Tuesday, 2 August 2011
The first 2 weeks
I’ve now completed my 2nd week of the placement, and so far it’s going pretty well – I haven’t managed to destroy anything yet!
My first impressions of the lab were very good – I was given a tour and saw how well equipped it is, and I was introduced to the other group members who are all very friendly. There is a good mixture of PhD students, postdocs and a couple of other summer students, and in total the research group has around 11 members – which I’m told is quite a lot!
My project is to work alongside my supervisor, doing some experiments looking at the interaction between a histone deacetylase (HDAC) and its binding partner. It took me a while to get my head around the project – the research in the lab centres around the structural biology of transcriptional corepressor complexes, which recruit and activate histone deacetylases to repress transcription of target genes through modification of chromatin structure. My role in the project is to help make mutations in the HDAC in previously identified key residues and regions of the protein thought to be essential for interactions with other components of the transcriptional corepressor complex. We can then look at the effect of these mutations on the binding and activity of the HDAC in order to confirm the importance of these residues and regions.
Although I had some prior knowledge about transcriptional repression and histone deacetylation, I hadn’t before looked in detail at the crystal structures of transcriptional corepressor complexes, and so I had to do a fair bit of paper reading to get my head around it all! The first week involved me being introduced to a lot of new techniques – I learnt how to do a MidiPrep to purify out plasmid DNA, how to make and run an agarose gel, how to set up and run PCR, how to do gel extraction – all things I had learnt about and seen bits of in practicals, but never done on my own. I also learnt the principles of mammalian tissue culture, which was completely new to me and quite interesting. I must admit I felt a bit useless for the first few days, not knowing where things were in the lab, having to constantly ask for help and keep having things repeated to me...but luckily my supervisor was very patient and helpful, and by the second week I was getting the hang of things and felt a bit more confident!
In the second week I was able to do much more on my own – I was left to introduce two mutations into a construct using several rounds of PCR with mutant primers, and this involved setting up the PCR, running a gel to confirm it had worked and then extracting the DNA from the band to use again in the next round. I was always surprised when the PCR actually worked! The process gets quite repetitive, but it’s all good practice and I’m getting faster at doing it each time.
I also had a go at some tissue culture – we had to grow and subculture mammalian HEK293 cells in preparation for transfection of our mutant constructs. Using sterile technique and working under a hood is vital in this process to prevent contamination of the cells, and I found that it really takes quite a lot of concentration to make sure everything remains sterile – you can’t go waving the pipette around and leaving lids off things, and everything has to be copiously sprayed with ethanol. So far my cells have stayed alive, which is promising!
All in all, I’ve had a very enjoyable first 2 weeks and feel well settled in to the lab now – I’ll keep you updated on how it goes!