Note: This blog post isn't done yet. It was basically a piece I started to throw all of my creative juices into while I kept my lab report as boring and academic as possible. However, what I'm doing in the lab (a 4000-level microbiology course) is anything BUT boring.
For the past two years, I’ve been eyeballs deep in Biotechnology studies at university. In those two years, I’ve gotten a summary of basically everything scientists know about biology… which isn’t really a whole lot.
Physics, mathematics, and even chemistry are pretty well understood these days. After all, scientists began to revolutionize mathematics as early as the 1400s, though algebra has been known about since the Babylonians started solving quadratics in 2000 BCE. Once Calculus was developed in the 1500s and 1600s, the study of Physics took off, culminating in the invention of the nuclear bomb. Alongside Physics, Chemistry was also experiencing a sort of revolution, since understanding atomic bombs and space travel required a solid understanding of Chemistry, too.
However, Biology- the scientific study of life- was pretty much left on the back burner for that whole time. Until the 1970s and 1980s, most of the technology required to study biology at a molecular level (where all of the fun stuff happens) didn’t exist. But even then, the technology to study biology at the molecular level wasn’t cheap or accessible enough for most scientists and researchers to obtain until the late 1990s and early 2000s.
Even still, everyone- from the general public, to the government, to the scientists themselves- were terrified of advancing biotechnology, especially after several major, high-profile fuck-ups that resulted in patients dying in horrific ways.
All that basically explains why the study of Biology and the development of Biotechnology have only started to see some major progress in the past 25 or so years.
Crazier still, I get to have a part in advancing this knowledge in one way or another, as a lowly undergrad, right now.
As soon as the Fall 2026 semester began, I was basically thrown out of the frying pan and into the fire by my Microbiology lab professor. A sweet yet very quiet woman with a thick Indian accent. Since everyone in the lab was desperate to be there, I did not get first dibs on a seat up front. So, I found (and still find) myself sitting towards the very back of the lab room, straining my ears to hear my professor's instructions.
Personally, I was expecting the first day of lab to simply be a lecture introduction to the class. Instead, right away we were told to take out our professional-style lab notebooks, don some rubber gloves, and start fucking around with E. coli.
Ok, it wasn’t that sudden. We were given about 10 minutes to refamiliarize ourselves with the micropipettes using water before we were asked to jam those into some actual samples of E. coli. But once we showed the professor that we could successfully configure and use each of the different micropipettes, she gave each of us our own sample of E. coli to work with, along with the DNA we wanted to jam into the E. coli.
Understandably, E. coli is a scary word. It’s a bacteria that makes people violently sick, and can even lead to death. Why, then, mess with E. coli, and was I just asking to be another Patient Zero for the next Covid-style pandemic?
First off, unlike Covid, E. coli is a bacteria that cannot get airborne unless one snorts an entire liquid sample of it and immediately sneezes it out. Even then, E. coli can’t really get into the system via the respiratory system (nose and lungs). It has to be ingested, or somehow get into one’s bladder. E. coli exists in everyone’s digestive tract and feces. It just doesn’t always cause infections.
In other words, every time someone takes a dump, they risk getting an E. coli infection in their bladder if they wipe the wrong way, though that risk is very low. It is even lower in the lab.
Every college lab in the United States follows extremely strict safety protocols. I had to take several other lab courses, along with two years of lectures about microbiology, before I was allowed to get into the E. coli lab. Had I done anything stupid in those previous labs, I would not have been allowed into the E. coli lab at all.
Long story short, as long as everyone wears gloves and nobody licks the sample, working with E. coli in the lab is safe. After all, nobody- especially those of us who took a handful of microbiology courses and learned about all of the terrible things that can go wrong- wants to get sick.
So, given that the risk of me starting the next diarrhea epidemic was (and is) very low, what was I trying to do in this experiment with E. coli anyway?
Along with a small vial containing a sample of E. coli cells, we were given another vial containing a little piece of DNA called a plasmid.
What is a plasmid? A plasmid is basically a tiny ring of DNA that can hang out in the environment without being inside of a cell. Plasmids are how bacterial cells protect themselves from viruses and antibiotics, but I’ll explain that a little later.
Anyway, what the hell is DNA?
While DNA is a popular buzzword, few people actually know what DNA is or does. Just like in the movie, Idiocracy, everyone knew the buzzword “electrolytes”, but nobody actually understood what electrolytes were (for those curious, electrolytes are basically any atom or molecule that can conduct electricity).
