'Does this mean I'll glow in the dark?'

Herein marks the beginning of my final week in the lab. The eight weeks have completely flown past and I can't believe it's coming to the end-- I feel like I should only be halfway through! Even scarier a thought is that in 3 weeks time I will be starting my third year and my dissertation... Cripes.

So last time I updated, there were question-marks here, there, everywhere. There is still no definite resolution to any of those questions yet-- but work has begun to solve the mystery. Because there is a lack of Zta banding in the blots (which is our positive control band) this raises questions as to whether something in the assay causes its' degradation or whether there was something wrong with the nuclear extract I was using. The solution to this is to create radioactively labelled Zta and run it through the procedure and see if it then bands (if it does, it means the nuclear extract had degraded). If not then there is something within the assay that causes this problem and the assay can be dissected to see where the degradation occurs.

After all this, before running the procedure again, the radioactive Zta can be doped in to nuclear extract to see that the modifications have worked. Hopefully then the next individual who tackles the project can get straight to work on isolating proteins (if there are any).

Of course, it's easier said than done because firstly we have to produce the radioactively labelled [S35Meth] Zta and I'm not trained to work with radioactivity. (Admittedly the idea of working with radioactivity brings up all sorts of 'Hulk' ideas in my mind and I'm not sure I want to mutate myself yet.) The first step is to generate this radioactive Zta-- through an in vitro transcription and translation system. This requires constant testing at every stage and it quite long and laborious... But it does look like we are producing Zta mRNA which is exactly what we need at this point in time.

With this being a long process, I've been helping out with some Maxi-Preps at the same time. And it's official: bacteria smell bloody funky. I felt like I could smell it on my skin for hours later and I kept getting really worried that customers at my other job could smell it on me too (I go straight from the lab to my other job some evenings). Anyway, I've managed to maxi-prep 6 separate colonies of bacteria with different mutations-- so I think I'm adjusting to the smell a little bit.

I've also done some mini-preps and restriction digests to compare different mini-prep techniques to see which generate the best results.

So yes, the last week has been spent doing bits-and-bobs around the lab whilst waiting for the next part of my own experiment to come into fruition. This week, of course, I have to start writing my report to go alongside my 8 week experience. Slightly daunting, but obviously everyone will be able to see how that goes when they get published.

Mutants, like X-Men, except in E.Coli.

As I mentioned in my last post, I've been temporarily helping out on a different project whilst my supervisor has been away. This time I was helping out with site-directed mutagenesis, transformation of E. Coli and preparing bacterial mutants for sequencing.

This was working alongside one of the PhD students in the lab, giving me a bit of an experience of what a PhD involves. Looking at it now, I've got to say that it's something that I would be highly interested in doing at some point-- but it is a decision that really shouldn't be taken lightly because it looks like a lot of hard work.

The first day of assisting on this project, I found myself learning the ins and outs of tissue/cell culture. The whole point of the tissue culture is to ensure that at all times there is an established cell line that can be used in the experiments. So I helped out examining the current cultures to see how well they were growing (that's a fancier way of saying that I was counting cells) and sterilizing all the equipment that was being used (as it's all highly open to contamination and when you're trying to grow a cell line this is exactly what you don't want).

After this, I was introduced to the procedures for site-directed mutagenesis-- using PCR. Nick (the PhD student) had designed and had his mutated primers already made, so it simply was just a case of adding the required components and setting the PCR to cycle through the conditions required to cause high numbers of the mutant plasmids to be replicated.

Once the PCR had completely cycled (4 hours later) we then removed the products and discarded the wild type plasmids using a digestion (in which the unmethylated wild type, normal plasmids were removed leaving only the methylated, mutant plasmids behind). We then transformed highly competent E. Coli cells with the plasmids and allowed the cells to incubate overnight.

The following day I was taught how to produce selective agar plates. Normally I would be completely up for doing any practical work-- but unfortunately when you produce agar plates you have to be fully suited and booted to stop any cross-contamination to the plates and you also have to have a bunsen burner going at full whack-- and it was probably one of the warmest days of the summer. Literally so sweltering, I don't think I've been that warm since my tent in Spain.

Anyway, to make the plates a selective medium, we added antibiotics to them which would ensure that only the E.Coli that had taken up our mutants would grow. If the colonies did grow then we'd know that we did, indeed, have a successful mutagenesis... Which we did! (For me that was a little bit of a novelty as I've not exactly had the best results thus far).

Over the next few days we repeated the above procedure several more times for different mutant lines (CIITA Forward, CIITA Reverse, CIITA negative, Zta Forward, Zta Reverse and Zta Negative) which are helping to amass a picture of the protein components of EBV.

Once all of the different mutant cell lines were made, I then took the remaining bacterial cultures and set about extracting the DNA elements from the cells via mini-preps. First for this the cells had to be isolated from their growth medium through centrifugation (the first time I'd ever used a large centrifuge opposed to a table-top one)-- which gave a pellet. The pellet was removed and then resuspended in 2 separate buffers (with Lysate Blu in which gave a colour change to confirm the procedure was being carried out correctly). These solutions were then placed into a table-top centrifuge and spun-- which caused the cellular elements to become debris leaving us with only the soluble components (proteins/RNA/DNA).

After removing the soluble elements I then carried out a separation procedure extremely similar to the procedure I'd used to separate the DNA from agarose a few weeks ago. Having done it before, by the time I'd finished the extraction this time around I felt like a tiny bit of a professional because obviously I could do it at a relatively steady speed without asking for any clarification.

Once the DNA had been purified, I then made a 1 in 10 mastermix of the DNA (diluting it with double-distilled H2O which, I didn't even know existed) which was then analysed via spectrophotometry. This gave us the overall concentration of DNA within the mastermix-- which I then used to calculate making a 50-100ug/ul solution. These resultant solutions have now been sent off for sequencing and they'll hopefully be back at some point this week.

Oh, also at some point over the last week this sign has appeared on our lab wall. It instantly became clear why a career in scientific research is for me:

Anyway, it's hard to believe I'm already halfway through my studentship. It's going extremely fast and before I know it I'm going to be submitting my report (slightly nerve-wracking in itself). Next week I believe I will be returning to my analysis of the OriLyt fragments as my supervisor is back, so I will obviously update to let everyone know what results I'm getting on that front.