Tag: ArmanK- Biochemistry

Day 8: The People that Make it All Possible

All good journeys come full circle. After a week of preparation and cell harvesting, Dr. Masoudi and I moved on to setting up column chromatography with nickel resin – the first task we undertook together when I came into the lab last Tuesday. We made some slight modifications to the overall procedure, but it began just as it had before when we transferred incubated flasks with E. Coli cells to smaller centrifuge containers. After we spun our 12 liters of cell/media mix, Dr. Masoudi and I poured out the unnecessary liquid remains, combined the leftover cells, and added in a buffer solution to both maintain pH and limit biological activity. What’s interesting about E. Coli cells, and something I had not registered when we first did this two weeks ago, is that these “gram negative” bacterial cells have two layers of membrane. The first, outermost layer contains a periplasmic interior that carries our precious protein, Nb6B9, in addition to thousands of minuscule nutrients. The second, interior layer more closely mirrors the cell membranes of eukaryotes, outlining a cytoplasm which has a DNA-filled nucleus. In order to break the periplasmic layer (and not disrupt the cytoplasmic layer), we initiated an “osmotic shock” that fills the periplasm of each cell with enough water to burst the outermost membrane, expose the protein while keeping the cytoplasmic layer intact (… theoretically). The violent rupturing of the cells was followed by another centrifuge session, where bottles of the broken cells were spun at 14,000 rpm for about twenty minutes. The intense pressure delivered by the high speeds was able to segregate the intact cytoplasmic layers from the disrupted periplasmic contents, meaning we had to take the soluble fraction (the liquid fraction) of the bottles to properly retrieve Nb6B9. Unfortunately, our soluble fractions were viscous. The only macro-molecule capable of making the solution viscous, according to Dr. Masoudi, is DNA, indicating that we had ruptured the cytoplasmic layers of some of the cells and made the extraction process a whole lot harder.

The list of things I absolutely despise is quite short: spiders, Sauron, Dreamworks’ Minions. But the one thing that always tops the list, the one thing that punctures my soul and leaves me dangling on a mountain of turmoil, the thing that casts an infinite shadow over everything that’s good and innocent in this world, is bleach. The lab had run out of clean spin-bottles for the centrifuge, and Dr. Masoudi assigned me the task of washing out some used bottles under a specific protocol. As you may imagine, the risk of bio-hazards in the lab is really high, and therefore the risk of contamination between experiments is also pretty high. Cleaning ordinary, 250mL spin bottles is no “scrub-a-dub-dub” and your done. It requires determination, perseverance, and grit. You really have to grab the bottle by its sides and scrub it with the force of a thousand pounds of pressure. In other words, I rinsed the bottle three times, soaked the bottles and their caps in a bleach bath, and used deionized water to eliminate any chance of cross-contamination. However, the bleach’s horrible stench made my five-minute job seem like an infinitely long ordeal, but in hindsight, it wasn’t all that bad.

Just like yesterday, I helped Dr. Li Yin out by washing some of her flasks and pipetting exact amounts of protein into a gel electrophoresis machine – the plastic contraption that divides proteins based on their molecular mass. It’s been a pleasure working with Dr. Li Yin so far, and I’m honored to have witnessed the phenomenal research that she and Dr. Masoudi are conducing to obtain the crystal structure of the beta-2 adrenergic receptor bound to our infamous protein, Nb6B9. I am looking forward to what the rest of summer has to offer!

The lab is a peculiar place. There are professors, doctors, graduate students, undergrads, and high schoolers that speed through the halls, joke around, and take the time to get to know one another. It took me two weeks to realize that it wasn’t only the chemistry in our compounds that made the science interesting – it was the chemistry between people too. The lab is a living, breathing place that depends on scientists and interns who are willing to make sacrifices for each other, who are willing to go extra lengths to not only be good researchers, but good humans as well. These past two weeks have been a formative experience for me, and although the research is overwhelming and engaging, I can’t wait to spend more time with the people that make it all possible.

