Refocus
Table of Contents
Sunday, February 23
🏃🏻 Travel (3/6) | ★★★★
ㅋㅋㅋ… In the morning, one of my friends took a polar plunge. He’d been goading me and several others since last night, but I decided to just record it. I captured an incredibly impressive video even though it’s a real shame I can’t share it here. After he plunged into the lake that looked like a sheet of ice, he quickly made his way back to the lodge, and I even cropped the footage to create a little emoji. What a fun friend!
I then returned to the lodge to pack up and took a quick rest before setting off for the Bay Area again. This time, the friend who drove on our way here had to load the ski equipment, so I ended up riding in another friend’s car. We chatted about all sorts of things and, as always, listened to various songs from our playlist(thanks to these, I ended up with over ten new songs added to mine. Thanks!). With no traffic, we arrived around 2 pm.
Once I got home, I can’t really recall much. I just took a deep rest, watched “냉장고를 부탁해,” “핑계고,” and did a meal prep, and went to bed early. I actually could have hit the gym, but I guess I wanted to take a break, using the trip as an excuse haha. From tomorrow, it’s back to working hard!
Monday, February 24
164.4 lbs (Day 59, starting from 162.6 lbs on Dec 28) | 🏃🏻 Done (1/6) | ★★★★
Outside of Work
Today was uneventful. I got a good workout in the morning and attended a class. Even though I didn’t feel particularly exhausted, I ended up going home and, without doing much in the evening, just fell asleep. I’m currently considering a way to leave my bag and phone in the library after work. I’m not sure how that’ll work out.
Lab Work
Last week, I checked the samples that had undergone dialysis and TEV cleavage via SDS-PAGE (the LC‑MS still isn’t working… I didn’t expect it to be this inconvenient). But the cleavage was not complete. For now, I’ve decided to add more TEV protease and let the cleavage proceed in the cold room for one more day.
Starting today, I began the process of introducing a different point mutation into the same protein. For this, I carried out site-directed mutagenesis: I added my designed primer to the WT template plasmid DNA, performed PCR, and then used a KLD reaction to obtain a plasmid carrying the desired mutation. After that, I transformed it into cloning-optimized bacteria and incubated them on a selection medium. Since the PCR had two different annealing temperature conditions, I split one plate in half for inoculation. I’m hoping that many colonies will grow by tomorrow.
Tuesday, February 25
165.0 lbs (Day 60, starting from 162.6 lbs on Dec 28) | 🏃🏻 Done (2/6) | ★★☆
Outside of Work
I got plenty of sleep, so my workout went well. In the morning, I attended a journal presentation class. One of my friends presented, and she really knows how to speak👍 Although the experiments didn’t go perfectly, I gave it my all. After work, I returned my rented ski apparel and then headed to Costco for some grocery shopping. I felt a bit more energized after eating some beef, although I ended up sleeping way too much… What can I say… haha.
Lab Work
Today was one of those days when almost nothing seemed to work out. I rechecked the TEV cleavage, and it still looked about the same. Determined to get some LC‑MS data somehow, I went to another lab to get it. The LC-MS pattern of our samples didn’t make logical sense(we have a hypothesis for sure). I proceeded with a reverse nickel column to separate the uncleaved from the cleaved protein; for the uncleaved fraction, I decided to try TEV cleavage again separately. But everything, including the TEV protease, eluted in the flow-through! What on earth is going on, haha… I suspected that pre-incubating the resin with the sample increased non-specific binding, leading to these results. Next time, I plan to secure binding by reloading the flow-through without any incubation. The resin manual from a vendor actually did that, and it seems we’re using far too much resin.
Previously, when I tried an overnight TEV cleavage again, I assumed the buffer exchange was complete and just added more enzymes while mixing. This time, I prepared a fresh buffer solution and ran the cleavage concurrently. More importantly, since I suspected that using TCEP last time might have affected the results, I switched back to using BME in the buffer and set up another overnight cleavage with that.
On a bit positive note, introducing a different mutation into the protein appears to be going well. I observed many single colonies, and for tomorrow’s mini-prep, I set up a total of six 5‑ml mini cultures overnight, three for each annealing condition.
