Few experiments in science are conclusive. So, it is very exciting when an experiment provides completely unambiguous results. Today that happened.
Molecular biologists use bacteria–specifically strains of E. coli that don’t make people sick (i.e., non-pathogenic)–to make lots of copies of circular DNA, called plasmids; one can think of E. coli as a photocopier for plasmids. Bacteria love to take up plasmids, copy them, and express them to make proteins with unique functions, and it is this ability that makes them so evolutionarily successful. Bacteria can transfer plasmids containing antibiotic resistance genes to each other, for example, and in this manner, can become “superbugs.” Staph is one pathogen that has done this so successfully in hospitals that we will soon run out of antibiotics to treat it.
Interestingly, molecular biologists give E. coli antibiotic resistance genes on purpose. It’s not because we’re bioterrorists. Rather, we want to be able to SELECT for those bacteria that take up our plasmid of interest and copy it. So we make sure that the plasmid DNA that we give the E. coli to copy also has a gene that codes for a protein that confers antibiotic resistance; ampicillin resistance is the most common. We take this plasmid, mix it with the E. coli, and warm the E. coli slightly. This creates little pores in the E. coli that allow the plasmid DNA to pass through. The process–called heat transformation–is not very efficient, and most of the E. coli don’t take up any plasmids. We don’t want to grow these E. coli because they are useless to us; we only want to grow the ones that took up our plasmid. So, we add some ampicillin, and only those E. coli that took up our plasmid DNA can survive. As a result, we end up with a bacterial culture that is loaded with our “photocopied” DNA. The resulting plasmid DNA can be used for many things; for example, DNA that codes for insulin can be copied in E. coli, purified, and then put into mammalian cells to cause them to make insulin, which can be harvested and given to diabetic patients.
I recently wanted to use E. coli to “photocopy” some plasmid DNA that I got from another researcher, but the researcher wasn’t sure which antibiotic resistance gene for selection was employed in the plasmid (not good record keeping). He thought it was either ampicillin or kanamycin. So, I transformed E. coli with the plasmid and tried growing the bacteria in both ampicillin and kanamycin. Take a look at the picture below. It’s pretty obvious that the bacteria survived only in the kanamycin (yellow cloudy suspension on the right), indicating that the plasmid coded for kanamycin resistance. The bacteria in the ampicillin died quickly and did not grow.
The strange thing is that, despite our suggestions otherwise, the Nature folks chose a not-the-most-interesting figure from the paper. Of course, I’m more than happy that they showed any of our awesome figures! But, instead of showing one of the super-resolution images that Hsiao-lu made, the highlight shows a proof-of-labeling image, which is diffraction-limited. That said, they did select one of the live-cell images. I suppose it could be worse: they could have picked one of the controls. Or not displayed a figure at all.
Thanks Nature. I don’t mean to look a gift horse in the mouth.
Well, I’ll highlight our paper here. And choose my favorite figure (it’s protein localization in a little-bity bacteria):
But what is a blog for anyway? Advertising! Here’s my most recent paper:
Sorry for the lame post, but I’ve been too busy to write cool things…
I’m coming back from a wonderful single molecule Gordon conference in the picturesque hills of New Hampshire. Richard Zare gave a fun talk that seemed to be a hit with the crowd. To commemorate, I thought I’d put out his seminal paper in the field of non-linear optics.
The authors make sure to consider the radical implications of their results:
UPDATE: Dan M writes:
The paper you refer to as Dick Zare’s article was actually written by Wayne Knox, not by Zare. Wayne persuaded Zare and Hoose to be co-authors so that the author list would be amusing. In fact, Wayne didn’t actually know Hoose before this; he had to hunt through various directories in order to find somebody in the field of optics whose name sounded vaguely like “whose”. The other Knox, by the way, is his dad, who was a biophysics professor for many years.
The basic idea is to use an an azide to disrupt the push-pull character of a known fluorophore, rendering it dark. Because the azide is photoconvertible to an electron donor (the amine), you can photoactivate fluorescence.
Anyway that’s all. Just wanted to talk about myself a little. Which is what a blog is for.
Just a picture of my nice column (thanks for the help and the hood-space, Nick):
Some photoproducts of a cool reaction that I’ll tell you about when it’s published.
