Lab tour transcript
What's up, you guys? My name is Conrrad Nicholls. I'm a fifth year PhD candidate in the lab of Dr. Richard J. Kuhn. And today I have the pleasure of giving you a virtual tour of his lab. Let’s go get started.
To start things off, I’d like to take you through our main lab area, our Biosafety Level One facility here in HOCK 283. Our BSL1 facility or Biosafety Level One Facility is considered to be a wet lab, meaning that anything pertaining to DNA, RNA, protein and anything to do with bacteria and expression purification, synthesis of any of those things is done right here. We even have our own chemical shelves in different reagents throughout the lab that help us make the specific cocktails that we need to do all of the reactions necessary for different cloning and different expression experiments.
Now the nice thing about it is that there’s plenty of room to work. We have three different aisles with four lab benches a piece, giving us 12 different spaces to operate. Each of the grad students, postdocs and researchers in the lab has their own pipette set, their own vortexer, their own lab, bench centrifuges, and many other tools at their disposal to make sure that the work gets done.
And also a nice feature about the lab is the copious amounts of refrigeration space and freezer space. We have freezers scattered throughout the lab and holding different machines such as the actor right here behind me. We have different machines and freezers holding antibodies and enzymes, as well as all of our different samples that we’ve produced over the years.
Let’s take a quick spin around the room and check out what else we have. So after walking into the lab, take a right and you end up over here at our chemical wall. Over here we have all of the different compounds necessary to make the different buffers and reagents that we need to successfully run experiments on DNA, RNA and protein extraction and purification and even synthesis.
So after hooking a left from the chemical wall in the back, what we see is our analytics bench. Now this bench contains a bunch of different machines that are very useful in determining what’s pretty much going on. What we have is our Odyssey gel and membrane reader, which helps us to understand our western blots or SDS page gels. We have a Spectrum Max ID 5, which I’ll get into in a little while.
And we also have our nano drop and vacuum chamber. And the nano job is very important because it allows us to understand how much DNA we have in our samples after collection, RNA, or even protein. That comes in handy because a lot of calculations can be done without knowing those minute concentrations.
So another very cool machine that we would like to highlight here in this lab is the Spectrum Acts IV 5. Now this machine is very high-tech, very high-end, and costs almost as much as a Lexus SUV. But it’s worth every penny, because it can do almost anything involving absorbance, fluorescence, luminescence, time resolved, fluorescence energy transfer, fluorescence polarization and much more.
For example, I currently use this machine to do ELISA assays on over 384 virus samples all at the same time. Now another grad student in this lab uses it to do specific enzyme activity assays, which monitor the activity of a specific enzyme over the course of 20 hours. Needless to say, this thing saves us a lot of time.
So on this side of the labs is where we keep all of our PCR or polymerase chain reaction thermal cyclers. And these machines are very useful because they allow us to introduce different deletions, mutations, and even insertions within particular DNA constructs. These constructs are plasmids that can then be put into bacteria through a process known as transformation.
Now this allows us to then clone all of those DNA constructs through the natural replication process of the bacteria. Now from this point, projects can diverge into a myriad of different avenues. But right here is where everything usually starts.
So right next to all of our thermocyclers is our chemical fume hood, where we can properly handle and store compounds such as hydrochloric acid and methanol that we use quite often in our lab.
Right next to that we have our hot water baths and different imaging stations for SDS page gels. Right now we have a few more freezers and of course, storage for all the different Eppendorf tubes.
And that room right over there, that we’re getting ready to walk into, is our gel electrophoresis room. Let’s go check it out. So straight across from our thermocyclers, we have our gel electrophoresis room. This room is very important because it allows us to better understand all the different fragments we produce through PCR. Over here to my right is our gel electrophoresis baths. They allow us to run DNA separation gels to better understand the size of those fragments and making sure that they’re all okay. But to see that, we have this machine over here to our left, that utilizes UV light to see ethidium bromide that we use to stain all of our DNA fragments.
So our last stop before heading out is this room just to the right of the entry door of the lab. In here, we have more refrigerators and more freezers for storage of different reagents. We also have our different antibody freezers, like this one right here.
And we also have a bunch of centrifuges over here to the left. Now these are definitely important when it comes to separating those different bacterial cultures when trying to express and purify different protein samples. All in all, our one facility serves as a foundational work room for many of our projects and has proven to be invaluable in the training of both undergraduates and new graduate students alike.
But the fun doesn’t stop here. Let’s go check out the SRT. So our Biosafety Level 2 facility is also considered to be a wet lab. However, we don’t work with any bacteria down here. And most of our DNA and RNA work is done upstairs. However, once we need to do something with that DNA and RNA, we bring it out here where we work primarily with mammalian and insect cell lines to study almost 13 different viruses in the lab at any given moment. These viruses come from a myriad of different families. But primarily we work with capacity viruses, alpha viruses, flaviviruses, enteroviruses, as well as rhinoviruses that cause the common cold.
Now to work with these viruses, we need to wear the proper protective gear. So we have safety glasses, lab coats, as well as gloves whenever we’re down here. Now, whenever someone works with one of these pathogens, they need to work with them within a specific biological safety cabinet, or BSE. The Kuhn lab utilizes four separate rooms containing two biosafety cabinets in each. Each of these rooms has been designated for specific virus families only, preventing cross-contamination and potential release.
