Schistosomiasis, a neglected tropical disease, affects 200 million people worldwide and 700 million people are living in risk areas. It is second only to Malaria, yet there is far less research currently being done to combat it. Jose Brandao-Neto, a senior beamline scientist at Diamond, is collaborating with a group in Brazil and as a result, during July, a number of different novel proteins will be studied on one of our macromolecular crystallography beamlines (I04-1). These proteins are being grown and crystallised just over the road from Diamond in the Oxford Protein Production Facility (OPPF), which is situated in the Research Complex at Harwell (RCaH).
Juliana Torini from the University of São Paulo in Brazil is currently over in the UK as part of the collaboration and here she blogs on her time at Diamond.
Juliana on beamline I04-1
Day 1 (Wednesday 27 June)
After a long journey, I finally arrived in the Oxford Protein Production Facility (OPPF). In the morning, I got off the bus at the wrong stop, so had to walk a long way, but it’s ok. Arriving in the lab, I met Louise and Yamini, the latter, introduce me to colleagues in the Lab. She introduced me to Petra and Maria Rosa. Maria is from Portugal, so she speaks Portuguese! It’s very good, because when I don’t understand something, I ask Maria, and she translates for me. In the morning, I just observed Yamini working in the lab, she prepared medium for growing cells. At lunch time, Jose found me. He’s the Scientist collaborator with my group in Brazil. We had lunch together with Maria and Petra, and some other scientists. In the afternoon, I picked some colonies for growing, Yamini helped me. Was my first time working in the OPPF! At the end of day Maria gave me a ride to Didcot. We went to supermarket together, she helped me to buy a plug adapter. This was very important to me because now I can turn on my computer in my hotel room and speak with my parents in Brazil through skype and check my emails. Maria dropped me off at the bus stop and I went to my hotel in Abingdon.
A purification assay in the OPPF
Day 2 (Thursday 28 June)
Today I got off at the right bus stop, much better! I arrived at the OPPF and waited for Yamini to arrive. Today we expressed 4 L of three different proteins. Were used 24 shaker flasks and 12 L of the Power Broth medium, crazy! I had lunch with Yamini but today I didn’t like the food and didn’t eat much. After lunch I started to really miss Brazil and the people, but when I started working again I forgot this. During the afternoon, while I waiting to make the induction of the expression , I went to get to know Diamond. I saw the ring, the I04-1 Macromolecular Crystallography beamline and the Small Angle X-ray Scattering and Diffraction beamline, but this last one I don’t remember the number, I think B21 but I am not sure. After I came back to the lab I induced the expression but had a few bottles that had to be induced later, so Yamini said she would do this for me because if I stayed I would miss my bus. I spoke with my parents in Brazil through skype and went to sleep.
Day 3 (Friday 29 June)
Today I harvested my cells, Yamini helped me. The pellets were so big, I never saw them like this. At lunch time, I tried fish and chips, but didn’t eat the chips because without chips, because the fish satisfied me. In the afternoon, I attended a seminar about pressure in the crystallization, very interesting. After this Maria gave me instructions of the how I get to Oxford on the weekend. Today I went to take my bus earlier, but I took the wrong bus again! I took the bus to Wantage and not to Abingdon. It wasn’t a problem because after the bus went to Wantage it went to Abingdon but it took two hours.
Day 4 (Monday 2 July)
Today in the morning I watched Jose’s seminar, he spoke about my project and was very cool. Meanwhile Yamini equilibrated the gel filtration column for me. After the seminar Jose and I talked about the enzyme’s biological activity from our project. After that, we started the purification, we defrosted the pellets and put DNAse and protease. We are using the cell disruptor for cell lise, I have never used this before, it is very practice. Then we centrifuged and applied the samples in the Akta express. This machine is extraordinary! Don’t have words to describe it. In Brazil we have a Akta but it don’t express, then we have take care of the purification all day, it is very tiring. Here the Akta express make all, we put the sample in the machine and we can go to home and in the next day it’s all ready, it’s so amazing!
