by Regina Gul
My placement this summer was at King’s College London in the Neuroscience department for the duration of two weeks (17th August – 28th August 2015). Before I start this entry, I would like to mention just how wonderful everyone was at making sure I was well accommodated with their work, that I knew what was going on and that I was as involved as possible to make the most out of this opportunity. With that said, I’ll start off talking about my first week which was spent with Dr Caroline Formstone before writing another entry about the work I did the Dr Matthew Grubb and his team (shout out to Elisa and Adna!) during the second week.
Dr Formstone is interested in how the early CNS was formed, the formation of neural tubes, how neurons set up their circuits and If neuroepithelial cells have polarity systems that allow the neurons to move towards the same place constantly.
Neurons move down the cerebellum in the back of the brain and what Dr Formstone is trying to find out is if this movement is generated by the neurons themselves or by the neuroepithelial cells. These cells have adhesions between them, both at their apical and basal ends to make sure that nothing gets through and it even allows the epithelial cells to communicate with each other. Now, the protein found in the adhesions varies – there is a variant 1 and variant 2. The differences between these two variants are that after the transcription of RNA, the last exon shown is either E14 or M14. The ratio of E14 to M14 is general 50:50 but this can be manipulated to get more or less of one. With this, we could predict whether it is the protein that causes the neurons to migrate towards the tail, whether it is this “parental guidance” given by the epithelial cells that leads them on.
With these different honing systems, it means that these variants are placed at different parts of the cell. But this is just a hypothesis, even though we know there are different proteins at the basal and apical ends of the neuroepithelial cells, we cannot be certain of this.
So in my first week, we wanted to carry out the project that the students from In2Science had previously started with her. What they had done is that they had managed to get onto the stage where they amplified the last exon (E14 or M14) using a PCR (polymerase chain reaction) and cloned it into the plasmid vector which is specifically constructed to allow protein expression at high level. The variant 2 of PDZ domain motif is found in proteins as a way of targeting a protein to a particular part of the cell and what we’re trying to find out is if the PDZ domain targets a particular part of the cell and what proteins are involved in the distribution process. Essentially: does it organise the migration of neurons down the cerebellum?
Throughout the week we grew agar jelly and BL21 bacteria to their exponential phase (when they grow fastest) and DNA as well as a digest so we could run this on a gel. The gel is mainly to help us ensure that the DNA is fine; it creates a ladder due to a marker and then when the DNA is added you can see the different bands and how much of it there is. We also added IPTG which helps transcribe the RNA which can then be translated as a protein and so we added this into some cell membranes with GFP (green fluorescent protein) to allow us to later see the green when left overnight. About 3/20 cells showed the GFP tag!
My first week was really interesting and fascinating, I learnt to see just how life as a scientist would be and got to be in an environment where I used many machines and learnt about simple lab skills that you can’t just learn out of a textbook. Things like using a shaking incubator, a centrifuge, running a gel, growing agar jelly, growing bacteria, or even simply pipetting with different equipment than we do at school became ordinary!