Biology for Writers: Some Awesome Genetics Techniques I Use in the Lab

Good morning, everyone! Welcome to the first Biology for Writers post of the new year! Today, I wanted to talk about some common techniques used in genetics and molecular biology, particularly those I use in the lab, since those are what I’m most familiar with. As a result, the list will probably be skewed toward plant biology, but I will include other techniques I’m somewhat familiar with too. If you’re interested in a specific discipline or something you don’t see listed here, let me know in the comments, and I’ll be happy to tell you what I know or help you find some resources! Let’s get started!

DNA/RNA Extraction

Why? If you’re working in genetics or molecular biology, you often need to isolate a nucleic acid, be it DNA or RNA. These molecules are useful for learning about genes and the various proteins, including enzymes, they produce (more on this another time).

Why DNA vs. RNA? Well, the DNA contains a gene’s complete sequence, including introns (which are removed in the messenger RNA that determines the protein’s amino acid sequence) and regulatory sequences that control when the gene is turned on or off. So if you’re interested in the pure molecular genetics of an enzyme, you might extract DNA. DNA is also useful if you’re trying to sequence a new genome (the complete DNA of an organism); genomes are often useful for future research.

DNA
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RNA, on the other hand, might be useful if you want to, say, produce a human protein in bacteria. To do this requires gene cloning, which is much easier with only the coding sequence (the part that actually codes for the protein–i.e. messenger RNA) rather than all the extra introns and “junk DNA” that comes along with a complete gene. So for things like genetic engineering, it’s better to extract RNA and back-convert it into DNA (more on this in a minute).

How? Extracting DNA/RNA is, in a nutshell, getting it out of the cells of your plant or bacteria or whatever organism you’re starting with, and separating it from the proteins, fatty acids, and other stuff that also belongs in a cell. (If extracting DNA, you also want to separate it from RNA, and vice versa.) The first thing to do, then, is to break open your cells. In plants, you usually start this by freezing your leaves in liquid nitrogen, which helps minimize molecular degradation, and grinding them up in a mortar and pestle. Then, a lysis (cell-opening) buffer is added to fully lyse the cells. (Buffers are solutions with a specific pH that “buffer” against big changes in acidity.) During this process, nucleases, which degrade DNA, are inhibited by the cold temperature from the liquid nitrogen. Other chemical inhibitors may be added as well.

Next, a series of centrifuging steps separates the DNA (in various liquid buffers) from proteins, lipids, and other cellular detritus (often in the form of a pellet at the bottom of the little test tube). DNA in the top liquid, or “supernatant,” is removed into several different tubes, until ultimately it is collected without the liquid on a little pad within a special tube. The DNA is then resuspended, or “eluted,” in super-sterile water (a small amount, like 150 microliters–a microliter is a millionth of a liter). This serves to make the DNA more concentrated. You can then measure how much DNA you extracted with an instrument called a fluorometer, and if you got enough DNA, you’re all set to go on to the next step!

Polymerase Chain Reaction

Why? Once you’ve extracted DNA, what do you do with it? If the answer is “isolate a gene,” your next step is polymerase chain reaction (PCR). PCR is an “amplification” technique that takes advantage of DNA’s structure to make many copies of your gene of interest. Then, you can move on to DNA sequencing, genetic engineering, or something else.

How? Let me first make a small digression into the basics of DNA’s structure. DNA is made up of four kinds of nitrogenous bases (let’s just call them A, T, C, and G) that join together in two long strands. The bases pair in a complementary manner to form that familiar double helix shape; A only pairs with T and C with G. When I talk about “DNA sequence,” I’m talking about the order of bases; AATCG is different from ATACG, for example. “Complementary sequence” refers to the sequence of bases on the other strand of the DNA; each DNA strand is the opposite of the other. For example, TTAGC is complementary (opposite) to AATCG. Got it? Great! Let’s go on to the technique.

PCR exploits DNA’s structure to make many, many copies of a gene or other sequence of interest. You can find a great fact sheet about PCR, including a helpful figure, here. Basically, if you know the sequence of your gene of interest, you can order custom short DNA molecules, called oligonucleotide primers (or just primers), of 15-25 base pairs each which are complementary to the ends of your gene. In a tiny little test tube, you mix together DNA, water, primers, and free nucleotides, and stick it all in a machine called a thermocycler, which can be programmed to heat and cool the tube for certain times. The heating profile basically does the PCR.

There are three basic steps to PCR. First, denature the DNA (separate the strands) by heating to about 95 Celsius. Next, drop the temperature to about 60 Celsius for annealing (of primers to DNA). The last step is extension (addition of new nucleotides to make a new DNA molecule), which happens at around 70 Celsius. Together, these steps make one cycle. PCR is usually repeated in about 36 cycles, which because there are more templates with each cycle, makes thousands of copies of the gene of interest, hence “DNA amplification.” PCR is a very useful technique and is indispensable in most genetics laboratories.

