a wild type background.  Additionally,  more phenotypes (in this case disruption of the brown color) are never bad things.

4) This one is a little inside baseball...but I'm teaching upper level microbial genetics next spring with a lab (about 80 students in the  lab). As part of this lab, I want to have the students perform transposon mutagenesis and go over basic ideas like screens, selections, phenotypes. This little project gives me the opportunity to fine tune this procedure in P. stutzeri using an easily scoreable visual readout and provides a good base for figuring out what experiments I can have the students perform next April. 

5) I'm going to sequence it:) However, given how much of an overkill it is to sequence bacterial genomes using Illumina's HiSeq technology, I'm waiting to collect 23 other bacterial genomes to sequence along this one (I've got about 17 right now) in a single lane. I'll be blogging about those results too, so don't worry.

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