Anniversary Post: A Change of Direction

I’ve been away the past few weeks. Visiting home, family and friends in the UK. Received some much-needed rest and recuperation. It was an odd feeling, being away from Israel and my lab, particularly the alleviation of responsibility that comes with traveling.

My recent vacation has also served as an important opportunity for soul-searching and provided much intellectual and emotional distance from science and my PhD. I’ve come to somewhat of an acceptance recently, vis-à-vis this blog. That this little project of mine has served its initial aim as a reflective outlet for my rocky transition into life in Israel and as a PhD student. I think an entire year is enough time for me to objectively consider the usefulness of continuing with writing, at least in the capacity I have been thus far.  The progress reports I’ve been religiously offering have not been as inspiring or as conducive to growing my organisational capacities as I have hoped. Don’t get me wrong, I am proud of the effort and commitment I’ve made towards this end, but I think I’m reaching a point where this kind of writing is producing diminishing returns. What I mean my this is that without serious contemplation beyond what is necessary—for what essentially amounts to glorified journal entries—I am not convinced that continuing this blog will be increasingly useful. I’ll make a new post later on outlining why exactly I think this is the case, and perhaps it will serve as a jumping off point for the new direction I want to take this project. Briefly: a big part of why I began this blog was, in a way, simply to find my voice. Writing has always been, in the back of my mind, something I felt I would enjoy seriously pursuing, given that I had the will and desire to express myself. The only stumbling block I felt was a lack of practise, namely, the failure to take the perennial advice that the best way to become a writer is to write. With this blog I have somewhat broken through this limitation, admittedly self-imposed, but nevertheless a personal doubt as to whether I could truly see myself as a writer.

Though ultimately, this project has served as a precondition for the beginning of an effort to direct my creative energies towards something more sustainably productive and outward-looking. I’ve recently exploring some of my more innate motivations to life & intellectual pursuit and am excited to explore them further on this blog.

I know no-one is reading, but nevertheless: stay-tuned.

SLL

Week 50: Rosh ha-Shana

In a few days, the Hebrew year 5780 will be ushered in with Rosh ha-Shana, literally “head of the year”, the Jewish holiday that—you guessed it—celebrates the start of the new year. For me, this also holds some significance. Last year, I arrived in Israel a month after “Rosh ha-Shana”, so this holiday marks my own personal journey in Jerusalem. I have now spent almost 52 weeks away from home and away from my friends and family back in the UK. It feels surreal to say the least, and actually, if I’m being completely honest, I’m not altogether sure if I like it here. There are many frustrating aspects to life in Israel, the religiosity of its populace (at least in Jerusalem) means that there is no public transport on Saturdays. Food and general day-to-day living is unreasonably expensive. There is this tense atmosphere that seems nigh-unescapable. Politics and religion are intertwined to a palpable degree. And living in Israel has made me aware that here in the ancient land of the Abrahamic peoples, trust is hard won and friendships aren’t made overnight. I’ve definitely grown a thicker skin living here, but frankly, I’m a little afraid of the callouses and the threat of becoming too cold and unemotional.

Overall, it’s difficult to say exactly what I have gained by doing a PhD in Jerusalem as opposed to remaining in the UK or even Europe. But perhaps I am too early in the process to make any claims. The challenges I have faced scientifically are likely not too categorically different from the issues faced by grad students anywhere else in the world. But I suppose the general attitudes and perspectives of my co-workers and colleagues make my experience unique. The cultural immersion too is something I appreciate. But you know, in couple I may be singing a completely different tune.

Lab Stuff

As seems to be the trend, I will be leaving not with a bang but with a whimper. My ChIP experiment did not work. At the end of the DNA extraction step I was left with basically no material as measured with Qubit (a high-sensitivity DNA concentration reader). I’ll have to regroup and reassess. As I’ll be leaving for home next week, I’ve halted operations in the lab. I’ll need to do some hard thinking about what exactly I want to achieve and the best/most efficient way to accomplish my goals. In particular, at which point, if any, should I give up on this project as to not waste time on threads that lead nowhere. Over the next few weeks I plan to sit down and write (and read). I hope I can come up with something .