Wednesday, February 26
166.4 lbs (Day 61, starting from 162.6 lbs on Dec 28) | 🏃🏻 Done (3/6) | ★★★★
Outside of Work
I got a solid workout this morning and then attended my Reaction Mechanism class. We jumped into stereoselective Aldol reactions, and wow, it’s getting more challenging by the day, haha… After a long day of experiments, I joined a Reaction Mechanism study session where we discussed some problems, and later, we headed to Raising Cane’s for dinner. The meal combo my friends recommended was a win, and the sauce was great. it’s so good that I’ve heard they even sell it separately, much like Chick-fil-A’s sauce. It almost felt like I hadn’t had chicken in ages😁, which made it taste even better. I wish there were more fast food spots like this near campus. It seems like I have to go out a bit to find them.
Oh, and on the productivity apps, I have used Todoist, but I saw one of my friends using Notion in a really cool, structured way with checklists. That got me thinking about what might work better for me. Since I can’t give up the Google ecosystem because of its calendar, I started exploring Google Tasks. I ended up splitting my tasks into three categories—Lab Schedule, Tasks, and Thoughts—which feels fantastic(for now at least). Now that I can substitute my notebook with a “Thoughts” section, I don’t need to juggle multiple apps anymore. I even added a Google Calendar tab using an Obsidian plugin and split my screen: one side shows the calendar and tasks, while the other displays my notes (like lab records) in Obsidian. The whole setup feels so clean. I highly recommend it!
Lab Work
TEV cleavage still isn’t working. When I checked the LC‑MS, it seemed I might have added too much protease, and everything just turned into a mess. I decided that continuing to struggle with this is a waste of time, so I’m starting over from scratch. For this (second) protein prep, I took a colony from a plate that had been streaked three weeks ago. So, for the next attempt, I transformed the fresh plasmid. This time, I’m determined not to stop purification after pellet lysis. I suspect that the precipitation we saw is now causing the TEV cleavage issues. Since the synthesis is nearly complete, I think I have enough time in the next two weeks to try again.
On a brighter note, the other mutation(which I’ll refer to as E→C from now on, while the previous one was M→C) has progressed well. I completed the mini-prep and sent the samples for sequencing. I’m excited to see the results tomorrow!
Thursday, February 27
166.2 lbs (Day 62, starting from 162.6 lbs on Dec 28) | 🏃🏻 Rest (3/6) | ★★★☆
Outside of Work
Last night, I passed out again pretty quickly and ended up waking around 3–4 AM. I decided to skip my workout today. There were no classes today. Oh, and I have a journal club presentation on March 25. After several discussions with my coach, I finally settled on the paper. It’s about an in‑cell active warhead that can target arginine—one of the amino acids—in a covalent and specific manner. Since I need to prepare for this diligently, I plan to post about it on my blog after the presentation, just like last time. Besides this, nothing else much happened… I went home and quickly fell asleep again. What’s going on with me?
Lab Work
M→C: The plate I incubated yesterday looked good, so today I started a starter culture. I might need to go out briefly on Saturday.
E→C: At first, I got confused about which sequencing data format (file extension) to open from the vendor, so aligning in Benchling didn’t work well and I had some trouble. I even wondered if I had designed the primer incorrectly since I only considered the protein of interest and not the entire plasmid sequence, it wasn’t exactly optimal. Fortunately, with help from a smart friend rotating in the lab, I managed to resolve the issue, and after discussing it with my mentor, we concluded that the plasmid was constructed properly. I then transformed it into a strain optimized for protein expression and incubated it on a selection plate. I plan to take it out tomorrow, store it at 4°C over the weekend, and then proceed with expression.
I also checked the NMR for the first time in a while. Even though the LC‑MS was down last time, the NMR of the major compound isolated from one of the test reactions was properly assigned. Tomorrow, along with a new reaction, I’m planning to run another reaction to synthesize the other enantiomer.
By the way, there’s a postdoc I often ask biology-related questions who, while not my direct mentor, is always super friendly and even works out with me in the mornings. He goes every day from Monday to Friday—what an amazing person! He’s almost like a second mentor, always looking out for me. Recently, he’s been struggling with a particular reaction for about a month, so I took some time to discuss it seriously with him (it’s an unusual vinylalumination reaction, but the reported yields are pretty good). I hope our discussion helped, even a little bit.
