Slog

You might be wondering where I’ve been lately – at least, I hope my blogging absence has been noted by someone! I have been working, every night, for free, on the EXPERIMENT THAT WILL NOT DIE. They don’t tell you, when you decide to become a scientist, that occasionally the rules of nature are suspended such that a vengeful demon can screw with your experiments. But it’s possible. I think that what I am about to describe should be required reading for every person contemplating graduate school, in order to prepare you for the long walk through the valley of frustration which is science.

The EXPERIMENT THAT WILL NOT DIE started innocently enough. About 18 months ago, I was discussing some experiments with my friend Zach. I suggested, innocently enough, doing some simple gene expression assays to see how gene regulation in E. coli had been affected by 33000 generations of evolution. It would be easy! I knew that the experiment would involve some cloning, which is one of my least favorite things to do, but just a little.

When microbiologists say cloning it is nothing so earth shattering as replicating a whole sheep. In bacteria, because the cells reproduce asexually, they are essentially making clones of themselves all the time. What bacterial cloning means is putting new genes into a living cell. The process is actually quite simple: first, you decide what genes and promoters you want to add to the cell. Then you use a PCR reaction to make a copy of that particular gene or promoter region. PCR is akin to photocopying: using enzymes and small, single stranded pieces of DNA that complement the edges of the region you’re interested in, it is possible to make billions and billions of copies of a gene. Once you have that, it’s a few simple more reactions to insert the new gene and promoter back into the cell.

That’s right. Simple. The first few clones I got quite quickly, and started producing data. But then Zach convinced me to make a new clone with an evolved promoter that was a bit longer. Sure, I said. I will work on that right away.

It is normal for the occasional thing to go wrong in molecular biology for no reason, which explains why we happen to be among the most superstitious of scientists. For example, an enzyme could go off and need to be replaced, or competent cells (bacterial cells treated with chemicals to make their cells walls porous, and thus capable of taking up DNA from the environment) can die off and need to be remade. Machines can break down in the middle of an experiment. And quite often, reactions just stop working for no discernible reason. All of these things can happen. What is unusual is when they all start happening at once. To make things more difficult, suddenly, I was tired all the time, nauseous, and clumsy. I was pregnant. This presented a whole slew of new complications, but I kept working on Zach’s clone. And working.

In July I started a new job in Calgary to be closer to my husband as we prepared for the new baby, but I did not forget about Zach and the elusive clone. I went into my old lab after hours to work on it. Enzymes failed, PCR reactions failed, competent cells died. At one point I accidentally left the frozen stocks out overnight, rendering them useless, an act that is by far the stupidest thing I’ve ever done. By September I was starting to panic – the baby was due in early November. Finally, I got a clone. I confirmed that it was the right sequence of DNA by checking its size. Correct size! We’re off to the races! Insert the new clone into six strains of bacteria, screen and do the expression assay!

The expression assay looked great at first. I had solid data from every clone but one – Zach’s new clone. This could be the result of two possibilities: either it was a non-functional promoter, which means that it wasn’t my fault, or the clone was wrong, in which case, the bad data was my fault. I decided to sequence the clone (which I should have done in the first place) to see if it was the correct sequence.

Two days later I got the sequences back and was horrified to discover that the cloned sequences did not match any known sequence in E. coli. I was stumped and horrified. The insert was the right length, so how could it be the completely wrong piece of DNA? I played with that sequence for hours, wondering if it was some weird chimera of random bits of DNA from the E. coli genome. Then, I had an inspiration. The lab that I was working in also worked on other species of bacteria, so I checked the sequence against some other species of bacteria. And wouldn’t you know, it matched up perfectly with some chunk of Salmonella DNA. And I remembered that an old colleague who worked on Salmonella used to use the bucket I was using to hold little tubes. It turns out, that bucket was contaminated with Salmonella DNA that had remained intact despite YEARS of autoclaving.

To illustrate how incredibly improbable this is, imagine that you are looking for a passage in a book. To find the right passage, you know the sentence that starts it and the sentence that finishes it. This is what primers are for: they are short pieces of DNA that complement the beginning and end of the sequence you are interested in. So imagine now that you are looking through a book with your sentences to find the right passage, but there are the ashes of some other book kicking around. And it’s in German. But somehow, your sentences match up and you end up copying that charred bit of German out, thinking it’s the right passage. Oh, and it’s also the same length as the passage you were looking for, so it has to be right. When I told my old PhD supervisor what happened, he just shook his head and advised buying a lottery ticket.

By this time, it was two weeks before the baby was due and I knew that I wouldn’t be able to get the experiment done in time. I started asking around for help, and in fact, offered a number of graduate students $100 to finish the cloning for me. But then, Darling Hannah came early, and for a couple of months I didn’t think about the clone.

By January, I was concerned about finishing, so got a friend of mine to take a stab at it (with the $100 bribe.) He tried for a month, but couldn’t get it either. I implored Zach to help, and sent him the primers: in one month he had a working clone and sent it back to me. I was thrilled – all I had to do now was insert the new clone back into six strains, and add it to the reporter/strain combination I already had and was waiting, frozen, in our -80 freezer. I paid my sister’s nanny to look after Hannah for a week and went in to do the transformations. They came out beautifully, and I went and tried to revive the strains I had already made. Oh no, not so fast – those frozen strains were dead. I’ve never had a strain just up and die in the freezer, but these ones did.

This is where things started to get really crazy. From here on in, I will use point form to go through the problems of the summer:

1. I retransformed all those combinations of strains and reporters. But the competent cells had also died.

2. Remade competent cells.

3. Retransformed. Finally got a complete panel, and moved on to the assay.

4. Set up three assays to run in three expensive machines overnight. Two broke down, one didn’t maintain its temperature.

5. Came in to set up experiment, but machines were again down.

6. Came in, set up experiment and finally got a decent set of data. Unfortunately, there were two combinations of reporters and strains that were wrong, so I’d have to retransform.

7. Went back to original frozen stocks to determine if they were also wrong. I could tell if the strain was incorrect because the contaminating bacteria grew a lot faster. So, I did a growth curve overnight to check. Growth curve from frozen culture showed that it was fine. I congratulated myself on this rare bit of luck and set up the big assay.

8. Last week, went in to collect data from what I thought was the last assay. Looked at the data. The strains had not even grown. This indicated a problem in the medium.

9. Went in and remade the medium. Growth of from frozen culture was fine, but second day of growth didn’t happen. This indicated that the problem was in the sugar I was using for the medium, because the bacteria were probably able to grow that first night off of the glycerol that’s added to frozen stocks as a cryoprotectant.

10. Went in and remade the medium again, this time with a new sugar stock. I got things to grow on the second day, so once again I set up the assay.

11. Inexplicably, two of the strains didn’t grow at all, and the other two had growth curves that varied widely from well to well. this indicated that maybe my earlier determination that the original frozen stocks were correct was wrong, probably because I did that growth curve using the bad medium. So now, it looks like I have to go in and retransform all the strains. Again.

At this point, this stupid experiment that should have taken a month has taken 18 months and cost me at least $300 in combined childcare and parking costs, and I’m not even getting paid to do it. Everyone is asking me why I don’t just throw in the towel, and to be honest, I don’t have a good reason other than complete stubbornness. Now, it’s a matter of pride: I can’t let this stupid, simple experiment beat me! Hopefully, in two weeks it will all be behind me, an unpleasant memory. More likely, 50 years hence someone will find me in my old lab and have to pry the pipet from my cold, dead hand. I think both the Experiment Demon and I are extraordinarily stubborn, and we’re both in it for the long haul.