Day 7: Nice Mice, Diced in Ice

Today, the lab took part in a “Data Club” where different researchers present their findings to each other. This morning’s speaker worked with a peculiar compound that binds to the inside of receptors in some neurons. These synthetic compounds amplify the abilities of a different compound, Neurotensin, that binds to receptors on the outside of cells, cooperating to inhibit the effects of psycho-stimulants like cocaine and methamphetamine. Their research with lab mice indicates that the presence of the compound in cells with Beta Arrestin, a transducing protein that helps internalize receptors, vastly reduces craving for addictive drugs, thus potentially breaking the cycle of addiction. There is still substantial work left to be done to confirm the effects of the compound; it’s possible that these positive effects only take place in the cells of mice. However, there is quite a bit of potential for this compound being made into an oral drug, and I’m definitely looking forward to the next Data Club meeting.

For most of the morning, Dr. Masoudi and I waited for vacant spots in incubators to house our bacteria cultures, so I ended up working with Dr. Li Yin in the meantime. Dr. Li Yin set up two columns for chromatography, similar to the ones that Dr. Masoudi and I set up last week with the exception of Dr. Li Yin’s use of an antibody as a filtering agent. After thoroughly washing the columns to flush out any “non-specific binders,” I helped Dr. Li Yin pipette equal volumes of protein-rich solution into the columns where, at their bottom, the antibody resin bound to her target receptor and rejected any other unwanted proteins. Theoretically, the protein that we want should have stuck to the antibody resin. This quick wash protocol is followed by elution, where a “FLAG Peptide” is implemented to compete against our target receptor for attractions with the antibody. The receptor is promptly taken out of the system and dropped into a new container. The contents of this container will be later purified with size exclusion chromatography (the results of which I will get to see tomorrow!) I also helped Dr. Li Yin by organizing, pouring, and washing flasks of insect cell solutions. Not so surprisingly, the wash protocol in the lab is incredibly thorough. Each individual flask is rinsed three times, bleached, and “autoclaved” – where glass objects are put under 120C conditions at high pressure. Nothing survives. Some of my time was also spent spinning our retrieved insect cell solutions in massive centrifuges and separating the lipid-based cell membranes from the unneeded soluble fractions. Although tedious work, it was satisfying to peel the lipid layers from the bottom of each flask, transporting them to smaller containers that were also spun at high speeds and eventually placed in liquid nitrogen. The super cool liquid (in both senses) evaporates around -190C, constituting a temperature so low that water sometimes doesn’t have time to crystallize orderly. The result is a “flash-frozen” solution of insect cells that preserves the structures and prevents any biological processes. We also played with the rest of the liquid nitrogen (safely) by pouring it on the flour and watching the water vapor around it condense 🙂

One of the more dramatic parts of yesterday, something I forgot to mention in the last post, involved dead mice. One of the new M.D.s in the lab had euthanized two mice, dissected them, retrieved tissue from their femurs, spun the results, and transferred the remains to cold containers. Thus, nice mice diced in ice. Yikes.

The liquid is ostensibly volatile because nitrogen boils at room temperature. The atmospheric pressure is not great enough to prevent the nitrogen gas molecules from breaking their inter-molecular attractions and leaving the body of liquid. However, the white gas surrounding the container isn’t nitrogen, but rather water. Because the escaping nitrogen is below freezing, the water vapor particles in the air condense and form a surrounding fog.

Day 6: Readily Receiving Receptors

Have you ever locked yourself in a freezer? Neither have I, but I imagine that it’s something like spending thirty seconds in the lab’s cold room. The microbiologists and chemists of the Lefkowitz lab need the cold room at temperatures near freezing to impede natural processes and preserve the trillions of proteins that line their metallic walls. Dr. Masoudi had to leave for a bit, so I spent the early morning reading up on G-Protein Coupled Receptors and helping Dr. Li Yin, Dr. Masoudi’s co-researcher. Dr. Li Yin was kind enough to let me do a majority of the hands-on work for her protein identification process, but this included heading into the cold room to occasionally refill the plastic column she uses for chromatography with a wash solution (it’s freezing in there). This form of chromatography works by employing an antibody that has a high affinity for the receptor that we want to extract. Everything else in the solution passes through the column without strongly interacting with the antibody in the resin. Eventually, Dr. Li Yin will pass a molecule through the same column that has an even stronger affinity for the antibody than our receptor, removing the receptor from the column. We also spent time grinding the cell membranes of moths – not something I thought I’d write in my lifetime. These moth cell-membranes hold the GPCR that we want, but in order to prepare them for filtration we need to homogenize the cell solutions and eventually mix them with some detergent. These soap molecules will act as a shield for the receptor, sustaining the structures for crystallization.

