It’s no secret that graduate students spend many hours each day in the lab, doing the exacting labor of research science, but what exactly are they doing in there? To answer that question, we shadowed Kim Armstrong, a student in the laboratory of Max Essex.
Kim is investigating HIV drug resistance mutations. She chose the project because drug resistance will become an increasingly important problem in Africa. As more and more people are enrolled in Botswana’s national program, which provides free antiretroviral (ARVs) drugs to anyone who needs them, and as people remain on their drug regimens for longer and longer periods of time, the percentage of people who will develop drug resistance will grow over time. Very little research has been done on drug resistance in HIV-1C, the dominant HIV subtype in Botswana. With a better understanding of the biological mechanisms of how drug resistance develops, researchers will be better able to use current drugs and to design new ones.
Here is a minute by minute account of how Kim spent Wednesday, April 22, 2009.
4:00: Kim wakes up at her home in Salem where she lives with her husband Ivan and daughter Samantha, age 12.
4:20: She heads out for a 2 ½ mile run through Salem’s historic district.
4:57: Back from her run, she showers and dresses for work.
6:46: Kim boards the commuter train to Boston and reads the newspaper.
7:30: She catches the Green Line subway at North Station and gets off at Brigham Circle.
8:05: Kim arrives at the Essex Lab at the Harvard School of Public.
8:07: She makes a pot of coffee in the kitchen next to the Lab.
(Note: To make the most efficient use of her time in the lab, Kim multitasks by running several experiments simultaneously, while also conducting basic tasks associated with her work. We have separated each task by color.)
Brown Task: Making Media to Grow Bacteria for Future Experiments
This is a basic task. Kim makes media which will be a food source for bacterial cultures. The cultures will contain a clone of HIV.
HIV is an RNA virus. When it replicates using its own reverse transcriptase, it makes mistakes, thus no two HIVs are exactly the same. To get around this problem, Kim makes a molecular DNA clone of HIV that can be replicated in bacteria to produce large amounts at low cost. This gives her a lot of identical DNA and virus that she can use in experiments. With the cloned virus, she knows what she’s starting out with and that identical virus will be used in repeat experiments. She uses the DNA form because it’s more stable and easier to work with. The bacteria will contain a DNA clone of HIV with a drug resistance mutation that she is studying.
8:15: Kim puts on blue nitrile gloves made of synthetic latex. She gets four 2000 milliliter (ml) Erlenmeyer flasks and places them on her lab bench. This has been her work spot since April 2002.
8:17: She turns on the radio and fills each flask with 500 ml of distilled water.
8:20: On the balance, she measures out 7.75 grams of Difco ™ Luria Broth Base Miller, made mostly of yeast extract, to add to each flask. The media will be used to grow bacteria.
8:25: She covers the top of each flask with aluminum foil.
8:26: In a room across the hall, Kim runs a couple of inches of water into a plastic tub. She places the flasks in the tub, then loads the tub into the autoclave for 45 minutes. The autoclave sterilizes with a combination of heat and pressure. It sounds like a dishwasher when it’s running.
8:29: She takes off her gloves and washes her hands.
8:30: Coffee’s ready. Kim walks to the kitchen and pours herself a cup.
9:50: Kim retrieves her sterile media from the autoclave. The flasks are hot, so she wears orange gloves. The media will cool and be stored on the bench top until needed.
Blue Task: Isolating Plasmid DNA to Use in Experiments
The purpose of this task is to isolate molecularly-cloned HIV plasmid DNA from large bacterial cultures. The plasmids will eventually be used to create virus in cell culture.
The sample that Kim is using in her experiments came from a patient who failed the first line regimen of HAART (Highly Active Anti-Retroviral Treatment) in Botswana. The patient had five drug resistance mutations. Kim wants to test why other drug resistant mutations didn’t occur. To test this, she will take out the drug resistance mutations that naturally evolved and replace them with a different set of resistance mutations to see if there is any change in viral fitness. Her hypothesis is that the most fit mutations would be the ones that naturally evolved. She’s testing this by putting in other mutations to see if they replicate as well as the original mutations. This step is the last phase of a five-day process.
8:45: Kim puts on another pair of blue nitrile gloves. She gets her culture out of the incubator, where it has been since 2:00 p.m. yesterday. The culture contains bacteria and media in which to grow the bacteria. The bacteria contain a plasmid that contains the entire HIV genome. From these bacteria she hopes to isolate large amounts of plasmid DNA. She pours culture into four centrifuge tubes.
9:25: Kim removes her cultures from the centrifuge. There is a walnut-sized glob of bacteria stuck to the side of each centrifuge tube; all the rest is the media in which the bacteria were growing. She pours the extra media back into the Erlenmeyer flask, then adds bleach to kill any residual bacteria in the media. A glob of bacteria is all that remains in each tube.