Simply put, DNA is a large molecule called “Deoxyribonucleic Acid”. There are four different types of smaller molecules that make it up: Adenine (A), Guanine (G), Thymine (T), and Cytosine (C). These letters form long chains with each other to make the helical structure of DNA. Aside from DNA, there is RNA which stands for “Ribonucleic Acid”, which usually hangs out as a singe strand. RNA and DNA are both made of A’s, G’s, and C’s. However, instead of using Thymine like DNA does, RNA uses Uracil (U).
Why is it important to know this? Because, DNA and RNA work together to make products, which are proteins. RNA essentially copies down the instructions provided by DNA, turning G’s into C’s, T’s into A’s, and A’s into U’s. Proteins are the structures our cells create, which our bodies rely upon to stay alive and healthy.
Our skin is made of various proteins, as are our muscles and our hair. Our eyes are made of proteins, as are our stomachs and our hearts. The list goes on. Health problems arise when the correct proteins aren’t being made.
How do DNA and RNA work together to make a protein?
DNA is like the “boss”. DNA contains all of the instructions (genes) to form a living body. RNA is like the “secretary”, which writes down everything the boss (DNA) wants to make. RNA then leaves the boss’s office (AKA the nucleus of the cell), and goes to the factory which are little things in our cells called ribosomes. The RNA tells the ribosomes (AKA factories) to make the products (proteins) the DNA wants to be made.
This is called the Fundamental Theory of Molecular Biology:
DNA→(transcription)→RNA→(translation)→Protein
Everything that goes on- both right and wrong- in biology, relies upon the shoulders of DNA, RNA, and proteins.
When DNA forms into little rings outside of living cells, we get plasmids. Plasmids exist everywhere in nature, and are how bacterial cells become resistant to viruses and antibiotics.
How do bacteria use plasmids to become resistant to viruses and antibiotics?
Basically, when a bacterial cell dies following a battle with a virus or an antibiotic, it leaves behind little bits of DNA- many of which are plasmids- that can tell other bacterial cells how it died. The surviving bacterial cells can then take in those plasmids and incorporate the plasmid DNA into their own DNA, so that they can survive what the other cell didn’t.
This process is known as cellular transformation.
How exactly does cellular transformation work?
First, a living bacterial cell must come across a dead bacterial cell that has left behind a few plasmids. The living bacterial cell then reads the DNA from the dead cell and “learns” about what killed the cell, and how to not die from the same thing. If the cell determines that some of the information is useful, it uses various enzymes (AKA Molecular Scissors) to cut open the useful DNA plasmid so it is a string instead of a ring.
Then, the bacterial cell cuts its own DNA open with the same enzymes, and through a series of very complicated DNA-healing steps, can integrate the plasmid DNA into its own DNA. That way, when the transformed living cell comes across the same thing that killed the other cell, it can defend itself from it.
Many plasmids come equipped with molecular scissors that can chop up DNA and RNA from outside sources, the most notable one being an enzyme called Cas9. Enzymes such as Cas9 can read DNA and RNA that the bacterial cell comes across and takes in from its environment. If a virus attempts to hijack a transformed cell, the cell’s new Cas9 scissors can be used to chop up the viral DNA/RNA, preventing the virus from hijacking the cell.
Since 2012, scientists have figured out how to make Cas9 cut specific portions of cellular DNA, so that new DNA sequences- made by the scientists themselves- can be incorporated into a cell’s genome, permanently.
This technique- colloquially known as “CRISPR”, which stands for Clustered Regularly Interspaced Short Palindromic Repeats- has been used to cure genetic conditions such as Sickle Cell Anemia and an unknown metabolic syndrome in a baby named KJ in 2025.
Indeed, CRISPR has been, is being, and will continue to be used to literally cure genetic conditions!
Back in the lab, I was tasked with getting my E. coli cells to take in a plasmid that contained two genes: a gene that would give them resistance to the antibiotic Ampicillin and a gene that would allow the E. coli to glow green under blacklight, called the Green-Fluorescent-Protein (GFP) gene.
The GFP gene comes from bioluminescent jellyfish, and was first used for microbiology purposes in 1994. GFP helps scientists mark a specific protein or organelle within a living cell, and see what it does under a blacklit microscope in real time. It also allows scientists to measure the length of specific fragments of DNA in conjunction with another technique known as Gel Electrophoresis.
The GFP gene was directly stuck to the Ampicillin-resistance gene, guaranteeing that if my E. coli cells could grow in an agar plate laced with Ampicillin, they would also glow green under blacklight.
How was I going to force my E. coli cells to take in the GFP plasmid?
While the process is, on paper, simple. In practice it’s very tedious, but still easy enough for a lowly undergrad like myself to do.