Until next year 😉

Week 49: Developing Transferable Skills in a PhD

Last week I mentioned being in a bit of a rut. Haven’t crawled out of it just yet, but now I’m feeling a bit more comfortable down here. As I mentioned today to someone, I’ve been having good days recently, however, my threshold for good is slowly becoming a day where nothing bad happens, i.e. experiments don’t fail—which seems to be the default mode of operation. What gets me through the day is taking refuge in a kind of prescient perspective that all of this will be ok eventually.

I’ve been thinking lately about transferable skills. In particular the skills that a PhD is supposed to help you obtain; for instance, research, problem solving, formal writing, etc.  Since I’ve been getting into programming lately, specifically using R and making graphs and learning commands in BASH, I’ve been shifting my expectations of what I want to get out of my PhD. Assuming that the trend of biology transitioning towards a more big data heavy, computational field continues, it might be wise to similarly tailor my day-to-day activities in anticipation of this. I’m operating under an assumption that learning wet-lab skills will have diminishing returns as technology and techniques improve, and that the more abstract skills like writing and thinking will be more valuable and robust to disruptive technologies and intra/inter-disciplinarily trends. Therefore, it follows that I should be minimizing my time spent holding pipettes and babysitting stem cells, and maximizing my time reading papers and writing code. I haven’t yet thought through how exactly to achieve this, but I suppose it will involve some planning my part—to ask research questions that can be best solved with the fewest experiments or to choose experiments which require the least amount of optimization. In this way I can use my time in the lab most efficiently and kind of redundancy-proof the majority of the skills I’ll need to pick up.

Lab Stuff

Actions speak louder than words, so I’ve been spending more time in front of my computer than getting my hands dirty this week. Tried to finish off a ChIP protocol and have been culturing some cell lines to perform some more experiments on. The rest of the time has been spend trying to think about what graphs and analysis I will need to do to finish off a story about pluripotency I’ve been trying to compile into a paper. I have a few nice graphs which seem reasonably convincing, I will write up everything in October, but I feel the need to add one or two more figures to tie things together and produce a comprehensive and cohesive piece of work. Some of this may require some wet-lab validation of results. There are a few roadblocks to achieving this so I hope my computational analysis can stand on its own ground as much as possible.

Week 48: In a minima

It’s been a week of ups and downs. I’m in the minima of a slump as far as I can tell, or rapidly approaching one. On the one hand, I’ve crawled out of a couple dead ends regarding options moving forward. But on the other, I am feeling like I have a mountain of work ahead of me before I can make some sense of what it is I am actually trying to achieve. Yes all of this is quite abstract and airy, but without getting stuck in the details, I am still trying to piece together the a fragmented story left behind by the previous PhD student of whom I inherited this project. Searching through his materials and cell lines, I encountered some discrepancies and also some unconvincing data. Thankfully, most of it is usable and I have made some great headway into analysing it and producing some interesting results. But the frustration comes from wanting to clean my hands of using material he left behind, and starting afresh with my own data and constructs and cells. It is as much a symbolic wish as much as a practical one. I feel like I want to make an independently intellectual contribution in one way or another. It all boils down to the impatience of wanting—as soon as possible—to begin a project of my own design and complete it.

There are holidays here in Israel in the month of October, it is the new year according to the Hebrew calendar, and the following weeks are generally a time for a number of feasts and celebrations. What this means is that Israel effectively shuts down for the whole month. I’m planning to take much needed respite from lab work and visit home for a few weeks.