I also had the opportunity to make some stock solutions and use high-tech pipettes to deliver them into tiny aliquot containers. The LB media that I made is a combination of benzonase, a highly reactive enzyme, and protease inhibitor that prevents the denaturation of helpful cellular proteins. We soon moved on to her Western blots that showed the presence of a receptor using a set of primary and secondary enzymes. The secondary enzyme that was involved actually derives from rabbits! Another one of the enzymes she used was horseradish peroxidase that, yep, comes from horseradish.

A majority of the day was used gathering information to understand these receptors on a broader scale. Dr. Masoudi and I went over the sequence that some of these receptors undergo, from the moment an agonist – adrenaline, morphine, a photon – binds, to the recycling of these receptors in the lysosomes of cells. There’s quite a bit of literature on GPCRs, but barely any information on the mapping of these macro-molecules with X-Ray crystallography, and even less info on our synthetic protein Nb6B9 (FUN FACT: Nb6B9 was originally made from the antibodies of llamas).

A llama

Finally, Dr. Masoudi prepared our bacterial colony by taking a set of cells that (hopefully) incorporated the Nb6B9 gene into their DNA. We’ll know for sure that the gene was successfully incorporated after the DNA is sequenced by a lab at Duke, but for now we’re hoping that the colony we’ve set up will grow to produce as much Nb6B9 as possible. To avoid any fungal infections, Dr. Masoudi uses a lamp to ward off aerial imposters, but he also uses ethanol to clean the table. We really only realized how precarious the situation was after we had finished setting everything up.

Ever seen a tornado?

Day 5: The Case of the Festering Fun Fungi Felon

One word: contamination. It’s like muttering the dark lord’s name. It’s like cursing in front of your mother. You say it and the whole world crumbles around you. The E. Coli flasks that Dr. Masoudi, Dr. Li Yin, and I had put together did not show growth over the weekend, meaning that some sort of alien substance had ousted our benign bacterium. Initially, Dr. Masoudi thought that the unwelcome stranger was Phage, a horribly persistent virus that would require a thorough bleaching of our workbench, but something had caught his eye before we went to bleach Dr. Li Yin’s station. At the bottom of our LB medium, a solution that we evenly poured in each of the E. Coli flasks, there grew an innocuous speck of fungi that competed for nutrients against our bacteria. It was this seemingly minor contaminating agent that forced us to pour 12 liters of prepared bacterial solution down the drain.

After washing our flasks and going over protocol again, we had to prepare new bacterial colonies that will house our precious Nb6B9 protein. We made two separate solutions: the first solution contains our LB medium and agar, a solidifying agent, while the second solution only has LB. The first solution was poured into about twenty petri dishes that serve as the houses for our bacterial colonies; we followed this by adding in the antibiotic kanamycin. Colonies will form overnight once we introduce E. Coli cells that have a particular plasmid, a set of DNA that is both resistant to our antibiotic and has the target gene for Nb6B9 production. We ensure that the plasmid enters the cell envelopes of the E. Coli with a half-hour cooling process followed by a prompt “heat-shock” that loosens up the cell membrane, increasing it’s permeability. Antibiotic exists in the petri dishes to weed out the bacteria that won’t produce Nb6B9 at our desired capacity, leaving the successful colonies that we will grow and eventually harvest. It’s a long process, but it’ll be rewarding once we extract our precious protein.

In the meantime, we checked up on the results from our Western. The membrane that has our protein imprint was taken to a special scanning device that emits different wavelengths of light to expose the presence of different bio-molecules. For example, DNA is detected using short-wave ultraviolet radiation. We had proteins though, and we forced the molecules to react with a substrate that leaves a bio-luminescent product. We turned the scanner from an ultraviolet machine to something like a photographer’s “dark room” so that we could see the faint bio-luminescence. What was found was a single strip of protein between 7 and 12 kDa that matches the identity of our Nb6B9 protein, indicating that Nb6B9 didn’t form any dimers (combinations of itself) and can be filtered out when using carboxypeptidase (the enzyme that we used to differentiate Nb6B9 from its constituent peptide chains).

