38 weeks til thesis

Well, the start of this week was a bit of a let down. It’s ok. It happens… Then it was a nice big high and it all worked out fine in the end. Rollercoaster week – aka week 38 til thesis.

The Low Point: Despite completing the data collection for Aim 1 (woot!), I feel like I didn’t quite do what I set out to do. After all the preparations of last week, the twenty two repeats, the waiting three days for freeze-drying – the final bit of data I was working on ended up being irrelevant. *sigh*

I was planning on comparing the solubility of the freeze-dried powders in water. I had done a cursory version of this investigation previously and was really excited by the results, so I had a fair idea of what to expect. But when it came to weighing out three sets of ~20mg of jumpy, statically-charged powder into 2mL tubes, for 20 different samples… lets just say, the set up took a LOT longer than I thought it would.

The plan – based, as always, on a published protocol – was to gradually add high purity water (solvent) to the known weight of powder (solute), mixing between each addition, until such a concentration was reached whereby all the solute was dissolved and the solution was transparent.

The alternative method is to have a known volume of solvent, and gradually add more solute until the solution is saturated and cannot dissolve any more. At this point, you would re-weigh the solution and work out the exact weight that was added, and from that you can work out final concentration. Can you imagine trying to get tiny amounts of statically charged powder into a small container that is already wet, so that if you add too much, there’s no going back… Well, that’s why I decided against that method…

So, with great anticipation, I added the first aliquot of water to my first sample and mixed.

It gelled 😐

giphy (5)
#accurate (via giphy)

It was starting out at the highest concentration, too high to maintain solution and instead formed such a thick gel that I could not draw it up in my pipette. It was 50% w/vol, so this wasn’t too surprising. The downside was that, once it gelled, I couldn’t add any more water to it.

Bummer.

I would have to add a larger initial volume of water. It worked! I was cheering and seat-dancing (I’m sure you know what I mean!), tilting the liquid side to side to watch it run around the curve of the tube – until it stopped moving and I was faced with gel again. It seemed no matter how much water I added, what was initially a glorious golden solution would end up gelling. I even took one sample down to 5% and it formed a sol instead of a gel – which is better, but not much better. I got my hopes up so high then was devastated.

wk38_powdertosolution
Top: the powder takes the shape of its container during freezedrying. Bottom: the golden liquid that induced seat-dancing… until it gelled.

From my initial solubility investigation, this was supposed to my most soluble version. And it was not playing ball. What was I doing wrong?

Like a good little PhD student, I went and unloaded my worries… I mean… chatted to my supervisor about it. He reminded me that in my preliminary cell experiments I had managed to make low (≤2%) concentration solutions just fine. He asked “What do you really need it (the solubility) to be for your intended purpose?” (Read: does it even matter that you can’t make high concentration solutions with this powder? What do the cells respond to best?)

This sparked a memory filed way WAY back in the vault. In first year, I had read the stats for a commercial preparation of a similar silk compound to the one I’m targeting, and they had only used concentrations up to 0.3% on cells. The solutions I had managed to make for my preliminary cell experiments were at least five times that dose!

I was assuming that the solubility had to be amazingly high to be valuable and useful. That’s what I was doing wrong!

The High Point: I was actually doing great 😀 I had just forgotten where my goalposts were, got disoriented and disappointed. I got so caught up in ‘maximising’, ‘optimising’, ‘comparing’ and having to be ‘better than’, ‘bigger than’, ‘greater than’ the current alternatives. I forgot that at the heart of this investigation were some really basic needs I was aiming to meet: 1) the needs of my intended application for this compound, and 2) the need to keep moving forward with the rest of my experiments (finishing Aims 2 and 3). The low concentrations I had already managed to make were all I really needed. Finding the exact maximum solubility for each sample was irrelevant for my application. Cool to have – for sure! – but not essential, especially since my PhD is in the School of Medicine and not Protein Chemistry. So I am now doing something very much outside of my character and cutting and running, leaving Aim 1 far behind me.

I feel so much better now that I’ve gotten used to the idea that my data is complete as it is. It really bugged me at first – that I didn’t get solubility data at n=5 after all the time and effort I had put into prepping samples for it – but in the end, I have all I need to write my first paper.

And that’s what counts.

Aim 1 is DONE!

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It’s happy dance o’clock! (via giphy)

Plus, the remaining powder I had made for (the now unnecessary) solubility testing can be used in Aim 2 instead, saving me some serious prep time.

So, double WIN!

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I am approximately  ^this^  happy right now (via giphy)

Now, to write it all up and submit to a journal for peer review and – hopefully! – publication 😀

N.B. I was posting 1-2 weeks in arrears until now. So there will be a several posts within a few days of each other, but all will be up to date from now on. It was getting too confusing looking at the correct current week for my Gannt chart, and then writing about the previous week on here as though it was currently happening, then planning ahead for the next week’s work and blog post, and switching between the different frames of reference.

Keep it simple. Keep it moving. Make it happen. 🙂