Lab Stuff

I have simultaneously been sub-cloning and treating some cells of mine fro mycoplasma. I was unsure if the knock-outs I had been generating were bonefide knock-outs, as I had found traces of my ‘knocked-out’ protein via Western blot in the cells I’d been growing. This was strange as the dna I’d sequenced of these cells did not indicate that the cells were heterozygous for my protein. What it did look like was that there were traces of contaminating cells that had my knocked-out protein. At first I considered the possibility that this was protein from MEFs, which are inactivated fibroblasts that are necessary to culture embryonic stem cells on to retain their pluripotency. However, I went through a more stringent MEF depletion regime of plating and re-plating on gelatin, and still this pesky band still showed up in the Western blot. I am not sure exactly what is happening, perhaps a solution is to plate my cells without MEFs using 2i media.

In other news, I am slowly returning to the ChIP-seq protocol I’ve been having difficulties with. I have some crosslink-ed cells that are waiting to be processed. I hope to finish this and send the samples off for sequencing by the end of the month. I also tried a co-ip recently, not entirely sure if I succeeded, there was an issue with not being sure whether the beads that we have for ChIP can be used for co-IP. The jury is still not out on this, but I think I’ll be postponing this set of experiments for a little while as they are not an urgent priority.

What is a priority is getting together a bunch of figures that tell a story using RNA-seq data about my protein and its involvement in pluripotency. I think I have enough data for a solid manuscript. I think I’ll begin drafting this paper in earnest in October.

 

Week 47: Mistakes are good

A bit late for this week’s entry. If I may, I’d like to attribute this to some domestic duties I had to attend to on the weekend. Namely, cleaning my filthy, filthy apartment. Now that that’s over and done with it’s amazing how much an effect it had on my mood, seeing my home clean, or at least, the cleanest its been for a long time. I feel like it’s kind of lightened the load of my general day-to-day anxiety.

I have many updates to share. Most importantly perhaps, is the change in my scheduled secondment to Paris. It has come as somewhat of a relief, as I’m not entirely ready in terms of what I want to achieve and what I need to prepare before I depart. I still have some cell lines I would like to generate and quite a few experiments I want to perform. Instead of Paris, I suppose I will take the opportunity to visit home for a few weeks, seeing that I’m already in the mindset to fly away.

I’ve been decidedly more conscientious recently of my activities in the lab. What I mean by this is just general carefulness and avoiding shortcuts or any habits I’ve fallen into out of laziness. The idea that it is better to do something slower than to repeat the whole experiment all over again is slowly becoming engrained in my skull. It’s been a tough process don’t get me wrong, and I think making mistakes is essential to having this occur.  One of the few times my undergraduate honours project supervisor actually spoke to me, he gave me this piece of wisdom: try and make your mistakes now. It’s a variant of that tech-bro-y adage “fail early, fail often”. But I’m seeing the truth in this more and more each day. I think in science one often has to learn the hard way.

No Lab Stuff this week, I’ll give this and the following week’s update next time. 

 

 

Week 46: Another Brick in the Lab

The weeks are really flying by. And this one went like a blink of an eye .It was the a scheduled staff vacation so most of my lab weren’t at work. But I was still coming in and I believe I was reasonably productive. It’s peculiar how the much of an effect the environment can have on your mood. Not sure if this can be a generalised phenomenon or a quirk specific to my own easily influenced psyche, butI feel a little more restless when there are more people around working and feel the need to match the observable levels of activity. And when no one is around, like this past week, I feel all the more lethargic and easygoing, although I also find I have a hint more focus. Not sure which feeling I prefer, and the jury’s not quite out yet on which style is more conducive to productivity.

But anyhow, as it will be September in the next few days, I’ll need be wrapping up some experiments and freezing some cell lines in anticipation of my secondment in Paris. Everything is still really up in the air with regards to this brief interlude to my work. I have to do some serious thinking and reading about what I want to accomplish in October. I’ll be using a high-resolution single molecule microscopy technique to visualise the intracellular dynamics of beta-catenin, a multifunctional protein implicated in pluripotency. I will have to think about what questions I want to answer specifically, and what data I will need to satisfy or disprove my assumptions. I also have the seedlings of two papers, one of which I can begin to sculpt a manuscript out of. This is what I plan to accomplish in my downtime over the next two months. As I’ll also be starting another programming course at the end of October, the next 6-7 weeks or so is the most free time I’ll have for a while.