9:33: She adds resuspension buffer to the glob of bacteria to resuspend it in the tube. The buffer solution
10:11: She adds a lysis buffer to the four tubes and shakes them again. The lysis buffer breaks down the cell membranes, allowing the DNA to be released. She sets the timer for five minutes.
10:13: She sets up four filters in a Styrofoam holder.
10:16: She adds a neutralization buffer to each tube that stops the lysis reaction. She shakes each tube with her hand.
10:19: To filter out the bacterial cellular debris, she pours the contents of each tube into its own filter and sets the timer for ten minutes.
10:25: She adds equilibration buffer to four columns. The buffer prepares the column to bind DNA.
10:44: She adds wash buffer to the columns to wash the DNA that is collected in the filters.
10:57: She adds more wash buffer to the columns that holds the isolated DNA.
11:09: After transferring the columns to 50 ml centrifuge tubes, she adds elution buffer to the columns to change the pH and get the DNA unstuck from the filter.
11:24: She adds isopropyl alcohol to the purified DNA. It should cause the DNA to form a solid pellet after it has been spun in the centrifuge for an hour at 4500 rpm at 4°C.
12:30: Kim puts on another pair of gloves. She stops the centrifuge.
12:35: She pours off the isopropyl alcohol so that all that’s left in each tube is a small thin pellet of DNA, about the size of a paper hole punch. She transfers each pellet to a microcentrifuge tube. She washes each sample with 70% ethanol to remove the isopropyl alcohol. She puts the tubes in the centrifuge to spin for five minutes.
12:50: She retrieves the DNA pellets out of the centrifuge. She pipettes off the ethanol. She does a second wash of the DNA.
12:55: She returns the DNA pellets to the centrifuge.
1:00: Kim removes the tubes from the centrifuge, rinses her DNA samples once more with ethanol, and spins the samples one last time for five minutes
1:10: She pipettes ethanol off the DNA pellets and lets them dry in the microcentrifuge tubes for about 15 minutes.
1:16: She mixes the solution to run a gel. DNA gel electrophoresis is a technique used to separate DNA by size using an electric current applied to a gel matrix.
1:25: Kim adds TE to the dried DNA, then puts the microcentrifuge tubes on the 37°C hot block.
1:27: She gets an enzyme out of the freezer. The enzyme will allow her to check her sample to make sure that she has isolated the correct DNA.
1:30: Word spreads among the graduate students that there is leftover food from a lunch time talk on “Innate Immune Responses to Leishmania Parasites” that was not well attended in a classroom down the hall. Kim gets a plate of pasta salad and fruit from the buffet leftovers and eats in front of her computer.
2:16: Kim dilutes her samples so that they are all at a concentration of one microgram of DNA per microliter of TE. She dilutes the samples by adding more TE.
2:25: After preparing the digests, she puts them on the 37°C heat block.
2:56: She prepares the gel to check her DNA.
2:57: Kim takes her digests off the heat block and spins them in the centrifuge for seven seconds.
3:00: She pipettes the DNA samples onto Parafilm taped to the counter.
3:05: She loads DNA into the gel and finishes preparing the gel.
3:27: She must re-purify a small amount of the DNA to send out to Harvard Medical School for sequencing. The DNA is suspended in TE, which contains EDTA, which binds to magnesium, which is necessary for a sequencing reaction. Because the EDTA interferes with the sequencing, she must get rid of the TE.To get rid of the TE, she puts the sample in a new tube and dilutes it with buffer. The DNA binds to the glass filter. She washes it once with alcohol and then elutes it with water to get the DNA off the filter.
3:35: She puts the sample in the centrifuge and spins it for one minute. She pours the excess buffer off the sample.
3:39: She adds an alcohol wash to the sample and spins it for another minute. She then pours the wash off the sample. The filter with DNA bound to it remains.
3:40: Kim spins the sample again for one minute to make sure that the filter the DNA is stuck to is dry.
3:41: She extracts the filter and puts it in a clean tube. She adds water and spins the tube. She gets the DNA and discards the filter.
3:57: The gel has finished running. Kim puts the gel in the gel imager and sees that the DNA is the correct size. She takes a photo of the gel.
4:00: Kim removes her gloves. Sitting at her lab bench, she pastes the photo of the gel into her lab notebook and labels it.
5:04: At her computer, Kim fills out an on-line order form for the DNA that she is sending out to be sequenced. The computer gives her an order number.
5:10: Kim writes her order number on the DNA sample, which she has packaged for drop off.
