The Genetic Coloring Book
No matter how hard we squint, we can’t read DNA directly. Most of the time, we can’t see it at all. The DNA samples that we typically work with in the lab are indistinguishable from clear water in a test tube. As you might imagine, this makes it difficult for us to know what exactly we’ve got or if our manipulations are working. So in this course we present some common tricks that we use for visualizing DNA.
Sometimes, we can detect DNA sequences indirectly by looking at what they do. Certain genes have clearly observable effects on the organisms that carry them. These are called genetic markers, and they are extremely handy for letting us track DNA with the naked eye. Sometimes, they are also useful or cool their own right.
This time our genetic markers of choice are chromoproteins. These are nothing but small proteins that take on a visible color when they are expressed and fold. They come in red, yellow, blue and all the colors of the rainbow. We know when a strain of E. coli has been transformed with a chromoprotein-expressing gene because the colonies show the lovely vibrant color. Our inspiration for using these cool proteins came from the 2013 iGEM team from Uppsala, Sweden, who collected many different chromoproteins for the BioBrick registry.
Jake Wintermute, Living Art, 2017
Bacteria, chromoproteins, agar, Petri dishes
We can also visualize DNA directly, outside of a cell, by mixing it with a chemical stain and running it out on an agarose gel. To do this correctly, we need to understand some key chemical properties of DNA. DNA is negatively charged, so we can pull it toward a positive charge in an electric field. DNA is long, so it moves very slowly though a gel matrix. DNA is double stranded, so we can stain it with specific pigments that fit in between the base pairs. We’re still not technically looking at the DNA directly (we’re looking at the staining pigment) but the result is beautiful and information rich.
Unit 1: Pretty Pretty Proteins
Synthetic biologists love proteins and you should too. They have everything you could want from a biological part. They’re powerful, performing most of the critical functions in any living system. They’re modular, meaning they can be moved between organisms and (usually) still function the same way. And they’re easy to identify: DNA that codes for a protein has a specific set of well defined features that can be automatically detected with DNA sequence editor software.
In Unit 1, we will be looking at chromoproteins at the DNA sequence level. We’ll explore the features that make it easy to recognize a DNA sequence that codes for a protein. Then we’ll go into the sequence editor software and see how we can pick out these features automatically and connect them with information about their identity and function.
Unit 2: Looking at DNA with your Actual Eyes
Most of the time, the DNA sequences that we’re interested in will not be associated with genetic markers. In this case, we need a way to detect the DNA molecule itself. More than that, we want to get some information that helps us to identify a specific piece of DNA. Just saying “yep, that’s DNA” is usually not enough. Imagine, for example, that you’re trying to confirm that the clear liquid in the tube labeled “plasmid pUC19” is in fact plasmid pUC19 and not some other plasmid.
You’ve heard of DNA sequencing, but often that’s a little bit too fancy for everyday use. We’ll cover that in a later lesson. Instead, we’re going to show you a cheap, quick and dirty way to identify a piece of DNA using an agarose gel. This is a technique for separating pieces of DNA by size.
When we put DNA inside a gel and apply an electric field, the negatively charged DNA moves toward the positively charged anode. Short pieces of DNA move more quickly through the gel matrix, resulting in the size separation. When we stain and visualize the DNA, its position in the gel will tell us how long it is: 500 base pairs, 800 base pairs or whatever.
Elijah Wood shows off the sizes of pieces of DNA from his own genome.
Art by DNA11.
Unit 3: The Visual Signature of a Plasmid
So who cares is a piece of DNA is 500 or 800 base pairs long? It doesn’t seem like a ton of information, and it’s not. Imagine asking someone about the book they were reading and they said “It is 437 pages long.” It doesn’t tell you very much about the book, but at least it helps you distinguish it from other books. Now imagine they tell you the length of each individual chapter of the book. It is possible that two different books have exactly the same chapter lengths, but it is unlikely.
In Unit 3, we will be cutting DNA into pieces at specific places. These are the “chapters” that we can compare to positively identify a specific plasmid. The physical cutting of DNA is done with restriction enzymes, which recognize specific short DNA sequences and cut no where else. Restriction enzymes were extremely important in the history of synthetic biology and are still used extensively today. In a sense, restriction enzymes a bridge between the digital world of DNA sequences and the chemical world of real DNA. They allow us to look in a DNA sequence editor and make a specific, targeted change. We will talk about restriction enzymes both on the computer and in the test tube.
Unit 4: Cell Pushing
So you have found your plasmid, positively identified it using an agarose gel, and transformed it to produce beautiful purple cells. What next? There are two very standard protocols that you will follow almost every time you create something interesting in synthetic biology.
First, you will streak for singles. This means taking a blog of cells and smoothly smearing it out on a Petri dish. As the cells get more and more spread out, you eventually reach a point where individual cells begin to separate from each other. These individual cells, at the tail end of your streak, will produce single colonies. It’s the same principle that we used when we were counting the CFUs in yogurt: single colonies come from single cells. We like to work with single colonies from single cells because we know that all the decedents of a single bacterium are genetically identical. You will almost always want to start your experiments with a single colony because it represents a pure, clean, uncontaminated genetic line.
Second, you will make a glycerol stock for the freezer. Bacteria, like all living things, eventually die. E. coli will last for about a month in the refrigerator. If you try to start a culture after this point, you will find that nothing grows. If you want to work with your cells for longer than this, or keep them forever, you will need to freeze the cells. This is very easy to do, and requires just a few little tricks to prepare the cells for the deep freeze. Specifically we add glycerol to reduce the formation of ice crystals which might otherwise cut through the cell membrane. Naturally, you will want to make your glycerol stock with a genetically pure single colony.
At the end of this lesson, we’ve looked at DNA in many different ways. We’ve played with the DNA sequence editor a few times now. You may feel empowered now to dive into the software by yourself and explore it’s capabilities. I recommend you spend a few hours just clicking on things and seeing what happens. All the major sequence editors have good tutorial videos, if you prefer a guided approach.
We’ve cut DNA up into pieces and run it out on an agarose gel. The main concept that you need to get at this point is “the bands tell us the size of the DNA pieces” and “different plasmids are cut in different places, thus producing different bands.” But obviously there is a lot more going on here. You may want to know more about the chemical properties of DNA or how it runs in a gel. You also might be curious about how restriction enzymes work and how many different sequences they can recognize.
Finally you may be intrigued by the idea of bacterial art, or living bio-art in general. Here are some cool links on that subject.
I love this billboard for the movie Contagion.
But how come microbes have to be the bad guy all the time in movies?