Monday, 12 September 2011

'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.

Sunday, 4 September 2011

Giant question-marks hanging over our heads...

I've got two weeks worth of work to power through, as I didn't update over the bank holiday weekend (which has nothing to do with laziness-- more to do with the impossible logistics of updating whilst moving house). Probably best to get cracking then!

As I said in my last post, I had all my samples prepared and ready to run on the SDS-Page and Western Blot for the beginning of the week. I had 6 samples in total:

-OriLyt 3 DNA with Nuclear Extract 1
-OriLyt 4 (negative control) DNA with Nuclear Extract 1
-OriLyt 3 DNA with Nuclear Extract 2
-OriLyt 4 (negative control) DNA with Nuclear Extract 2
-OriLyt 3 DNA with Nuclear Extract 3
-OriLyt 4 (negative control) DNA with Nuclear Extract 3

First I set these samples to run through SDS-Page-- a procedure used to separate proteins out in order of their molecular weight. The procedure should allow us to separate all the proteinous elements in a Nuclear Extract that bind to our OriLyt DNA, therefore giving us an idea of what elements associate with the origin of lytic replication (giving us the replisome of EBV).

After the SDS-Page was finished, I then had to transfer the separated proteins from the gel onto a nitrocellulose membrane for Western Blot analysis. This process is sort of like running an SDS-Page almost, it uses the same idea (running a current as a means of development) but in this process you create something dubbed the 'transfer sandwich' continuing the trend of 'bizarre items/names in science'.

Once the transfer has finished, I then had to block the membrane before amplifying the proteins using antibodies-- or, more scientifically put, I then subjected my membrane to a Western Blot. This was then developed via ECL which, this time, I got to make up myself.

Making up the ECL made me feel a little like a crime scene detective-- as ECL involves Luminol. For those who haven't been brought up on CSI like I have, Luminol is a chemical that is able to fluoresce blood and so is used to detect where blood traces have been (though the visible evidence has been removed). It makes a lot of sense that this is a key component of the ECL reagents that allow you to develop Western Blot samples.

When we developed the blot the first time, we could see that there were very weak bands appearing (figure 1 below). The problem with this is that the bands only appeared in the lanes containing OriLyt 4, NE1 and OriLyt 3, NE3-- so why did they only develop in these regions? And even then, what were they because they definitely didn't appear in the region we expected them to.

  Figure 1- Western Blot 1, with the two bands highlighted.

When I showed these results to my supervisor she was just as baffled as I was, so we decided to reblock the sample and reprobe it again to see whether it was a definite result. When we reprobed it, we got a much clearer blot (figure 2 below) which showed the same results-- but also showed clear bands in OriLyt 3, NE1 and OriLyt 4, NE 3 as well... Further adding to the mystery.

Figure 2- Reprobe of the original western blot, showing further bands. Green arrow indicates where bands should theoretically be found for Zta.

That brings us neatly around to what this week was about, although I've been a little ill and so I've been in-and-out of the lab. We decided that the lack of bands in our nuclear extract lanes could potentially be caused by it being too weak to be detected by the ECL-- therefore we decided to scale up the experiment (by eight).

Repeating the previous procedures we then got yet another mysterious western blot... But, the bands that have developed have once more developed in the same place as previous-- indicating there is something there (opposed to procedural error). Of course, there is still a giant question-marks hanging over us as we try and figure out what it is and why it is there (one of my fellow lab-peers has laughed and said that maybe I've discovered another form of Zta; that's wishful thinking and a half).

Figure 3- Amplified western blot using NE3. Shows bands in OriLyt 3 (right highlight) and a weaker band in OriLyt 4 (left highlight). Question marks are bands in which the appearance was previously dismissed as beads-- but cannot be anymore as there were no beads added to these samples (Nuclear Extract 3- far right; Supernatant 3- right; Supernatant 4- left).

So right now, we have more questions than answers.

Sunday, 21 August 2011

Flying solo in the lab obviously means you're going to destroy something...

As predicted, this week I returned to my analysis of the origin of lytic replication in EBV-- although this time I was doing everything completely by myself. As exciting as it is to be told that you're now 'flying solo', it's also quite daunting. After all, what happens if I get confused? What if I don't get results? What if I blow something up? 

Okay, so blowing something up is extremely unlikely in this instance. But who knows, maybe I was going to be the one to short circuit something and burn down the entire lab. (I have a very over-active imagination, it has to be said).

