Life lessons for synthetic biologists

1. A serious lesson

"Biology may or may not care about the physicist's insatiable desire for elegance." -- Jeff Hasty

Figure 1: In other words, sometimes this happens. From Hasty et al, Physical Review Letters 2002 | doi:10.1103/PhysRevLett.88.148101

2. Another lesson that is just as serious

Humor in lab is essential, of course. However, if you just heard the great story about the giant biohazard bag full of innocuous things in your PI's car... finish laughing before you load your gel.

The idea, of course, is to prevent this from happening. [Original photo source]

I, for one, welcome my new bacterial overlords

Over the past 24 hours, and yes, I really do mean the past 24 hours, I've been constructing a growth curve in my lab. The idea is this: there's an easy way and a hard way to tell how many bacteria there are in a culture. The hard way is to take a sample of that culture, spread it out on a plate, and count the colonies by hand (assuming each colony arose from a single bacterium of the original culture). The easy way is basically a more sciency version of "just look what color it is".

Figure 1: Shall I compare thee to a summer's day? Thy color is more lovely and more linearly related to the pH of the solution. [Source]

The bug I'm working with ferments sugars for a living, so as it grows it makes its environment more acidic. This is really handy, because we have wonderful molecules like phenol red that change color according to pH. In this particular case, yellow means acidic and red/pink means basic, so as the bacteria grow, the ratio of yellowness to redness should tell us something about how long the cells have been growing and how many there are. Here's a paper by another group that used this method, although they did so much other (amazing!) stuff that you'd have a hard time finding the details (look at figure S4 on page 41). So it really does boil down to "just look at what color it is", except that if you need to be precise, you use a spectrophotometer instead of your eyes.

The trouble is that the yellow/red ratio doesn't actually tell you anything about the number of cells in your culture, unless you take the trouble to do it the hard way and the easy way at the same time and figure out how they correlate. This is called doing a growth curve, and it's something that has to be done for every different bug if you want to measure it by any method that's easier than counting spots in a dish the day after you wanted to know.

So... while I can't say I have been in lab for the past 24 hours (I took a couple hours off last night to go get food and a nap), I have been running this same experiment for the past 24 hours and taking samples more or less every hour. It's tiring in multiple ways. I could barely make myself come back from my dinner-and-nap break because I was in physical pain from being tired. Also, having to go do something for 15 minutes out of every hour is hell on your ability to get anything else done.

When I said I had blocked out the entirety of my IAP for this research position... I guess I really meant the entirety of my IAP. All hail science!

Do I get a Home Improvement merit badge now?

I didn't grow up tinkering or making things or using tools, and this has always sort of bothered me -- especially since coming to MIT, which is full of tinkerers and makers, even in the more theoretical majors. And, the DIYbio movement notwithstanding, mainstream biological research is not exactly hands-on or building-intensive. I've learned to program a little and taken an excellent hands-on circuits class, but that's about it. So you can imagine my trepidation going into Help Week, in which the pledges of my fraternity kick the initiates out and devote lots of time to home-improvement projects.

My pet project was to install a towel bar in the women's bathroom so that people have someplace to put their washcloths that dirty water won't get all over the shelves. At the beginning, I knew absolutely nothing about how this was done. I spent some time deciphering the pictorial instructions that came with the towel bar, then went to the Internet and learned about using plastic anchors to put screws in drywall.

The trouble started when I realized I didn't know what size hole to drill in the wall. The internet said to use a bit "a little smaller than the diameter of the anchor", but it didn't say how much smaller, and the towel bar package didn't specify a bit size appropriate to the anchors that were included. So I just guessed too small to start with. After enlarging the holes once or twice, I attempted to hammer the anchors into the wall. The first one went in fine, but the other three didn't go in straight. It was here that I discovered I was using craptastic cheap anchors -- when I hammered on the last one, it bent and broke rather than going into the wall.

So now I had to figure out how to get them out. I had an idea, but I wasn't sure it would work, so I went back to the internet. It suggested (a) pulling them out with pliers, (b) pushing them all the way into the wall with a screwdriver, or (c) cutting them away with a drill in the process of enlarging the original hole. I was able to pull out three with no trouble, but the fourth wouldn't come out. So I pushed on it -- no progress. So I drilled on it -- also no progress. (Why? Was my drill bit dull or something?) So I went back to the original idea I had before consulting the internet: screw a screw halfway in, then pull it out using a regular hammer. Thankfully, that worked.

