So you want to do "experimental evolution"

Rich Lenski and his lab are getting a lot of well deserved publicity lately because they have published yet another awesome paper from their long term evolution experiment (LTEE). The success of the LTEE has no doubt sparked a bunch of researchers out there to go "hmm...I can do that!". I'm guessing that I was in third grade or so when Rich started the LTEE, and I have only been tangentially associated with the Lenski research lineage (who in my own experience are as smart and helpful as their mentor), but I've set up long-ish term lab passage experiments a couple of different times with different systems. There are a few things I've learned along the way that I think would be helpful to share with others jumping into the experimental evolution game, and hence this post. Please feel free to add suggestions to this list, or to contact me off-blog if you'd like to talk shop. The best tribute I can have for Rich is to provide as much help for the community as he and his students have for me over the years. I say this every time, but thank you very much!

1. Let the question guide your experiment.  We all have our favorite microbes (OFM), and the reaction that I've seen time and time again is to want to perform an evolution experiment with OFM just to see what would happen. I can assure you that OFM will evolve and adapt to passage conditions and will do so quickly, but what does this really tell you? My first piece of advice colors everything from here on out, and it's to focus on finding a question to ask and only then find the best microbial system to work with. E. coli works great for understanding general evolutionary principles, and in fact one of the most important questions to ask yourself should be "why not do this with E. coli?", but this would be a terrible system to study sporulation. Find the question that excites you and then find the system, it's easy enough to set one up if you know what to look for.

2. Once you've got the system, make sure you can measure fitness. A major piece of the LTEE is the ability to compare phenotypes and genotypes of cells from one generation vs. all others. For any evolution experiment to work, however, you need to be able to demonstrate that that evolution takes place. Competitive fitness assays are just one way to do this, but they are a very powerful test because they enable direct comparisons between strains. In order to carry out competitive fitness experiments, you need to be able to distinguish two strains from one another within a single culture under conditions that closely approximate passage. Rich's experiment directly competes strains that differ in arabinose utilization (Ara+/Ara-), which under the correct plating conditions enables you to visualize different strains by color (red/white). In many cases, such a simple phenotypic comparison isn't easily accomplished. In my first stab at an evolution experiment I was investigating the effect of natural transformation in Helicobacter pylori. Out of necessity, I designed my competitive fitness experiments slightly different than Lenski's because I was using antibiotic markers. Instead of directly competing evolved strains against each other, I would compete evolved strains vs. an ancestral "control" strain which was doubly marked with kanamycin and chloramphenicol. This isn't quite as elegant as I'd like, but I wanted to avoid confounding my evolution results with compensation for these phenotypic markers (in Rich's case, he spent a lot of time demonstrating that the Ara marker is a neutral change under his passage conditions, this often isn't the case for antibiotic resistance). At first I simply tried to plate out the same competition onto non-selective media and kan/cam media, but found that the variance in ratio of evolved/control strains was way too high to be reliable for fitness estimates. For instance, in some cases there would be more colonies on the kan/cam plates than on the non-selective media. To get around this issue and control for such plating variance, I decided to first plate the competition out on non-selective media and then to replica plate to the kan/cam selective conditions. This change allowed me to actually measure fitness using antibiotic markers and all was happy and good for the time being.  It completely sucked to replica plate everything, but it was the only way to get reliable numbers.