Day 5 (Tuesday 3 July)
I arrived in the lab, and wanted so much to see my results, but had a meeting in the office so I had to wait. Today I was a little sad because is my mom’s birthday and I wished I was in Brazil so I can give her a hug, but it’s ok I’ll soon be with her! At long last we were able to see the results of the three proteins not expressed, so the other two were to concentration for later, the tag be cleaved. Today I went to the hotel earlier, when I arrived all stores were open! So I could so some shopping and I bought a birthday card for my mother. Arriving in the hotel I called her and I said Happy birthday! She said, she is very proud of me and this moved me.
Day 6 (Wednesday 4 July)
Today I ran the SDS gel to see the cleavage. One protein was good, but the other one no, it needed to be purified again. The good protein was concentrated again and stayed in the freezer. The not good protein was purified again. While this was happening, I went to the I04-1 beamline with Pierre. Pierre is the crystallization man, I was introduced to him today. When I got to the beamline I saw Jose and I was introduced to Alice. We spoke about the project for the crystallization, when an alarm rang, we were a little concerned because we didn’t know if the alarm was true or false, so we were stuck in the beamline for some minutes. After this I came back to the lab, and I saw that the other protein wasn’t purified again, so was necessary to abandon it. I was so sad, but it’s ok because we have another one, and we’ll starting the crystallization tomorrow.
Day 7 (Thursday 5 July)
My first report was ready this morning, Jo made it. I’m so happy to see my work here in OPPF. After, me and Pierre learned to operate the crystallization machine, Anil taught us. And after lunch we started the crystallization. The first time we put the plate in the wrong place and the needles warped! My heart soared, I thought we had broken all the needles, Pierre was also concerned. Yamini showed us that the needles had not broken or warped. We just needed to change the position of the plates, was a relief. We laughed a lot! So we started the crystallization, we crystallized the AK1 protein, in 4 different conditions. I prepared 3 ligand and put the protein to incubate overnight, tomorrow we’ll have AK1 protein in complex with Adenosine monophosphate (AMP), Adenosine diphosphate (ADP) and Adenosine triphosphate (ATP) ready to crystallize.
About eighteen months later was the day I18 took first light, and I had the obligatory bottle of champagne in the fridge in the second floor kitchen. We opened the shutter, an image appeared on a fluorescence screen, but it had a strange shadow down the middle; several seconds later the shutter closed due to a vacuum trip. We tried again but the same thing happened. A thermocouple on a position monitor blade had fallen into the direct beam path during installation and this caused the trip. The champagne stayed in the fridge that day. However learning from Robert the Bruce, the second attempt at with beam commissioning went much better after the problem had been rectified.
As Director Research at EPSRC I was able to observe first-hand the case being made for Diamond in the late 90s. Making the financial case was not easy, but the deal was sealed by the Wellcome investment. Then the “politics” of location began. “Whitehall wisdom” determined that it should go to Harwell, but as a separate company rather than as part of RAL. A first-rate Board and a brilliant CEO has overseen what must be the most successful major facility built in the UK ever (certainly in my 40 year involvement with the research councils). When I retired from EPSRC end-2003 Gerd Materlik asked me to join the team on a day-a-week basis to help make the case for funding extra beamlines and build links with industry. It was a privilege to witness the construction of this remarkable new facility. Congratulations to Gerd and his team for a fantastic achievement.
I arrived at RAL shortly after the decision to locate Diamond there had been taken and my first job was to assure staff of their future. The legal basis of the company still had to be determined and this took a considerable amount of time and effort before it was finally decided. The original intention had been to site Diamond behind ISIS which would have made it out of sight and would have then prevented the construction of target station 2. I argued for siting it where it is currently located so that it could be seen by all travelling down the A34. UKAEA who own part of the land would give no assurance that the land was completely clear of wartime debris and we agonised whether to have it checked over first and eventually took a risk since the cost of checking was so high. Then the building began.