Day 4: A Protein more Stingy than Mr. Krabs

Today was national doughnut day. Per the laws of office jobs, someone brought in two dozen doughnuts and they all disappeared within a matter of minutes. You could probably model the amount of doughnuts in the break room with an exponential decay graph. I barely got half of a doughnut, but Dr. Masoudi and I had to carry on with our forlorn bellies. Our empty stomachs pretty much paralleled our experiment (which was fruitless). The electrophoresis analysis that we ran yesterday told us that we needed to start the entire protein-harvesting process all over again. Dr. Masoudi got out a new batch of E. Coli cells, introducing a bacterium to the flasks and keeping the containers in incubators. We also set up a protein-identifying process called a “Western” that creates a protein imprint on a membrane using an electric current – I have yet to see the results of this experiment. We ran a second electrophoresis experiment today as well, and we received a new and unexpected result. The gel that holds our data works by showing us the distribution of peptide chains based on molar mass. Yesterday, we assumed that the thickest, boldest line indicated our target protein, Nb6B9, even though its molar mass was a bit off. The new setup tells us something completely different; that thick, bold line was actually a contaminant that attached itself to our nickel resin during filtration and was never successfully washed out. In our new analysis, a faint second line pops up below a bolder line and is between the molar masses of 5,000 and 17,000 kiloDaltons (the first and second notches) – this line better matches Nb6B9 which is approximately 12,000 kDa. The new trial has pretty much dispelled our previous preconception that the bold line was our target protein, supporting the idea that our filtration methods resulted in an unsatisfactory percent yield.

Besides this bad news, the rest of the day went pretty well. I’m getting to know everyone in the lab a bit better, and I’ve even observed some other chemists setting up X-ray crystallography. In this practice, proteins are crystallized for stability and X-rays are shot at the configurations to create images of the crystallized structures. Hopefully Dr. Masoudi and I will soon get the chance to crystallize and examine our stingy Nb6B9 protein when its attached to a cell receptor (if the protein ever decides to cooperate). Fingers crossed!

Here’s an end-of-the-week haiku:

 

Insect receptors

Patiently wait for stingy

Nb6B9

Day 3: In Pursuit of the Protein

We were unlucky today. Filtering out a relatively lightweight protein with various buffer solutions is already a tedious, time-consuming process, but knowing that you’ll likely have to do it all over again is just heartbreaking. We began the day by running mixtures of acid-base buffers (a solution known as HEPES) along with solutions of tiny carbon-nitrogen rings (imidazole) to extract as much of our precious Nb6B9 as possible. I got in a lot of good practice using micro pipettes, especially when we needed to place little volumes of protein indicators in our filtered solutions. The initial results we got from the indicators weren’t pretty – the image below shows a gradient of color from light brown to blue and back to brown. This seemingly harmless, variegated collection told Dr. Masoudi and I that only a few samples of our solution had a high concentration of protein, meaning we had lost quite a bit of Nb6B9 along the way. Our electrophoresis analysis of the results was even more disappointing. This analysis works by introducing a specific binding agent, called SDS, to each of the proteins. Proteins composed of longer amino acid chains will invariably bind with more of the agent, making the larger structures more net-negative and the smaller structures less net-negative. When a current is applied to the sieve, the protein samples travel different distances based on their relative negativity; the larger proteins have a more difficult time reaching the bottom of the cylinder compared to the smaller proteins. Through this process, we were able to identify that we had some Nb6B9 but not nearly enough. We’ll probably have to go through the entire filtration process again to increase our yield…        🙁

Regardless, there’s still hope for tomorrow! Stay tuned!

 

Day 2: Adventures with E. Coli

Hustling to make it to the lab on time, I ran into Dr. Masoudi. We had a short discussion about our protocol today and we soon proceeded to get some E. Coli cells that were injected with a specific compound known as “IPTG.” This special protein turns on the promoter for a gene in E. Coli’s DNA that produces a nanobody called Nb6B9. Needless to say, I was utterly oblivious this morning. It took me a while to better understand what exactly was happening, but when I got a hang of things we started rolling. We began by vigorously shaking the bottles of E. Coli using some machines that bounce up and down upon contact with the bottle; my hand was numb after using this “vortex machine.” After the E. Coli had been disrupted enough and mixed with some buffer solution (basically a stabilizing solution), we placed the bottles in a freezer room to prevent any unwanted reactions. But Dr. Masoudi and I soon came across a pretty severe problem. The E. Coli bottles were filled with foam that made it nearly impossible for us to extract the cell solution. Usually, this wouldn’t be such a major issue, but the next step in our procedure was to use a gargantuan centrifuge (giant spinning device). As the bottles rotate at 14,000 revolutions per minute, the pressure against the sides of the bottles greatly increase and any space not occupied by dense solution (any of the area with foam) increase the risk of collapsing the bottle, ruining the centrifuge, and costing the lab tens of thousands of dollars. Did some famous person say something along the lines of “successful people take risks”? Because that’s exactly what we did. We balanced a couple half-filled bottles and popped them into the centrifuge, hoping to the gods of science that we wouldn’t hear an explosion. The centrifuge continued spinning for a good ten minutes without any problems, so we used the same procedure for the rest of the bottles. The well-mixed E. Coli solutions were poured altogether in the same flask, but another problem arose: the liquid was viscous. Because we needed to filter the E. Coli mixtures through a paper with fine, minuscule openings, a viscous liquid that binds together powerfully wouldn’t be able to meander through the holes. Only after adding a special enzyme, benzonase, did we have a watery liquid that we could us in our setup.