In fact, a form of PCR called reverse transcription-PCR (RT-PCR) is used to back-convert extracted RNA into complementary DNA (cDNA). RT-PCR uses primers that are specific for messenger RNAs, enabling the isolation and amplification of only the coding sequence of an organism’s genes. This is the next step from RNA extraction (see above).

Gel Electrophoresis

Why? This is a sort of confirmation technique. Once you have your extracted DNA/RNA or your PCR products, you want to make sure you did your previous techniques right, so you run an agarose gel. This helps you determine the size of your DNA fragments (or genomic DNA in the case of an extraction), so you know you can proceed with the next step in your research.

How? Gels are made from agarose, a compound found in seaweed, and buffer. You pour molten agarose into a rectangular mold with a comb stuck in, so when it hardens and you pull the comb out, you have a flat, rectangular Jell-O like surface with a line of wells at one end. After pouring buffer over the gel so it’s under the surface of the liquid, you mix your DNA/PCR product with a blue loading dye and put one sample into each well (called a “lane” once you’ve run the gel). You should always load a size standard, called a “ladder,” into one lane, and it’s good to run a positive control (you know the result will be what you want) and negative control (you know the result will be the opposite of what you want), so that if something’s wrong with your samples but not the controls, you know it’s the samples themselves, not your PCR.

Once you’ve loaded all your samples, the next step is to turn on the electric current and let the gel run. Because DNA has a slight negative charge, it will migrate toward the positive end of the gel (away from the wells in which you loaded it). As it moves, it will bump into all sorts of molecules within the gel, i.e., the gel itself will get in the way of the DNA’s movement. This allows for size separation of DNA fragments; smaller fragments will bump into the gel less and therefore move a greater distance in the same time. When you look at the gel under ultraviolet light, you see bright “bands” that indicate where the DNA is; the closer to the wells, the longer the DNA fragment. Comparing these bands to the ladder size standard (which shows up as many bands of known sizes) allows you to identify the size of your PCR product, so you can tell whether you got the right gene. This makes gel electrophoresis an immensely useful technique.

 

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Biology for Writers: What Scientists Do All Day

Hello, everyone! It’s the second Monday of the month, which must mean I’m doing a biology-for-writers post. Okay, so I know “what scientists do all day” doesn’t have that much to do with scientific topics of interest to writers, but it’s finals week and I wanted a post that would be quick to write but still informative. And hey, some of you might have scientist characters and be wondering what research looks like, or what different stages of academic science look like. (Science also happens in industry–pharmaceuticals, anyone?–but I’m most familiar with academia, so I’m going to focus on that!)

Life Stages of Academic Scientists

(I’m going to talk a little bit about this first, so I can talk about how research looks for the different stages of scientist in my next section.)

People don’t become scientists overnight. Especially in academia, there’s really a hierarchy, and you can find every stage of scientist working alongside each other in a lab.

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The first step is to get your undergraduate degree in a science field (which is where I’m at right now). If you’re an undergrad scientist, you’re probably taking a fairly heavy course load to complete all your general education, major, and elective courses in four years. Undergrad costs a lot, too (at least in the U.S.), so you might be working on the side to help cover that. If you think you want to go to grad school, you’re probably working in a lab, helping a grad student with their research and/or assisting with lab maintenance things (yes, someone has to wash the flasks and beakers). If you’re extra motivated, you might have your own research project, but you get a lot of help from the professor who runs your lab since you’re still so young and inexperienced. And the project can’t be too big, because you really don’t have that much lab time when you’re also taking four classes and working. But you do what you can to get you into grad school.

The next step is to get one or more graduate degrees. If you want to work in industry, or you don’t have enough research experience yet to get directly into a PhD program, you get your master of science degree first. If you want to teach at the college level and run a lab (i.e. be an academic scientist like your faculty mentor), you need a PhD (short for Doctor of Philosophy). Master’s programs usually take two to three years, and PhD programs in the biological sciences average around five years. You take some courses, but not as many as you did when you were an undergrad, and once you’ve selected a faculty advisor, you get right to work in their lab on your thesis project. In order to graduate with your degree, you need to write a thesis (basically a book on your research) and defend it orally to your faculty committee, who will then decide if you get your degree or not. In a PhD program, you also have to pass a comprehensive exam somewhere around halfway through your degree, which will determine if you get to go on to “candidacy” (full-time dissertation research) or not. Now I’ve gone into a lot of details, but basically grad students do a lot more research than undergrads, because their degree depends on it.

