If you give a bacterium a plasmid…
Updated: Jul 16, 2020
In the lab, we have been busily making SARS-CoV-2 over the last weeks, to use in our experiments. I've written before about how we make HIV-1 in the lab and about how we get bacteria to do most of the "heavy lifting" for us. But today, I thought I'd come back to this same topic with a new spin, in the light of this brave new corona-world we now live in.
When we build viruses to use for our research, our starting place is pure genetic material: little circular bits of DNA called plasmids that can be engineered to encode different pieces of the desired virus. To make SARS-CoV-2, we use several different plasmids, each with different parts of virus on them, for safety and for scientific elegance. Picking and choosing what plasmids to later combine gives us remarkably precise control over the virus that we are creating. For example, in our last few experiments we have been using a version of SARS-CoV-2 that cannot replicate (i.e. make more of itself after it infects cells - it just enters cells and then gets stuck) and is luminescent (so if cells are infected, they basically glow). This makes the virus not only safer, but also easier to work with.
How is it easier to work with? You can get an idea of what I mean if you compare this infection readout by luminescence to my infection readout when I work with, for example, non-glowing HIV-1. When I infect samples with a normal HIV-1 virus for my doctoral research (which has focused so far on finding new host-directed antivirals against HIV-1), I measure infection by permeabilizing the cells I've infected (basically poking little holes in their membranes), then adding a fluorescent stain that only binds to virus proteins (thanks to the little holes in the cell membranes the stain can get through to bind the viral proteins, which should be inside the cells), and then measuring fluorescence on a specialized machine. The whole process usually takes about 4 hours of rather hands-on time. If instead I were to use a virus that is already luminescent on its own, the readout is simply measuring if the cells are glowing or not, and it only takes about half an hour.
The part that does take a little more time, is making the virus in the first place. Luckily for us, when an outbreak or pandemic like SARS-CoV-2 happens, many scientists will quickly start working on the same virus, and scientists often are kind enough to share the plasmids that they develop. In the group that I'm in, we've been lucky enough to have a few different research groups share their plasmids with us, thereby giving us the blueprints to make our own vials of SARS-CoV-2 to use for our research. But, science proceeds slowly, and to make any breakthroughs we'll need to do a lot of experiments, and use a lot of virus. So, the first thing we need to do, is make more plasmid 'blueprints' for all the individual pieces of the SARS-CoV-2 virus that we want bacteria to make for us.
You see, if you give a bacterium a plasmid, it will basically act like a photocopier, pumping out many more versions of that same genetic material. This gives us a nice big stockpile of DNA to work with. After we've made many copies of all the different plasmids, each encoding different pieces of the SARS-CoV-2 virus, we can later insert some of that DNA into a human cell line, which are immortalized human cells (another cool topic that I've written about here) in the lab. These human cells will read the 'blueprints', and from them create the exact virus we've instructed them to - whether it's plain old HIV, or a luminescent SARS-CoV-2.
By the end, we'll not only have a nice stockpile of plasmids, but also a nice stockpile of vials and vials of virus to use in our experiments… Which hopefully I'll be able to tell you more about sooner rather than later!
PS. As a little side note for the Canadian readers among you, I had a proud Canuck moment today. As my colleague and I were preparing to transform bacteria (yes, that is the technical term, 'transform'), we were rooting around in the store room at the lab looking for all the materials we would need. I asked her if she had found the hockey sticks already. Apparently that's not the technical term for the L-shaped spatulas used to spread bacteria in petri dishes. The technical term is, much less interestingly, "L-shaped spatulas". I think the Canadian way is better ;)