However, I was now without anchors. A quick search of the hardware closet found no extra anchors, and I was just about to start getting upset when I realized they must have been taken upstairs by the person who was working on fixing the banister. Sure enough, there they were -- actual quality plastic anchors with actual mechanical strength, able to withstand hammering, plus matching screws. And get this, the package even specified the appropriate drill bit size! I attacked the wall for the third time...

...and luckily, before I hammered the new anchors in, I thought to test the depth of the screws. Turns out they were too long for the wall. What to do now? I went back to the package and looked at all the mysterious numbers written all over it, and eventually decided that "#10" must refer to the size of the screw shaft. Then I looked through our Big Box O' Screws and found some more #10 screws that were shorter.

It was amazing how much better the new anchors worked. They went smoothly into the wall with no trouble, and I had the towel bar on the wall inside five minutes. Hey! I did a thing with tools and screws and stuff! I performed an act of home improvement by myself! Why isn't there a Science Scouts badge for biologists who step outside their academic bubble like this?

Figure 1: Hells yeah. That towel bar ain't going nowhere.

Bitesize Bio is shiny!

I just discovered Bitesize Bio, an awesome site full of discussions of common molecular-bio lab questions and problems. It has an RSS feed but is also set up with categories and menus for non-blog-style browsing. The ~5 posts I've read have all been informative, well-written, and interesting. I got a couple about interesting new techniques, a couple about theoretical questions and their practical consequences, and a couple about being a good grad student or a good mentor. It's really shiny, especially for a new lab member like me who wants to know the reasoning behind the magical incantations we sometimes do. "Wash with 0.75mL Buffer PE"? What does Buffer PE even do? ...OK, that's a cheating example -- Buffer PE is a proprietary mix from a commercial kit (but I'm told that after you add ethanol to it, as you must, it's just an improvement on straight ethanol).

For example, I just read an article about touchdown PCR, a neat hack on traditional PCR.

Sometimes in a PCR reaction you'll have trouble with the primers binding in incorrect places, because there happens to be some random sequence in your sample that's sorta-kinda complementary to your primers. Then you get nonspecific amplification of random crap, which can drown the gene fragment you actually wanted to amplify. What do you do?

You can calculate the optimal annealing/melting temperature (Tm) of your primers, and make sure to do your annealing step at that temperature. But random salts and stuff in your reaction can affect the Tm, so the calculated Tm is only an approximation. And at any temperature reasonably close to the Tm, even if it's not optimum, some annealing will happen. Maybe not much, but some. That's thermodynamics for you.

The idea of touchdown PCR is that there's a sweet-spot temperature where, statistically, it's too hot for nonspecific annealing, but just cool enough that the correct annealing can happen. You can't know exactly where this temperature is -- and you wouldn't want to run your whole reaction at that temperature anyway, because you'd only get a small amount of primer annealing and your yield would be low. So instead, for your first cycles you use an annealing temperature significantly higher than the calculated Tm for your primers (about 10 C higher). Then, in subsequent cycles, you gradually lower the annealing temperature. So in early cycles, only a few primers will anneal, and they'll almost all anneal to your actual sequence of interest, and not to random other sequences that are close-but-a-little-off. By the end of the cycle, you've amplified your correct sequence by a little bit relative to incorrect sequences. Run through several more cycles, and by the time you get to a "standard" annealing temperature where nonspecific annealing can happen, you've hopefully amplified the correct sequence quite a bit already, so nonspecific annealing becomes less of a problem because there are just more copies of the correct sequence available for the primers to bind to.

When I read about that, I was blown over. It's such a clever thermodynamics hack! -- it takes advantage of the fact that chemical reactions (DNA base-pairing or anything else) have fuzzy, stochastic behavior. For a given reaction, there isn't a sharp cutoff temperature where it goes from "too hot to react" suddenly to "OK now reaction proceeds fully". It's fuzzy. If it's too hot, a few molecules will react. Get a bit closer to the optimal temperature, and more molecules go. If you have two competing reactions with slightly different optimal temperatures (like specific and nonspecific annealing!), it's like having two bell curves overlapping, centered at slightly different values. You can find a value where one bell curve is acceptably high and the other is very low -- and then once you've amplified your chosen sequence N-fold, the game changes and you don't have to worry about the incorrect sequences nearly as much. It's like magic!

References (from original post):
1. Roux KH. Genome Research. 1995. 4: S185-194.
2. Mattick JS et al. Nature Protocols. 2008. 3(9). 1452 - 1456