This last room on the right, however, has been reserved for cell culture work only. Our BSL2 facility comes equipped with six minus 80 degrees Celsius freezers that are capable of storing and preserving all of our different virus samples from all of those different virus families for many years, so that members of the Kuhn lab and future generations will have plenty of virus samples to use and to study.
Now our freezer room also contains several centrifuges and ultracentrifuges. They’re used in the preparation and purification of all of our different virus samples. For example, this centrifuge here is used to separate cell debris from virus, any immediate collected alpha cells, spinning upwards of 15 thousand times the force of gravity. So because our group makes all of its own media to use in cell culture work, in virus preps ourselves, without buying it from an outside source, you have to keep everything in this walk-in refrigerator.
So lastly, our Biosafety Level 2 facility is a completely closed system due to safety reasons. We don’t want any biological agents getting out and causing harm. Therefore, no biological waste or dirty dishes such as media plates or bottles are allowed to exist BSL2 for cleaning. But that’s all right, because our BSL2 facility is completely standalone, which comes with its own autoclave system, and its very own dishwashing system. Now both of these systems together are very useful in terms of making sure that no agents are able to escape the lab and cause any harm to anyone unsuspecting within Hockmeyer. Well, that pretty much sums up everything down here in BSL2.
Now I’d like to conclude our tour with a little sneak peek at our Cryo-EM facility. Well, before we get there, let’s take a stop over at BSL3. I’ll tell you a little bit about that. Unfortunately, I’m not allowed to show you guys the inside of this laboratory due to security reasons. However, I can tell you a little bit about what goes on inside BSL3 and about the people who work inside.
So our group works with three specific viruses within the BSL3 laboratory. One being West Nile virus, the second being yellow fever virus. Both of those are flaviviruses like Zika and Dengue. However, we also work with the Sars Cov-2 virus or the culprit behind the COVD-19 pandemic.
Now to work inside BSL3, users have to wear a very complex set of personal protective equipment. This includes a full body suit or a Tyvek suit, a Tyvek hood or cowl, a false face respirator with its own air supply, two pairs of gloves and standalone booties that never leave the lab. Now, to get to all that stuff though, users have to go through several interlocking doors to make sure that no contaminants are capable of coming out. And before they’re able to leave, they have to take a shower and rinse all that nasty stuff out just in case anything else got underneath all of those layers of protective clothing. So far so good though, nothing’s ever gotten out. But you can never be too careful. So I’m sorry that I can’t take you in, but it’s probably for the best. We wish these guys luck – they’re in there right now working.
Our last stop today is right here in the Purdue University Cryo-EM facility. Now of course, this facility is a shared one throughout Purdue’s campus, but especially here within Hockmeyer Hall, due to the ubiquitous nature of cryo-electron microscopy in protein studies. However, the Kuhn lab, alongside members of the lab of the late Dr. Michael Rossman, have cornered a specific niche within the Cryo-EM ecosystem dealing specifically with viruses.
But to preface briefly, most Cryo-EM work done here at Purdue University and Hockmeyer Hall is done on proteins, purified proteins, and protein complexes to better understand their structure and ultimately their function. The benefit of Cryo-EM over a more traditional method of protein structural determination, such as X-ray crystallography, is that Cryo-EM eliminates the need to produce crystals entirely, allowing for a more robust refinement of proteins or protein complexes that cannot easily be crystallized.
But, before we get too deep into all of the cool things we’ve done as a group with this awesome technology, let’s talk a little bit about how it works. In a cryo-electron microscope, an electron gun shoots a stream of electrons through several magnets until the stream passes through the frozen sample. Then a specifically designed high-tech camera captures the electrons to form an image, which is then fed into a computer to be ultimately analyzed by the user.
Like I said, most Cryo-EM facilities such as the one here in Hockmeyer are used to study proteins that are incapable of being crystallized. However, the Kuhn lab primarily uses this resource to solve the structures of numerous whole virus particles and their membrane embedded proteins, include Zika virus, Dengue virus, C15A rhinovirus, and several other alpha viruses and flaviviruses throughout the last 25 years.
To do this, once the camera has collected the images from the microscope, another computer algorithm sorts the images, and finds the tens of thousands of similar virus pictures, which is then sorted into different class averages. Another software uses all of these averages to build a composite, ultimately form an accurate high resolution 3D model of the virus that can be used for all kinds of different foundational studies.
Now the Cryo-EM facility houses three cryo-capable electron microscopes. A Talus F200 C, a Ti Crios G1 and a new Ti Crios G4, which was only installed in July of 2020. Unfortunately, I can only show you some of the footage of the Talus since the others are currently in use. Needless to say, the Kuhn lab is extremely fortunate to have such amazing tools at our disposal.
Altogether, Cryo-EM is an invaluable tool that has helped us and many other labs around the world answer those very difficult questions regarding viruses and their structures. I’m sure it’s going to be playing a huge role in many more years to come.
Well, unfortunately, that concludes our tour today. Now I hope that you are able to enjoy seeing a little bit about what we do every day here in the lab. But if you have any more questions regarding anything that you saw in the video, please feel free to reach out with a timestamp included, and we’d be happen to answer all of your questions. Until next time.