Image Credit: fineartamerica.com
I’d like to talk a little bit about my “qualifications.” Academically, I’m currently at an undergraduate level of biology, but that’s nothing to write home about; I can’t really say that I’ve been overly excited by what’s being taught. My professors are talking about the CRISPR-Cas9 system as if it was invented yesterday (spoiler alert, it wasn’t).
What I want to talk about instead is my experience in biological research. I’ve done a thing or two if I say so myself. Here’s a short list of the laboratory techniques that I’ve been taught:
- Polymerase Chain Reactions
- Gel electrophoresis
- DNA subcloning
- Bacterial transduction
- DNA isolation
- Human cell transfection
- Tissue culture of cells
- Cell haemocytometry
- Infection of HEK293T for lentivirus production and purification
- Con focal microscopy
Well, that probably doesn’t mean that much, but I thought it was worth mentioning.
My personal experiences might be slightly more interesting/comprehensible, so let me tell you about my life as a student scientist so far. Stay awhile and listen maybe.
Summer 1: YIP @ UNC
That’s how it ended. No, no, not the whole thing, sorry. I’m not dead yet. That’s how the first half of it ended. This picture was taken at the concluding “conference” of UNC’s Young Innovators Program. I’m in this picture. It was 2016, and I had just spent pretty much the entire summer as an intern in the laboratory of Dr. Nathaniel Hathaway. Great guy by the way. I didn’t get to interact with him much as he was the Principal Investigator of the lab, or the Boss (which made him pretty busy), but he wrote me a college recommendation. I didn’t read it due to my waiving the right to at the urging of my college counselor, but I suppose it was a good one since I got into the school that I wanted to.
I’m greatly indebted to this program, as it gave me a superb introduction to laboratory science. In addition to teaching me the majority of the techniques listed above, it gave me the skills necessary to begin interpreting scientific literature on my own. As a side note, the labs were extremely nice, although I thought this was the norm until I saw other, less up-to-date labs.
So how about that lab stuff?
The objective of my research project that summer was pretty simple: compare the effectiveness of different transcriptional activators (which are proteins) in “turning on” a particular section of DNA, called the Oct4 locus, which is usually “turned off” in a cell. Oct4 locus just means that that is the spot where the Oct4 gene is located.
To test our transcriptional activators, we had to be able to do three things: deliver the transcriptional activator genetic instructions into our cells, make sure they got to the Oct4 locus, tell if the transcriptional activator had turned on the locus.
We used modified HIV to deliver genetic instructions into our cells. To make sure the transcriptional activator, once made, went to the Oct4 locus, we made our genetic instructions encode a protein “puzzle piece” with the activator stuck to it, and modified the genome of our cells so that the complementary puzzle piece (which is a sequence of DNA) was right next to the Oct4 locus.
The more technical terms are Gal4 (protein) and Gal4 DNA binding domain (the DNA). This is part of a system called the Gal4/UAS system, and it’s very handy as you can see.
How did we know how whether the transcriptional activator was doing its job or not?
Good question. There’s two things you need to know. Remember I told you that the Oct4 locus is usually inactive, meaning whatever the DNA there has instructions for isn’t being made. Secondly, the cells were modified in another way. Their Oct4 locus had been edited so that instead of the usual stuff, instructions for a special protein called GFP (Green Fluorescent Protein) were pasted in.
As the name suggests, GFP glows green, though only when exposed to ultraviolet light. So cells with activated Oct4 loci (plural of locus) would produce GFP, and glow green, providing a measure of how effective the transcriptional activator was (we pretty much assumed all cells were infected, as we used so much virus).
For your gratification, below is a diagram which hopefully explains the process better as a whole. Just an FYI, for the sake of simplicity, I skipped drawing the steps of reverse transcription (HIV has an RNA genome, which gets turned into DNA before host genome integration).
So uh...did you count all those green cells yourself? Is this what I have to look forward to as a laboratory intern???
Ah, thankfully not. We’d feed them into a machine called a flow cytometry machine to do that. I dug up some of the flow cytometry data from my project, take a look.
Each data point represents the measured values of a single cell (i.e size and fluorescence). The important bit here is that the dots/cells within that irregularly shaped polygon are those that were fluorescence within the frequencies normal for GFP. In other words, you can quickly tell how effective the transcriptional activator was by seeing how many dots were within that outline. All the graphs...