In the image above, the blue part of the column is nickel resin that binds to our oh-so precious protein, Nb6B9. The filtered liquid was dripping at a rate of about 2mL per minute, meaning the entire process took up to four hours.

The greatest lesson I’ve learned from my first two days in the Lefkowitz lab is that understanding is a requirement for appreciation. I thought I had a good handle on chemistry before I got into the lab, but I’ve learned that there is so much more to know, so much more to analyze, and so many more opportunities to take risks.

Day 1: Introduced to the Lab

Getting to the “CARL” building where my supervising post-doc works was a pain this morning. I had actually come to the Duke Medical Campus in Downtown Durham earlier in the year, so I was acquainted with the tortuous paths, high-rising brick buildings, and construction sites. Needless to say, I still got lost. Knowing that I’d get lost, I came to the lab one hour early, wandered around for thirty minutes, and (surprisingly) ran into my supervisor, Dr. Masoudi. Promptly, he directed to me to the lab’s manager with whom I signed some confidentiality paperwork. The lab is tucked at the top level of the building, where a plethora of groups are working on biochem projects. My specific lab, the Lefkowitz lab, deals with G-Protein Coupled Receptors that act as intercellular communication devices in eukaryotes (animal cells). These critical receptors come in thousands of different forms but they all work by wrapping around the cell membrane seven times. A “binding site” occupies the end of the receptor outside of the cell while a G-protein – a protein composed of three primary parts that can be ejected to communicate intracellular messages – is connected to the receptor on the inside of the cell. Whenever a unique body binds to the receptor outside of the cell, a “conformational” change occurs where the positioning of the macromolecule slightly alters and releases the G-protein. This complex process is the same process that cells undergo for nearly 40% of our prescribed medicine. The G-protein coupled receptor plays a colossal role in human health, and I can’t wait to get started in the lab!

Today’s labwork began with the expression of beta-2 adrenergic receptors in insect cells. The cells were placed in a solution that expedites receptor formation. The flasks holding these cells were put in a massive centrifuge – a device that spins vessels at high speeds to separate insoluble particles. We poured out the solution, extracted the cells while stabilizing their pH with a buffer solution, and labelled each of our solution-filled flasks. Dr. Masoudi emphasized that everything in the lab must be labeled. We used a smaller centrifuge (going at 4000 rpm!) to separate the insect cells from the buffer solution. Using ethanol and dry ice (the lab ran out of liquid nitrogen), we flash-froze our vessels and later placed our rack of flasks in a massive freezer. The cells in these flasks will eventually be used when the receptors are needed for X-ray crystallography. After our lunch break, Dr. Masoudi and I went over to a lab-wide meeting where different project leaders explicate the results from the past week. Besides X-ray crystallography, other researchers only a couple doors down use cryogenic electron microscopy to better understand the structures of these receptors.

Everyone in the lab is incredibly kind. One researcher called “Bullet” gave up his set of pipets so I could use them later on in the internship. Dr. Masoudi works with another researcher, Li Yin, who was kind enough to give up her work bench and desk so I could use it for labwork. Overall, the lab’s atmosphere is positive, but what I found to be most surprising is that this space is highly diverse; a large majority of the researchers here are first-generation immigrants. Dr. Masoudi just so happens to be a first-generation Iranian immigrant like my mom! It’s been such a phenomenal experience so far, and I’m so grateful for the opportunity to witness some crucial work in the field of biochemistry. Can’t wait for tomorrow!

 

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