Nothing like this did happen, of course. The only thing that I have managed to prove is that no matter how careful I am, I seem to be unable to get a successful PCR reaction. I've gone head-to-head with it now four times and got: 1) contamination like you wouldn't believe, 2) a positive PCR for one of the four samples, 3) no amplification at all, 4) a negative result on my gel. Round five will not be pretty unless I get some sort of positive result, it's starting to aggravate me.

PCR is not the only thing that I've been doing this week (otherwise, well, I may have lost it completely and be a gibbering mess of a failed scientist by now), I've been busy with my first OriLyt pull-down!

On Monday I was given the task of taking apart the two different assays that have been used in the past to do an OriLyt pull-down and decide which of the techniques give a better result and would be better to use. The first assay was the one suggested by the company that provides the Streptavidin beads (Invitrogen) and the second assay was designed by the masters' student that has just finished her project.

Although the basic outline of the procedures are the same-- the concentrations, timings and some procedural steps are different (where the text is normal, the procedures match, where there is bold this determines what Invitrogen suggest and italics indicates the masters' assay)

  1. 20ul of beads to magnet and removal of supernatant.
  2. Addition of 200ul of bead-bind wash buffer. Remove from magnet, wash.
  3. Repeat step 2 three times.
  4. Addition of DNA to beads/ Resuspension of beads in bead-bind wash buffer to 5ug/ul concentration (40ul)
  5. Addition of equal volume of biotinlyated DNA (diluted in H2O) to buffer.
  6. Wheel for 3 hours/ Wheel for 15 minutes to 1 hour.
  7. Centrifuge/ Apply to magnet and remove supernatant to rewash (once)
  8. Resuspension to desired concentration
  9. Gel-electrophoresis.

Once the gel had run and developed, I found that although both assays significantly reduced the DNA concentration from the original sample (indicating that there had been successful biotin-DNA binding to the Streptavidin coated beads).

 Sample 3 original (lane 2) with Masters' Assay output (lane 3&4)
Sample 3 original (lane 5) with Invitrogen Assay output (lane 6&7)
Sample 4 (lane 8) to allow concentration estimation

Looking at this, I decided that using the suggested method by Invitrogen would be the way forward as although we had the same approximate ratio of B-DNA binding, the procedure only required a maximum of 1 hour incubation on a wheel which meant that a full bead-DNA-nuclear extract preparation could be done within one day.

The following day I repeated the chosen assay with sample 4 just to ensure that it wasn't a fluke. Although the gel didn't run properly (soon to be discovered that there was contamination with the TBE-- overnight seemingly) there was still a positive result:

Sample 4 original (lane 3)
Sample 4 with bead addition (lane 4)

After this I was tasked to make up the buffers that are required for the second portion of the assay for the completion of DNA affinity protocol. This involved a lot of maths (which I quite frankly fail at doing every time) and weighing as well as some precise pH work (as the buffers have to be at pH 7.9). I was also required to work out the concentration of Did-C using spectrophotometry which was a bit of an eye opener as to how sometimes translating 'units' into usable concentration is more awkward than you'd think.

On Wednesday I spent the day working with the nuclear extracts to complete the assay. First I was tasked with spectrophotometry to determine the OD280 of the samples-- proportional to the amount of DNA/Protein present in the extracts. This was done so I could add a proportional amount of each different nuclear extract to the beads from sample 3 and sample 4 (I had three different nuclear extracts from 3 different cell-lines). 

After preparing the nuclear extract appropriately, they were then centrifuged and 3ul was removed to run on a gel at a later date. 30ul of each nuclear extract was then added to their appropriate beads (resulting with: NE 1 with Sample 3 and Sample 4; NE 2 with Sample 3 and Sample 4; NE 3 with Sample 3 and Sample 4-- amassing to 6 separate samples). These were then rotated together for 45 minutes before the supernatant was removed (which has been frozen to run on the same later date gel-- allowing us to pick apart the whole pull-down procedure if needed). The samples were then washed with a buffer before being added to Protein Sample Buffer and boiled.

This boiling procedure allowed my microcentrifuge tubes to gain coloured shoes.
These are geekily adorable. 

At this point all the samples have been frozen ready to be run on SDS-Page and a Western Blot early next week to show whether we had a successful pull-down or not. Right now I am keeping everything crossed that I have a positive result so I can go on and analyse the remaining 2 OriLyt fragments (Sample 1 and 2). 

The rest of the week was spent on aforementioned PCR-- but we all know how that went, so it's not particularly worth going in to any details when obviously said details aren't working the way they theoretically should

Sunday, 14 August 2011

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.