[besides the bottom middle and left, which are negative controls, meaning they were a culture of cells we made that we definitely knew weren’t going to have any green cells]
...depict data from cultures that we introduced the VP16 transcriptional activator on. The reason why you see pretty big differences is we added variable amounts of another molecule, called 5azac, which helped complement the effects of the VP16.
Besides just raw experience that I took away from the program, here's a few dearly bought insights and lessons from that summer:
Research takes time
When I was at the program, I interned (unfortunately for me, students only started getting paid the summer after I had participated) from 9 to 5. What did we do most of the time besides shiver in the intense AC? Wait. The various protocols we do throughout the day have a lot of down time. Samples need to spun in centrifuges, occasionally for hours on end. Cell cultures needed to absorb reagents, stringy soups of lysed E. coli needed to be filtered for plasmids. Another big one was running gels (the thing above), where DNA fragments are separated by size in a gelatin substrate, propelled by an electric current. Sure, we worked too, feeding cells, preparing PCRs, passaging cell cultures. But I’d say for about every half hour we were actively working, we had to wait one.
Don’t sniff the ethanol
You’ll notice that’s on the banner of my blog. For good reason too. That summer, I was taught how to do tissue culture using sterile technique. That basically means I was taught how to take care of dishes of cells without allowing any contamination to occur. Harder than it sounds. For sterility, you do all your business that requires taking the lids off of cell cultures within something called a biological safety cabinet (picture). No, I’m not trying to Rick-Roll you, I just couldn’t find any of my old pictures so I pasted a link to Google images.
There’s a vent at the opening of the cabinet which constantly sucks air into it when the sliding glass pane is raised. This prevents outside contaminants from entering.
It’d be a shame to go to all this high-tech trouble to keep things clean but carry germs inside with your grubby hands, so you always make sure that they're sterile before you put them inside the cabinet. This is where the ethanol comes in. I’d spray my gloved hands down with the stuff every time I was going to put my hands in the cabinet. Every time. Even if I’d only taken them out for a couple of seconds to do something like grab another 10 mL serological pipette. My preceptor was very strict about this.
That summer (and my career so far), I never contaminated a single cell culture, something I am pretty proud of. However, that came at the cost of deciliters of ethanol evaporating in my face daily. I ended up getting what I called “ethanol headaches” pretty frequently. It was halfway through the summer when I finally had the genius idea of actually not inhaling the clouds of gaseous 70% ethanol. Don’t sniff the ethanol kids. It’ll save you many headaches, literally and figuratively.
Beware of glass pipettes
Oh gosh. “DON’T recap used needles” is one of the major safety rules in laboratory science. Reason is, it’s very easy to stab yourself doing so.
I like to see myself as something as an innovator, so I stabbed myself once with a broken glass pipette. Not intentionally, mind you. I was sucking out the media (cell food) of cell cultures that had been infected with virus a few days ago. The media had already been changed once before, meaning any loose virus had probably been suctioned out along with the old media, so my preceptor let me do it this time. I managed to break the tip of the glass pipette (it’s very thin, almost as thin as the lead on a mechanical pencil). No big deal, I reached to pull the disposable pipette out of the plastic suction tube it was connected to when suddenly, my left index finger made contact with the now-very-sharp broken end of the pipette.
*Internal screaming*
Uh oh.
I quickly took my hand out of the biological safety cabinet, pulled my gloves off, and checked for blood. There was just a little pinprick of red.
*internal screaming intensifies*
So at that moment, I had just potentially exposed myself to the modified HIV. This isn’t actually as bad as it sounds as barring a few unlikely events, the HIV was modified specifically so it could not replicate; the genetic instructions for making new virus aren’t there. In other words, it’s very unlikely that I’d develop AIDS in the future.
And that’s assuming that there had been virus present at all: it would have had to avoid infecting and entering one of the cells in the culture, escape being flushed out with the first media change, and be present at the pointy end of the broken pipette. All considered, it’s very unlikely that any real exposure occurred, but that episode taught me to be cautious around glass pipettes, broken or not.
[Edit 6/3/21]: A 2019 HIV test came back negative, fortunately. Not today, AIDS.
Next up: not just hypersharp needles, virus-coated ones!
I spent most of last summer at a government institution that will remain unnamed. Ironically, given my previous experience with sharp glass bits and viruses, I was working with hyper-sharp glass needles whose pointy ends had been purposefully coated with virus. I’ll tell you guys about it next time.
Thanks for reading!
- BJW
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