– Could you remind us briefly what your project is about?
My thesis is about the crystallization of membrane proteins with an automated microfluidic pipeline. But what do these fancy words really mean? They simply describe that my work is associated with the process of crystallizing membrane proteins on microfluidic devices.
What are membrane proteins and why we need to study them? Membrane proteins are proteins located on the membranes of the cell. Depending on their structure, they are involved in a plethora of biological functions. For example, there is a family of membrane proteins (transporters) that are responsible for transporting molecules across the cellular membrane. They transport for example ferric iron ions (Fe+3) into the interior of cells. One of the model membrane proteins that I am working with is responsible for this function in human pathogen bacteria. However, in order to understand the function of the various families of membrane proteins, we need to know their 3D structure at high resolution. Nowadays, we can study the structure of membrane proteins by using for example X-ray diffraction. But, the premise for that is to have crystals of the targeted membrane protein. And this is the reason why the crystallization of these biological macromolecules is crucial. We need to find the right, optimal conditions for crystallizing each target and use X-ray crystallography to “read” their structure.
However, one might think: how can we optimize the crystallization of membrane proteins and which tools we need? Well, from my point of view the answer is microfluidics! I am working on developing microfluidic chips for crystallizing membrane proteins on them (on-chip crystallization) by using the microdialysis method. When the protein crystals are grown, scientists must harvest them and protect them (usually by cryocooling the crystals) in order to use them for X-ray diffraction measurements. The microchips that I am developing use materials that are compatible for in-situ X-ray measurements. This means that we don’t have to harvest the protein crystals and the crystals won’t be damaged during the steps of harvesting or cryocooling. Moreover, one of my goals is to develop a whole microfluidic pipeline where the process of crystallizing the membrane proteins will be automated and fully controlled. For example, I want to integrate temperature and chemical composition control into the microchips. This way, we will be able to study phase diagrams and develop protein crystals in precise conditions. Another advantage of microfluidic crystallization is to significantly reduce the amounts of protein samples to a few hundred nanoliters.
– What important milestone have you reached until now?
Lacking any previous experience on biochemistry, my first goal, when I started my thesis, was to learn as much as possible for membrane proteins and their functions. So, I concentrated on studying the two model membrane proteins that I am working with. I learned how to produce and purify these two proteins and I performed crystallization trials with traditional crystallization methods (vapor diffusion). Now, I am confident to produce and purify the amounts of membrane proteins that I need for trials with the microchips.
In parallel, I was working on learning microfabrication techniques in order to develop my microchips. I tried various protocols and materials and currently I did my first tests with the chips. And they worked! I performed an experiment and tried to crystallize Lysozyme and I got my first crystals on the chip. My next target is to crystallize the model membrane proteins that I am manipulating on the microchips.
– Did the ITN help you in the implementation of your project until now? If yes in what way?
I received a lot of help from our collaborator Jean-Baptiste Salmon (LOF, UMR 5258 CNRS-Solvay-Université de Bordeaux). I spent two months in Bordeaux during my first secondment within the RAMP network. There, I learned everything I needed to know about microfluidics and microfabrication.
I should also mention that the other students of the RAMP network, during our common workshops, helped me to understand concepts or methods related to membrane proteins. They were always very patient and willing to explain me various stuff, and for that I thank them.
– Would you recommend other students to apply to a position within a MSCA network such as RAMP? What advice would you give them?
Yes, I would strongly recommend that. Being a member of a MSCA network can provide additional experience and opportunities in contrast to other doctoral programs. What I really like about our network is the diversity of members’ scientific background. I am an engineer but I am collaborating with structural biologists or crystallographers towards a common goal.
But MSCA networks provide more than scientific opportunities. I am learning how to better communicate with people of various backgrounds, how to make new acquaintances and build my network. I learned to better deal with deadlines and administrative procedures. And last but not least, during my secondments within the RAMP network, I learned how to adapt to new conditions faster and more efficiently.
– Is there a topic you would like to share/collaborate within the network in relation to your research work? (Please, develop.)
My work is mainly focused on instrumental development. But as I already mentioned, these microfluidic devices are fabricated to facilitate the crystallization of membrane proteins and the study of their structure. We will test these microfluidic chips with the two model membrane proteins that we are producing in our lab and we hope that we will obtain well diffracting crystals. After that point, I hope that other members of this network would like to collaborate with us and use our microchips for crystallizing their own membrane proteins.