Lab Stuff

As I mentioned, I’ve been in the lab this week doing several different things. First of all, I am regrowing cell lines and supervising the clonal expansion of some putative knockouts. I’ve also done western blots on some of the grown colonies I’ve picked and found out some unfortunate, yet not completely tragic, news: it seems that the “knock-outs” I thought I generated were actually heterozygotes (at best). I don’t know what do with this information. On the one hand, my failure to make knock-outs nullifies all of the plans I’ve had so far about where to take my project. However, having a heterozygous knock-out may not be so bad, as long as these cells are actually what I think they are. I can still probably take these cells to France and do some imaging of them. No harm can be done, if there is an effect, it may still be interesting. There’s also the question if a homozygous knockout somehow pushes the threshold for whatever change is observable, and that a heterozygous knockout does not produce severe enough effects in cell to be noticeable. For instance, there may be some compensatory mechanism that buffers the severity of a lower dosage of my protein. In any case, I have learned something important and can make a more informed decision moving forward.

 

Week 45: A Surreal Journey

August is approaching its close, and I’m about to begin my eleventh month in Israel. It’s been such a surreal journey. I keep thinking back to my first time in Jerusalem, when I came here to visit the lab and for my interview. This seemed like such a strange & alien land— who would of thought that I’d eventually end up finding some semblance of a home in the arid, windy heights of Jerusalem. I’ve met a bunch of people, some of whom have already become friends and others who are quickly working their way into my life. I haven’t had the most social of a year, but I think I’m through the worst of the growing pains now and am ready to look a bit more outwards regarding my life and relationships here in Israel. My Hebrew has improved substantially, I’m still extremely awkward and my vocabulary is sparse, but I’m catching more and more words and sentences each day. I can carry out brief conversations in basic topics. My reading still needs a lot of work, but this should come with time as my vocabulary widens. I’ve also started singing a little in Hebrew, I heard this is one of the best ways to get a feel for the phrasing of a language, and to absorb subconsciously the grammar. I did this a little when I was learning French, quite some time ago now. Speaking of French, I might be heading to France in a little more than a month. I’m going to be working there thanks to my a program-sponsored secondment at the Institut Curie in Paris. I’m pretty excited! I hope I get wake up some dormant memories from all those years studying French back in the UK.

Lab Stuff

Just figured out I made an embarrassing and costly mistake. I used the wrong kinds of beads for my immunoprecipitation. I’ll have to start the entire ChIP experiment from the beginning, as I didn’t keep any frozen cells. On the bright side, I think I will use some freshly thawed cells to do it the second time round. I had a slight suspicion that the cells I was using weren’t the cells I thought they were. I’ll check them with Western blot this time to make sure they’re the knock outs I think they are.

With regards to computational work, I’ve been racking my brain to try and figure out some kind of connection between my protein and the pluripotency network, particularly where my protein fits into the puzzle. I’ve decided that there I two routes I can take with my research, both of which should hopefully lead to a paper with results and conclusions independent of one another.

 

Week 44: From Wet to Dry

Been a hectic week. On Wednesday I finished up a paper for a class in R programming and statistics. I took the class pretty seriously and put a lot of effort into the final project. It’s been quite a journey this past year — I went from knowing next to nothing about bioinformatics, to spending upwards of 10 hours a day on R this week trying to figure out how to get my code to work and learning how to make pretty figures with ggplot2 (the quintessential biological plot-making R package). I’ve mentioned this on multiple occasions to different people, but I’ve been finding programming immensely satisfying, particularly the ‘instant-feedback’ nature of computational work. It’s such a breath of fresh air from wet-lab and I like that I always can do something ‘different’, whenever I get sick of working in tissue culture or in the downtime between incubation-steps in my lately seemingly never-ending protocols. I would wholeheartedly recommend getting stuck into data analysis as soon as possible for anyone with a bio-background. The sooner you learn the basics, the more productive you’ll be on the whole I think. I’m looking forward to improving my skills and seeing where bioinformatics takes me.