Purple Task: Isolating the Reverse Transcriptase Gene
This particular reverse transcriptase gene is the same reverse transcriptase gene as the Blue Task except that all of its resistance mutations have been removed so that it resembles the wild type HIV (HIV that hasn’t been subject to the selection pressures of antiretroviral drugs). This experiment will determine if the drug resistance mutations that naturally evolved have a cost relative to its own wild type.
Once she has confirmed the sequence of a drug resistance mutant, Kim can place the reverse transcriptase gene into MJ4. MJ4 is the HIV subtype C molecular clone. In this task she will isolate only the reverse transcriptase from the rest of the plasmid. She does this by digesting the DNA with two restriction enzymes (ApaI and HpaI), and running the digest on an agarose gel. The gel will separate the plasmid into two pieces, with one being the reverse transcriptase gene and the other being the DNA vector which will be discarded.
This is the first step in a five-day process. Like the Blue Task, the end product will be a molecular clone of HIV with a drug resistance mutation.
11:38: In the small office that she shares with five other researchers, Kim sits at her computer and checks DNA sequences that have been emailed to her. She has (she hopes) inserted drug resistance mutations into reverse transcriptase. She’s checking to see if the correct mutation was put into the DNA. It had been. She is happy about this because it means a lot of her work in the lab has not been wasted.
12:07: Back in the lab, Kim pulls on another pair of gloves and gets DNA samples out of the freezer. She measures the concentration of DNA on the spectrometer to make sure she has enough DNA for each digest.
12:15: She adds buffer, bovine serum albumin, water and enzymes to the DNA samples. She’ll let them incubate for a full hour at room temperature.
1:15: She puts the digest on the 37°C hot block for an hour.
1:16: She mixes solution for a gel.
1:45: She adds ethidium bromide to the gel solution and pours it into the gel tray. Ethidium bromide is commonly used as a fluorescent tag in gels. When exposed to ultraviolet light, it will fluoresce, intensifying almost 20-fold after binding to DNA.
2:56: She prepares the gel. She is using the gel to isolate the reverse transcriptase gene.
2:57: Kim takes her samples off the heat block and spins them in the centrifuge for seven seconds.
3:05: She loads the DNA into the gel and finishes preparing the gel.
3:55: She removes the gel from the gel box and takes it to the dark room. Wearing a protective face shield, she cuts out the rt (reverse transcriptase) band. The UV light in the darkroom allows her to see the DNA in the gel.
3:57: Outside of the dark room, she puts the gel into the gel imager and takes a photo.
4:00: She labels her photo and pastes it into her lab notebook.
3:57: Outside of the dark room, she puts the gel into the gel imager and takes a photo.
Kim is testing the fitness of drug resistance mutations. When she inserted the mutations into her clone of HIV-1C, the virus became nonfunctional (dead). However, she knows from clinical data that these mutations occur in patients receiving treatment. To be able to test these mutations further, she is changing her clone so that it will match the average subtype C Reverse Transriptase. She is trying to figure out why she can’t replicate in the lab (in vitro) what happens to a person with HIV (in vivo).
In this task Kim will isolate plasmid DNA from cloned mutagenesis reactions. She is in the process of changing the Reverse Transcriptase of MJ4 to match the consensus sequence (from the Los Alamos database) of subtype C. MJ4 was cloned from a patient isolate in the late 90s and represents a single isolate of HIV. She is changing the amino acid sequence so it will be representative of the average subtype C Reverse Transcriptase. There are ten amino acids that need to be changed. These cultures are clones of the fifth change (fourth mutagenesis reaction). Once she isolates the plasmid DNA, she will use it as a template for the next mutagenesis reaction (Pink Task). This is the third day of a three-day process.
10:40: Kim gets the cultures out of the 37°C incubator and spins them in the centrifuge for ten minutes.
10:50: She gets digest buffers out of the freezer to thaw. A digest is used to check the DNA by cutting the DNA at a specific sequence so that she can confirm that she has isolated the correct DNA.
10:51: She retrieves her samples from the centrifuge and pours off the media so that a small clump of bacteria remains.
10:55: She adds suspension buffer to the bacteria and shakes it briefly on the mechanical shaker, called a vortex.
10:56: She adds lysis buffer to the bacteria. It turns blue.
11:00: She adds neutralization buffer to the blue sample. It turns white, assuring her that the lysis reaction has stopped. She spins the tubes in the microcentrifuge for ten minutes.
11:01: She measures out TBE buffer and adds it to the gel solution that she’s preparing in a glass jar. The TBE buffer is an electrophoresis running buffer. This buffer gives DNA a negative charge so that when it runs in the gel, it will flow towards the positive electrode. She adds agarose to the buffer and microwaves it for just over a minute to melt the agarose. Agarose is a thickening agent which creates the gel.
11:05: She sets up a gel tray. She takes the agarose solution out of the microwave, shakes it, and returns it to the microwave for another 25 seconds.
