shardBefore 9:00 Walking in!

The Shard is one of London’s most famous landmarks and I use it to direct me to Uni. I’m lucky that I can use the cold morning air as a wake-me-up.

9 o’clock, My Desk: I clearly control NASA from my dual monitors! My research is split between the lab and my computer. In the lab, I ‘hack’ DNA in order to generate engineered proteins of interest for experiments which are complemented by computer-based molecular dynamic simulations. I produce a lot of large data that needs to be processed, especially the X-ray crystallography files which are GBs in size – this is where two monitors and a lot of CPUs come in handy…

 

desk

After 9:00 Checking my cells. I take them out of the warmth of the incubator to have a look at them under the microscope. I’m checking for foci of cells to signify that my transfection has worked – this means the cells are producing antibiotic resistance and can survive while the other cells die. Once they have divided I hope that they will glow green since the transfected protein is GFP tagged. In a 24-well there are about 0.2 x 106 cells in one well of about 3 ml. Occasionally I will use the cells for an experiment but mostly I think of them as my protein-production factories, I ultimately want to obtain the greatest yield of my protein of interest. In order to this, I’ll eventually have to scale them from this small well into the large 1L vessel shown below. The fun starts once I have purified the protein but until then I’ll have to tend to my cells for about a month.

My other cells still requiring ‘splitting’ as part of their regular maintenance. I give them a morning wash with PBS and then split them up form their friends (dividing them into new flasks) so they still have food and space to grow.

10:00 I have a meeting with the other PhD representatives; Justin and Federico at the AMT café on the 16th floor of Guy’s Tower. We’re launching a Mentoring scheme for the PhD students in the department. It has this lovely distracting view on nice days:

view

11:00 FACS. An occasion when I do use the cells for an experiment, I’m measuring binding of one of my proteins to the IgE molecule expressed on the cell surface. This is a multi-layered experiment; first I add a blocker – this shields sticky proteins on the cell surface to reduce non-specific binding. Then, I add my protein and give it some time to stick, I then wash the cells to remove the excess. Currently I have no way of detecting if my protein has bound so I add a labelled antibody specific for my protein. This antibody is chemically labelled with a flourophore so I detect the coloured light emitted by it using a FACS machine.

12:30 Lunch! The experiment has incubation periods of 30 mins, during one of these breaks I think it’s a good time to take lunch with the other PhD students in the lunch room. I’ve got a packed-lunch and cup of tea.

13:30 During another incubation period I answer my student’s questions, I’m currently supervising an undergraduate and a MRes student who are making recombinant mouse protein (We’ve told E.coli to manufacture large quantities of mouse protein for us).

 15:00 As I complete and clean-up after the previous experiment, I analyze the data on my computer and then take a walk down to the X-ray crystallography lab. Here I check to see if my protein has crystallised – each plate has 96 different conditions, here’s what one condition has yielded:

cristal

Crystallising proteins can be a long process, its still considered ‘black magic’, I’ve set up a number of conditions to increase chances of success – and check regularly to see if any new crystals have grown. Here is one example of a successful condition – regular crystals that posses bifringent properties – this means they are most likely to be protein crystals! This image was taken with 10x magnification so they are tiny – perhaps 0.3mm in size. I will eventually take them to the particle accelerator in Oxford to generate one of those large gigabyte sized files that may reveal the atomic structure of the protein.

xray

X-ray crystallography – turning dots into 3D-atomic structures with a bit of help from maths.

16:30 Another cup of tea, I update my lab-book and plan the experiments for the rest of the week, however, I’m easily distracted and in my chatty-office we break out into a group chat.

18:00 I would be walking home along the southbank but today I’m heading off for fencing training.

 

Written by Veronica Ilkow