Sometimes it’s just entertaining to look into a microscope. I like this pic I took the other day:
It looks like a tiny sun. But it’s just a fluorophore solution that has dried up and left some large aggregates (which emit at a longer wavelength—the green is the normal emission—maybe Excimer will like this, at CBC’s new website).
You can also see a bleached region in the middle of the green from the peak of the laser excitation region, and a swath of bleached dye where I moved the stage up and down before the picture. You might even make out some single molecules in the center. Quite impressive for a simple digital camera!
This was actually before I aligned the beam for real experiments, so all the ring patterns are actually interference due to poor alignment. I just thought that the misalignment made it pretty, so I took a color picture.
No, not these.
Another, and the setup:
It’s a pretty simple experiment: bleaching a vial of dye to determine what the fluorophore photodegrades into. I bubbled some air to make sure I get the reaction with oxygen.
Several days ago, I wrote a note on my hand to remind me to do something (fix the house N2 system, although you wouldn’t be able to tell from my scribbles). I wrote the note in red pen (a Sanford Uni-Ball micro1) and it disappeared in a few days.
Figure 1. My hand in room lighting.
Or so I thought! Today—days after the mark faded completely—I was looking at some dye solutions under a UV lamp and (voila!) there were the marks again.
Figure 2. My hand under UV illumination. (click to zoom)
Pretty cool. Unless you’re worried about degraded ink remaining under your skin for weeks.
1 By the way, don’t use these pens, even on paper. I’ve been using them for about a year, and just yesterday I discovered that the ink is very much not waterproof. I spilled water across my desk. Now none of my to-do lists make any sense.
A new twist on an old favourite. To make the NoChromix volcano, add one package of NoChromix® to your favourite H2SO4 and stir. Insert a cherished piece of glassware contaminated with whatever the hell pissed off the bath, slam the sash, and enjoy!
(I know what you’re thinking, but I scrubbed the hell out of those filters, rinsed them with nitric, then triple rinsed them with H2O before putting them in the bath).
What is going on here? I woke up the last two mornings to look out the window only to see pigeons in queues in the field across the street:
This is strange. Upon closer inspection, it was revealed that they were eating along an old faded white line on the soccer field. I tried finding some mention of this phenomenon in The Google, but to no avail. The closest we found were blue jays eating house paint (here and here) and sparrows eating grit. Maybe this is the same thing: pigeons eating field chalk for grit or minerals. But they were only on one older faded line. Maybe the new lines are a different paint (to avoid this problem). Or maybe the field paint made some yummy type of grass grow there. Still unclear to me. Anyone else know what’s going on here?
Dylan at tenderbutton is always posting about his beautiful crystals. But I’ve one-uped him, because I did it the lazy way and with no usable results: I made crystals of my dyes by letting the waste beaker dry in the hood. I know, that’s a lab-safety no-no, but it was a mistake. And the results were cool!
Figure 1. The crystals under room lights.
Figure 2. The crystals under UV illumination. Note the heterogeneous fluorescence, a neato result of mixing a bunch of dyes and solvents together and letting evaporation do all the work.
I’m just finishing up a class on Fourier transforms, and the discreet transform has me all gooey about sampling. Have you ever noticed that computer screens shown on TV sometimes have a line moving though the picture? It’s a sampling error. The frame rate of the TV camera is similar to the refresh rate of the computer screen. As a result, the refreshing sweep is visible. It’s like strobing a light with exactly the same frequency as a spinning fan. The result to your eye is a stationary fan. Check out the link in Figure 1.
We use a few parabolic mirrors on our optics table to align and focus a couple of beams to a tight spot. It’s important that these mirrors be well aligned because power density is really important for our (non-linear) purposes.
The mirrors need to be replaced from time to time. Last week the big 2 inch mirror was removed, as shown in Figure 1. Replacing the mirrors can be a little
Figure 1. It’s dirty, also the coating is improper.
worrisome. Of course, there is no signal without the mirror in place, and proper alignment can be difficult because one of the beams is almost invisible. No worries, though. Once the system was working again, we got twice the previous signal, as shown in Figure 2. 2x is mighty fine.
Figure 2. Sort of hard to tell if this is good or bad. It’s OK.
Some Data I took recently with David Pearson – cool, huh?