It’s also been a while since I’ve posted some photos. I think the novelty of living in Jerusalem has worn off, it’s becoming increasingly rare I get the urge to pull out my phone to snap a picture. What was weird and wonderful in the beginning has now become mundanity tinged with annoyance. I guess this is a sign I’ve acclimatised *shrug*.

Lab Stuff

As I mentioned, didn’t do much work in the lab other than sitting in front of my computer. Started some sub-cloning of my knockout cell lines, will grow them up and then western blot to make sure they are pure KOs. Also I’ve continued with the ChIP-seq, just started the library preparation steps. The protocol following uses magnetic beads for DNA purification, I’ve managed to succeed with this step with reasonable amounts of DNA. Although, I think I may have mixed up my input samples with my ChIP-ed samples, I think I’ll have to do some quality control with qPCR to make sure I have the samples I think I have, and also to check that my antibodies actually worked (which is super important).

 

Week 43: Follow through

Decided to make a more concerted effort to learn Hebrew this week. It’s been a little over 10 months now since I’ve been in Israel, and I feel like I’ve settled in enough that I can  schedule my time in a reasonably predictable manner. What I mean by that is that I know roughly how long my routine is each day/week, e.g. travel times, meal prep, duration of experiments, how much attention the cell lines I’m culturing need, etc. As I wrote in the previous entry, I’m ready to start setting aside some time to get better at things I want to get better at—and actually follow through.

However, I want to postpone all of this until next week, and I have a bit of an excuse as to why: the final project of a course I’ve been taking this semester is due really soon. I want to concentrate on getting that done. In the meantime, I’ll start thinking about structuring a kind of study plan for weekends. I’m always tired in the evenings, too tired for the motivation to do more learning. Particularly as I’ve been having 10-12 hour days in the lab this week. When you factor in other admin, travelling and daily necessities, there isn’t really time for much else. I can take solace in my weekends being relatively free from obligation, I don’t have too much of a social life either so that makes things easier – for better or worse.

 

Lab Stuff

Been continuing with this ChIP-seq protocol. I’ve made it to just before the library preparation stage. It’s been a lengthy protocol, and unfortunately with its fair share of hiccups. Next week I’ll sit down and make a note of everything I need to know to carry it out, including where to get all the necessary equipment and who I need to ask. The next time I do this, it will definitely get a lot smoother. But I’m really glad I’ve made some headway. Whether this experiment works or not I’ve definitely learned a lot.

My transfections didn’t work. I’ve been trying to generate another CRISPR knockout after I discovered my old cell line was contaminated with mycoplasma. My plan B was always to continue treating this old cell line with anti-mycoplasma antibiotics while sub-cloning. There was a problem that in my knockout cell line, some wild type cells recolonised the culture somehow which my western blot and sequencing confirmed. I might still try and do another transfection, but I’m running out of time. I need these cell lines before October for an experiment with STORM imaging, but I’ll talk about this in the next entry perhaps.

Ciao bella!

Week 42: Habitual

The idea that there is a probational period required for ingraining habits is intriguing & comforting. I understand ‘habit’ as a repeated activity which requires far less activation energy to perform than activities that have not been integrated into a person’s daily/weekly life (intuition tells me beyond this timescale, repeated activities cease to be habits but become ‘traditions’).Perhaps being accountable   Of course this activation energy can even become negative, in the case of some of the more malignant habits, i.e. addictions. I’m thinking about this because of the positive/productive habits I’ve been trying to cultivate, and which of these have been successfully implemented into my life and which haven’t.

The common wisdom (and some research) tells us that it takes around 2 months for a repeated activity to set in as habit. Anecdotal evidence is hazy on this. I feel it’s all too easy to get set back into old ways, even after the 2 month prescription. But I haven’t really formalised and recorded activities aside from entries on this blog. From now I think I’ll try and hold myself accountable to some of the habits I want to form, by noting when I actually do each thing and when/how often.