Say now you’ve gotten your master’s and your PhD, and after eleven years you’re finally out of university schooling. Phew! What happens next? Well, if their research is outstanding, some lucky souls get hired straight to faculty positions. Most go on to postdoctoral fellowships (“postdocs” for short). The postdoc is the most senior member of the lab save the faculty member who runs it, unless it’s a really big lab and has a professional lab manager who reports to the professor. Postdoc fellowships usually last one to three years, and they work full-time, similar to grad students except no classes and no thesis defending. As I understand it, this serves as a time to build up one’s resume before applying to become a faculty member.

The highest tier is your PI (primary investigator), the faculty member who runs the lab. They determine what is studied in the lab and who works under them doing their projects. They also teach classes and, when they’re young, try to get tenure (which is basically where your university can’t fire you because you’ve proved the quality of your work). I’ve heard there’s a lot of pressure on young faculty to publish a lot of research in order to get tenure. Older professors (like my PI), who already have tenure, can be more relaxed since they are freer to study whatever they might be interested in–contingent, of course, on the availability of funding. PIs manage all aspects of the lab, mostly from their offices, although mine sometimes comes in and reorganizes things in the freezers or teaches students techniques. They usually teach classes for undergrads and grad students, and write up research articles for publication in scientific journals.

What Science Looks Like (In a Laboratory)

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With some understanding of the academic science hierarchy, we can now get a general idea of what scientists actually do all day. Naturally, this depends on the discipline, in that different scientists do different things. A neuroscientist, for instance, might do behavioral experiments or brain dissections on mice. I have less idea what ecologists do, but my guess is they go into the field to at least collect their samples. When I worked in a seaweed lab, I used to go to the shore and collect seaweed sometimes. Now, as a rice geneticist/biochemist, I split my time roughly 85% lab/15% greenhouse. Some of the things I’ve done in my lab are in bullet points below.

  • Lab Maintenance
    • Washing dishes
    • Making tissue culture medium (think Petri dishes)
    • Disposing of biohazardous waste (seriously not a big deal–I’m only in a Biosafety Level 1 lab)
    • Cleaning up ethanol spills (also not a big deal–it’s basically handcleaner)
    • Preparing/organizing work surfaces (we put this paper on our prep room bench to absorb any spills)
  • Experimental Things
    • Collecting samples (read: cutting up rice leaves and putting them in tubes)
    • Subculturing tissue cultures (the medium dries out every so often)
    • Checking tissue cultures for bacterial contamination (things have to be sterile)
    • Extracting and quantifying biomolecules
    • Data analysis (Microsoft Excel, anyone?)
    • Checking raw data files for quality control (on lab computer)
    • Pouring salt water on plants (so I’m a little sadistic once in a while)
    • Planting seeds
  • Common Genetics/Biochemistry Lab Equipment
    • Petri dishes
    • Pipettes (used for EVERYTHING)
    • Disposable pipette tips
    • Microcentrifuge tubes
    • Boxes with little dividers for freezing things in tubes, and tube racks
    • Freezers (4, -20, and -80 Celsius)
    • Autoclave (a kind of steam sterilizer; usually common to multiple labs)
    • Heat/stir plate and stir bar magnets
    • Small spatulas, forceps, scalpels, razor blades, “scoopulas” (cross between spoon and spatula)
    • Beakers, flasks, graduated cylinders, other glassware
    • Test tubes (although I use these less than the microfuge tubes mentioned above)
    • Centrifuges, spectrophotometers, and other larger equipment
A pipette and Petri dish with medium (Image source)
A lady with some cool lab glassware. (Image source)

 

So that post was a little bit longer than I meant it to be, and it didn’t even include anything about specific techniques that are often used in biology. If there’s interest in a post about techniques, let me know in the comments and I’ll drum up something for next month!

That’s it for me this week! Did you find the day-to-day of science interesting? Do you think it will be helpful to you? Would you like to hear about techniques? Isn’t lab glassware awesome? (I’m going to be that person with chemistry-themed dishes someday!) Are you interested in becoming a scientist? Let me know in the comments!

Biology for Writers: The Human Microbiome

Good morning, everyone! Happy Monday! It’s the second week of November (already!), so here I am with a quick post on a bioscience topic which may be of interest to some of the writers who read my blog (and don’t have that much background in science). Let’s get right to it!

The Overview

One of the biggest topics in science right now is the human microbiome. What is a microbiome, you ask? Well, it’s simply the entire set of microbes that inhabit the human body.