3. Carefully think about your passage conditions.  When you are performing a passage experiment, EVERYTHING MATTERS. Are you going to passage under batch culture conditions where there are such things as lag/log/stationary phase, are you going to passage in a chemostat, are you going to passage in vivo, etc...? Every change you make to your passage conditions can affect the results in subtle or not so subtle ways as selection will operate differently under different conditions. If you are passaging in vivo (mouse, plants, whatever), how do you control interactions between other microbes and your targets of interest or sample your focal microbe for freezing? Even the way that you passage your microbes in vivo can change selection pressures. For instance, motility will be a target of selection if you simply place your microbes on a plant leaf and select for infection BUT if you inoculate a leaf with a syringe (bypassing the need for microbes to invade), motility likely doesn't matter at all for infection and my guess is that you'll quickly get amotile mutants. Along these lines, always try to set up cultures using defined media even if you aren't quite sure that all components are necessary (plus, if you carry out LTEE long enough, cool things happen with the "unnecessary components"). With my H. pylori cultures, applicable to passage experiments with many pathogenic microbes, I was forced to use media which contained fetal bovine serum (FBS). The problem here is that every batch of FBS is different because every calf is different! I no doubt missed out on some fine scale evolutionary events simply because my H. pylori populations adapted to growth in different batches of FBS. LB is a little bit better, but remember that a major component of LB is actually yeast extract which can differ significantly from batch to batch and company to company. Something else to keep in mind is that LB media and other types of rich media provide a wider range of niches than defined media which can promote crazy scenarios of dependence between microbes (such as acetate cross-feeding).

What is your dilution factor going to be each passage? Even though effective population sizes are calculated based on harmonic means, differences in dilution can change evolutionary dynamics within cultures. Passage to densely and your cultures will spend more time at stationary phase than if you passage less densely (unless you time things perfectly). I always try to find the dilution scheme that allows me to catch ancestral populations just after they've started to hit stationary phase at some multiple of 24 hours. For H. pylori a 1:50 dilution achieved this every other day, for Pseudomonas stutzeri (in my experiment) a 1:1000 dilution achieves this every other day. I can't emphasize this enough, for your own sanity you want to design the conditions so that you can come in and passage at regular intervals!

4. How will you archive your populations? Another powerful characteristic of the LTEE is the ability to freeze populations to create a "fossil record". Carefully consider how frequently you want to freeze, and how much of a population you will freeze. The answers here will depend on the hypothesis you are testing. For frequency, consider that frozen cultures take up space that your PI can't allocate to other projects. One of my graduate school advisors still (maybe) has my H. pylori populations frozen down in her freezer (Sorry Karen! We're BSL2 now and I can finally take them off your hands!) even though she is not working with these lines anymore. As the generations pile up, you have to allocate more and more space. As for how much of the population you'd like to freeze, just remember that unless you freeze the whole culture you will be losing some of the population. This doesn't necessarily matter for high frequency genotypes but it does for the low frequency variants. Think of this as a good example of human influenced genetic drift just like an actual passage.

5. Catastrophes will happen. You can have the best planned experiment in the world, but that doesn't prevent your lab mates from "accidentally" (shifty eyes) knocking over your cultures. Before you start, make a plan for what happens if you lose a passage or if your freezer melts. For me, I always keep the previous passage in the fridge until the next passage is complete. Sure, it's a slightly different selection pressure than constant passage...but so is going into the freezer stocks. Also remember that catastrophes happen to everyone, even Rich Lenski, and it's a part of science. It sucks at the time, but exhale and move on. Trust me, you'll be much happier in the end.

6. Can you tell if you've cross-contaminated your experimental lines? Trust me again on this, cross contamination happens so figure out ways to identify it. I always try and alternate between pipetting and passaging phenotypically different strains. For H. pylori this meant having one set of strains be kanamycin resistant while the other set was not (had to perform an extra experiment after the fact to control for this difference). However, I was able to spot one instance where one of the lines had a low frequency of kanamycin resistant colonies. In the final analysis I threw out this line, which is why there are only 5 competent lineages in my Evolution paper. You might say "well Dave, I'm not that sloppy in the lab". That could be a very true statement, but I guarantee that if you run the experiment long enough you will have other people perform the passages. People make mistakes when they aren't as invested, haven't designed the experiments, and are reading from a written protocol. They don't mean to, but it's a fact of life.

7. Be curious. I suppose this works for every single experiment ever done...but curiosity is one of the most important characteristics for research. You will grow to love your cultures, to see them flourish and change. If you understand what to expect from your cultures, you can identify interesting yet unexpected events. Know what to look for and note any changes from this search image. That's where you find really cool results.