I’m going to list them sequentially by how established I feel each activity is in my life, and consider my motivations for embarking upon them, and also, whether or not I’ve been successful in converting them into bonafide ‘habits’.

(1) First of all, naturally, this blog [once a week]

I think it’s self-evident for undertakings like these that you are very aware when you’ve been lazy. Having a deadline sort of forces you to start thinking about needing to do something in advance, and when that deadline passes you feel a little guilty that you’ve let yourself down, and that guilt is usually enough to make you sit down and do it (it’s enough for me at least).

(2) Exercise [once a week]

For me it’s going to the gym and using the free weights, more so machines now since I’ve moved here. My motivations for going to the gym have oscillated between vanity and general health/wellbeing over the 6 or so years I’ve been a gym-goer. In between sometimes months-long respites, I’ve been rather consistent with this activity. However, after starting this PhD this habit has become a once-a-week, I attribute this to a lack of energy and convenience. I believe both these problems can be solved. But the issue has taken a backseat to more pressing matters. On the whole, I’ve readjusted my priorities to focus on my research, I still take my health seriously, but perhaps without so much of the vigour that I used to. Of course, it it becomes more convenient and I fix my diet, I think I’d like to bump this habit up in frequency.

(3) Reading [several times a week]

Not so much an intentionally cultivated habit per-say but rather a habit in the traditional sense, I think reading is something people just for into, so long as they have an interesting book at hand. I generally read before going to bed. This is probably where I could do with changing things up, maybe I should set some time aside for reading (and taking notes, as I will soon describe).

(4) Maintaining a ‘Index Rerum’ [whenever I read, weekly?]

Basically, note taking on books I read and transferring notes or quotes to index cards.

I’ve been slacking on this, and I think it never really picked up steam. This is something I picked up from productivity guru cum life coach blogger Ryan Holliday and a habit that I suspect underlies much of the brilliance of writers and interesting people. It might be wise also to set some time aside to compile from the books I’ve read or am reading, the sentences I’ve underlined using the system in the about link. I really have found much value in this activity, I’d like start taking it more seriously. It could be a great first trial in my experiment in formalising habit formation. I’ll think about this.

(5) Music (playing guitar/piano/singing etc) [whenever]

This one has been more or less been under the whims of my mood. I see little urgency in this activity, but I’ve always wanted to learn jazz piano properly. It might be nice to also try this out too.

(6) Learning Hebrew [daily]

Now this is something I feel could benefit greatly through an accountability system.  I might make a log of practice sessions/time spent on each and also create a document where I can put lists of vocabulary I want to learn. I’ll also get back on duolingo. Now that Hebrew classes have finished for the semester, I have to motivate myself to get some practice in.

Anyhow, I think that’s all for now. I’ll keep thinking about how to achieve these goals and also think of the best way to record everything and keep myself accountable for the two months it’s supposed to take to form a habit. It’s a nice little experiment, I think I’ll start in November when I return from my planned secondment to Paris. I’ll explain what I’m planning to do there in the next entry.

Lab Stuff

A lot of transfections and FACS work this week. I’m trying to redo the CRISPR-KO after I decided that the cell line I had succeeded on was no longer trustworthy (due to a mycoplasma contamination). I may revisit this cell line and try to treat them again with antibiotics after sub-cloning on a bigger plate. But I want to see if I can make a new cell line in the meantime.

Everything is now prepared for the ChIP-seq I’ve been planning. I will try and do this next week. It’s going to be a lot of work, the protocol is three days long and requires a lot of effort and attention, I’ll need to be super focussed. After this experiment I have a little bit of a reprieve from lab work. I can finish up this project I need to complete or one of my courses and also rewrite the draft of the paper that, if all goes well with the ChIP-seq, I hope I can submit it at the end of the year.