11:10: She gets the samples out of the microcentrifuge. The protein has collected at the bottom of the spun tube.
11:12: She dumps the clear fluid which contains the DNA into a small filter and puts it back into the microcentrifuge.
11:16: She records the recipe for her digest in her lab notebook.
11:17: Because some enzymes need protein to work, she adds bovine serum albumin and digest buffer to the tube and then adds ultra pure water.
11:21: She adds ethidium bromide to the agarose solution. It will make the DNA visible in the gel. She pours agarose solution into the gel tray. She changes her gloves after handling the ethidium bromide, which is potentially carcinogenic.
11:28: She adds the enzymes to the reaction mix. Enzymes are unstable, so they are kept cold in a -20°C freezer.
11:30: She adds the DNA that she was preparing (called a prep) to the microcentrifuge tubes that hold the reaction mix. These will sit for 30 minutes at room temperature, then another 30 minutes at 37°C.
12:45: She adds buffer to the digest. She tapes a piece of Parafilm, a square flexible film, to her lab bench.
12:46: She adds more TBE gel running buffer to the gel bath.
2:07: She puts on a fresh pair of gloves and turns off the gel.
2:27: She puts the gel plate into the gel imager to look at it. She prints out a photo of the gel.
2:30: Kim throws away her gloves and labels the gel photo. She is pleased with the results. The gel proved to Kim that she has successfully isolated the DNA. She puts on another pair of gloves.
2:40: She takes the sequencing primers out of the freezer.
4:50: She sets up sequencing reactions by pipetting DNA into one plate and primers into another.
5:04: At her computer, Kim fills out an on-line order form for the DNA that she is sending out to sequence. The computer gives her an order number.
5:10: She writes her order number on the DNA sample.
5:13: Kim drops off her DNA samples at the pick-up spot. She will receive the DNA sequencing information via email the day after tomorrow.
Orange Task: Pelleting Virus for a Reverse Transcriptase Assay
In earlier experiments, clones of certain HIV-1C viruses would not grow in cell culture. To be sure that the defect that prohibits viral growth is non-functional Reverse Transcriptase, Kim is testing the ability of the Reverse Transcriptase to perform its function of synthesizing DNA. This task is performed in the Cell Culture Room, which must be kept extremely clean in order not to contaminate the cell cultures. This is the second day of a three-day process.
3:10: Yesterday Kim had set up viral dilutions and mixed them with a polyethylene glycol (PEG) solution and incubated them overnight on ice. She takes the tubes out of the ice bucket in the fridge and puts them in the centrifuge for 45 minutes to separate the virus pellet from the PEG. She double-gloves when doing this task because she is working with potentially infectious HIV.
4:01: She removes the tubes from the centrifuge. Each contains a small white pellet which is the spun down HIV virus. She pipettes off the supernatant and resuspends the pellet in lysis buffer. She then added reaction mix, which will incubate overnight. Tomorrow she’ll get a color read out on a spectrometer which will measure the RT activity of the virus.
As in the Green Task, Kim is changing the Reverse Transcriptase to match the average subtype C sequence in order to test the viability of drug resistance mutations. She is setting up another mutagenesis PCR reaction to add the sixth amino acid change to the Reverse Transcriptase. PCR or polymerase chain reaction is a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions more copies of a particular DNA sequence. It is the same type of technique that forensic detectives use to determine who committed a crime based on tiny bits of blood or hair.
2:35: Kim adds water to dried mutagenesis primers to reconstitute them to one microgram of DNA per one microliter of water. She labels the concentration. “Pretty much everything gets labeled with a concentration and a date,” she says.
2:39: She checks to see if there is a PCR machine available. There is.
2:45: She measures the concentration of the isolated DNA (from the Green Task) in the spectrometer.
2:47: Diluting with water, she makes a 1 to 10 dilution of DNA so it won’t be too concentrated when she adds it to the PCR.
2:53: She takes a PCR kit out of the freezer. The kit contains a buffer, nucleotides, and a polymerase enzyme. She pipettes buffer into a small PCR tube.
2:56: Kim loads her sample into the PCR machine, which will run for about 2 ½ hours. She discards another glove.
5:18: Kim boards the Green Line train at Brigham Circle and reads Nature as the train makes its way through downtown Boston to North Station.
6:04: She boards the computer train at North Station.
6:53: She arrives home in Salem.
7:04: She changes her clothes and relaxes.
7:45: She helps prepare dinner.
8:30: She eats a dinner of steak, potatoes and salad with her daughter, husband, and mother-in-law as they watch a Bruins hockey game.
10:00: Lights out, Kim goes to bed. After a long day in the lab, she falls quickly asleep.