Friday, February 28
166.0 lbs (Day 63, starting from 162.6 lbs on Dec 28) | 🏃🏻 Done (4/6) | ★★★★
Outside of Work
Today, I woke up around 3 AM again, and I felt so bad. I just knew I couldn’t keep going like this. I ended up ordering a whiteboard from Amazon to jot down my daily action plan and check off tasks as I complete them. I really hope it works. The problem is, I have to come home for dinner, and once I shower and eat, I just don’t feel like heading out again. Starting next week, I’m seriously planning to leave my bag and my phone at the library after work. Some might say sleeping in isn’t a waste, but I have so many studies and preparations to tackle at my level (like the upcoming exit talk and journal club… ugh). Still, I managed to go to the gym and had a good workout. On the way, I spotted some unique purple flowers in bloom.
I attended my favorite Friday Research in Progress class. The first presenter was a grad student from the lab where I’ll be rotating next. Although their work is primarily biochemistry with little synthesis involved, it was fascinating to see how they methodically designed and executed experiments to elucidate a protein’s function. I can’t wait until I start my own thesis project. After a productive day in the lab, I came home and did some meal prep, but while waiting for rice to finish, I ended up dozing off again…truly remarkable😵
Lab Work
Biology:
- M→C: inoculated starter cultures into two 1‑liter TB flasks. After 2 hours, I checked the OD, induced protein expression, and then incubated at 18°C overnight. This is my third time doing it, and I’m already feeling somewhat comfortable with the process!
- E→C: I checked the plate and it looked good! I think using carbenicillin is smart since it lets us skip the step of adding SOC medium during transformation. I stored the plate in a 4°C freezer.
Chemistry: I ran two reactions, successfully purified products, and then put them onto a lyophilizer. I plan to check the NMR for both on Monday. As for the reaction the postdoc had been struggling with, I gave some tips. However, we checked that the reaction still wasn’t working well. We decided to try one more time using a different method of addition—not dropwise, but just dumping it all in at once. If that still doesn’t work, we’ll move on to a completely different methodology that I found on Sci-Finders.
Saturday, March 1
164.6 lbs (Day 64, starting from 162.6 lbs on Dec 28) | 🏃🏻 Done (5/6) | ★★★★
Although I woke up again at 3 AM, I managed to catch a bit more sleep and somehow squeezed in a workout with a friend. There’s something about regularly meeting up, exchanging thoughts, and catching up that really helps clear my mind! Even though my professor will eventually have ideas for my thesis project, I personally dream of undertaking a truly groundbreaking study.
Later, I headed to the lab briefly to centrifuge the protein-induced culture, collected the cell pellet, and stored it at –80°C. Starting Monday, I’ll kick off the purification! I even went to pick up the ceramic painting I got a couple of weeks ago. It turned out far more beautiful than I expected, and I’m really pleased with it. I suppose it went so well thanks to having a friend with a good aesthetic sense. If I’d done it alone, I doubt such charming pieces would have come to life(btw, Totoro was the perfect choice).
When I got home, I ended up writing nonstop on my blog. To be honest, I was so overwhelmed by everything piling up that I put my English studies aside and focused solely on getting the post done by writing in Korean first, then running it through GPT and doing a quick proofrea🫠 From next week on, I’m determined to balance my studies more consistently. Let’s do this!
That evening, a friendly postdoc from our lab invited a few rotating students and a couple of grad students over to his place for drinks. Even though such gatherings weren’t easy for me, everyone was so relaxed that I felt comfortable joining in! We enjoyed some delicious pizza and snacks along with beer and wine. After being away from alcohol for so long, I got tipsy pretty quickly and couldn’t even finish a small bottle of beer. As I’ve noticed in the lab, fun and playful people are just as entertaining outside of it. Come to think of it, if you can sense that vibe at work, it’s only natural, right?
What still saddens me is that there are so many parts of conversations I still can’t fully understand, especially when they involve cultural references or unfamiliar vocabulary, which makes it hard to react appropriately. It’s particularly disheartening when I miss a joke since I can’t exactly ask for a detailed explanation or have someone break it down for me, and humor that comes naturally just doesn’t translate that way. I’ve only been here for about six months, so maybe I’m expecting too much, but I can’t help missing my Korean friends. I’ll do my best to catch up as much as I can.