First, I had to thaw out and mix the E. coli DNA and the plasmids together in a single tube, just like I was mixing waffle mix and milk in a bowl to make pancake batter.
Immediately after mixing my E. coli and plasmids together, I cold-shocked them by putting their tube in ice for exactly two minutes, immediately put them in a hot water bath for exactly 30 seconds afterwards, and returned them to the ice for exactly two minutes following the hot bath.
After that, with much less urgency, I added a handful of buffer solutions to my E. coli + Plasmid solution to stabilize the shocked DNA, then spun them really fast and hard in a centrifuge to separate the junk from the DNA I wanted. The stabilized DNA (which included the plasmids and the still-living E. coli cells) floated to the top of the tube, while the unwanted junk (such as dead E. coli cells and excess buffer solutions) formed a nasty little pellet at the bottom.
I transferred the less-dense liquid containing my wanted stuff into a new vial, used a couple different wash buffers to purify the solution, then added a sugary, nutrient-dense goo to whatever was left of my living, hopefully-transformed E. coli cells.
Finally, I added that sugary, gooey mixture of transformed E. coli cells to three different agar plates to grow cultures of the stuff. One agar plate simply contained the agar (which is just a fancy term for unflavored Jell-O) for anything and everything to grow on. One agar plate was laced with an antibiotic called Kanamycin, which was pretty much guaranteed to kill everything that touched its plate. The last agar plate was laced with Ampicillin, upon which only my transformed E. coli cells would grow upon.
All three of those agar plates were put into an incubator to grow for two days.
While having multiple plates may seem redundant and even wasteful, each plate served a specific, important purpose.
The first plate- the one that wasn’t laced with any antibiotics- was my “positive control”, meaning that unless something went horribly wrong, something was guaranteed to grow on the plate. The second plate was my negative control, meaning that unless something went horribly wrong, nothing was gonna grow on that plate. The third and final plate was my actual experimental plate- the plate that would only grow the E. coli that I wanted it to grow- which was my successfully-transformed, glow-in-the-dark, Ampicillin-resistant E. coli.
To be continued...
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Note: I wasn’t sure where to put this piece. I sat down intending to write a continuation of my E coli story. But instead, this came out instead. I will keep this piece at the end of my E coli story, and may even try to integrate it. But, I'm not sure. My brain does weird things sometimes.
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Lately, I’ve been feeling exceptionally alone and afraid. I’ve come to the expected-but-still-very-scary realization that, as intelligent and knowledgeable as the “adultier adults” in my life are, I know a lot of things that they don’t. I chose to study biotechnology at university, which is a field that was entirely science fiction until the 1970s.
Even then, it didn’t get much attention until 2012, when scientists such as Dr. Jennifer Doudna, Dr. Feng Zhang, and Dr. George Church figured out the mechanism behind how bacterial and prokaryotic cells protect themselves from viruses and antibiotics, known colloquially as CRISPR, which uses an enzyme called Cas9 to chop up DNA for alterations. Up until 2012, we knew a lot about genes and genetics, but almost nothing about how to fix fucked-up genes and genetics. But the discoveries made in 2012 suddenly made gene-editing a reality.
A little over ten years later, I’ve become one of the very few people in the world to successfully harness CRISPR/Cas9 to make simple E. coli cells glow green under blacklight using a gene originally found in jellyfish.
Trying to explain that to literally anyone outside of my Biotech classes is damn near impossible, it seems. I almost feel like I’m doing some Manhattan Project shit in the 1950s, even though I can freely discuss what I’m doing. People just don’t understand it, including my own friends and family.
Over the weekend my dad, smartly, challenged me to explain what I was doing in my microbiology lab course in a way that even his scientifically-illiterate ass could understand. After some thinking, I managed to liken a cell to a company, with DNA being the boss, RNA being the secretary (or the human printer), and everything else being the product makers and managers.
Luckily, once I put cell biology in those terms a construction manager like my dad could understand, he understood the gravity of what I was doing pretty much right away.
“So, just so I understand, you basically genetically engineered a jar of E. coli to glow green, so you could see what it was doing, and how long its DNA was, in real time.”
“Yup!” I answered him, “And this is just the beginning of my cell biology projects. In about a month, I’ll be CRISPR-ing a mammalian cell.”
“So you really are on the cutting edge of this shit, huh?”
“Pretty much.”
“Doesn’t that freak ya out a bit?” my dad asked.
“Oh abso-fuckin’-lutely! But if I don’t do it, who else will?”
But if I don’t do it, who else will?
To be continued…