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Yes, that’s right–there are microbes in you, right now, and they’re even supposed to be there! As a matter of fact, there are more bacteria, archaea (basically extreme-environment microbes), and unicellular fungi in your body than there are human cells. Yes, you heard that right–we humans are more microbial than human. Blows your mind a little, doesn’t it?

So what do these microbes do for us? Are they good for anything, or do they just sit there? Well, my friends, I’m glad you asked. Let’s take a look at some of the parts of our bodies where our normal microbes are most influential.

The Mouth

We all know about dental plaque, and brushing our teeth so we don’t get it. (Seriously, I hope you all brush your teeth, ’cause plaque is disgusting.) But your mouth also contains 50-100 billion normal bacteria of at least 500 different species, mostly anaerobes, or bacteria that don’t require oxygen to live. (In fact, some anaerobes die when exposed to oxygen.) Streptococcus mutans and S. sanguinis, the organisms that cause plaque when they build up into a biofilm, are both “facultative anaerobes,” which means that they can either use oxygen or not depending on conditions in their environment (the mouth). Many other normal mouth bacteria can cause problems for the human host if there is bleeding in the mouth, or some other abnormal condition, bringing up an important point for the microbiome in general: Microbes that are normal inhabitants of our bodies (“normal flora” or “microbiota”) can be pathogenic if they are moved to a different spot or if something abnormal happens. These are called “opportunistic pathogens,” and we’ll see more of them as we move through the body.

The Gastrointestinal Tract

This is one of the places where the human microbiome is most important and best characterized; a lot of work has been done linking the gut microbiome composition to everything from diet to Parkinson’s disease. And gut microbes do a lot for us. For instance, we can’t digest vegetables without our gut microbes, and other microbes make vitamin K for us. That’s pretty darn helpful of them, don’t you think?

But when I say “gut microbiome,” what am I talking about? It turns out that not every part of the human body is colonized by microbes; accessory organs such as the liver and pancreas are sterile, and the stomach has a very low level of microbes due to the high acid content there (the stomach’s pH, a measure of its acidic content, is about 2–so very acidic). Not many things can survive the acidic environment of the stomach, which is why it’s great for digestion. One Helicobacter species, however, survives by burrowing into the stomach lining and secreting basic compounds, which neutralize the surrounding acid to create a neutral pH (good for life). This species can also cause stomach ulcers when it has lived in the stomach lining for a long time.

So if the stomach doesn’t have very many microbes, where is this gut microbiome I’ve been telling you about? Most of the gut microbiota live in the ileum (the last part of the small intestine) and in the colon (also known as the large intestine), where, as I mentioned, they help us digest our food, give us nutrients, and take up space so invaders can’t enter. This is known as a “mutualism,” in which both symbiotic partners benefit from their relationship. How do the bacteria benefit from us? Well, most parts of the body are at a neutral pH, which as I’ve already said is good for life, and they remain at a constant warm temperature, which allows microbes to grow.

I also just want to point out quickly that taking too many antibiotics can interrupt your gut microbes, and thus actually make you susceptible to sickness, since with space freed up by antibiotic treatment, pathogens can easily colonize your gut. Since this post is mainly about the microbiome, that’s all the space I can give to this extensive topic, but if you’re interested in learning more, feel free to comment below!

The Skin

The skin is one of our first defenses against microbes, since we are constantly sloughing off dead skin cells, but it is also colonized by many microbes. Similar to the gut microbiome, these take up space on your skin, preventing infection by pathogens. The skin microbiome also includes Staphylococcus aureus, an opportunistic pathogen which normally lives on the skin surface, but can cause problems when it penetrates deeper into the body. For instance, if you have a deep puncture wound, your normal S. aureus may get under your skin (literally) and cause nasty carbuncles and things when it infects you. So if you’re writing a novel and your character gets wounded, this is one thing that could follow that up if you want to give them extra torture be realistic about the consequences of wounding.

Find out More!

Here are some resources if you’d like to find out more about the human microbiome!

Human Microbiome Project–This would be a great resource if you want to find out some of the specific microbes that exist in each place on the human body (there are many, many more microbes than the ones I talked about today).

The Gut Microbiome in Health and Disease–A scientific paper about the gut microbiome. (Please note this is intended for a scientific audience, so it may be a bit dense.)

When Gut Bacteria Change Brain Function–An interesting general-audience article about how gut bacteria impact the brain.

I will continue to add more resources here as I find them! If you find interesting resources, feel free to let me know in the comments and I will consider adding them to this page.

That’s it for my first Biology for Writers post! Are you thinking of using the microbiome in your story? If so, how? Is there anything else you’d like to know about the microbiome? (I’m definitely not an expert, but I can point you to more resources if you’d like!) Are there other biology topics you’d like to see addressed in this monthly post series? Let me know in the comments!