This morning we spent our time understanding PCR which is a method of copying parts of DNA in large quantities. Prof Rebekka Wachter gave us a introduction to PCR. It is very new technique. In short DNA is heated to break it into single strands(denaturation or melting). Then primers ( short strands of DNA with specific code) latch onto the single DNA strand(annealing). Then a special enzyme, DNApolymerase, which works at high temperature, links onto the DNA/primer complex and starts adding new amino acids. This cycle is repeated many cycles(30) and 2^30 or 10^9 new strands of DNA are created.
Nan, a graduate student, let us start a PCR lab.
Finally, with Petra Fromme, we put small seed crystals into our PS1 dialysis vessels.

This morning we spent the morning with Prof Petra Fromme preparing reaction vessels to create crystals of PS1. PS1 crystallisation uses dialysis to reduce the salt concentration to create crystals.
There were lots of different stages. We had to find the exact molarity of the protein. We did this by finding the molarity of the chlorophyll which we did by using a spectroanalyser.
We had to make dialysis tubes. The salt solution will pass through a membrane to a high salt conc leaving behind protein in a higher salt conc.

We spent the morning working on x-ray diffraction. Raymund had set the machine to work overnight for 8 hours to take 102 images. The images were analysed with HKL2000 software which made sure they were all consistent. Then using the software suite CCP4 he created an electron density map, the an atom model and then finally a ribbon model.
In the seminar meeting Han described his projects. He was trying gene shuffling on a protein H1057 and was also crystallising a mutant green fluorscent protein(GFP)

Here are two photos of philocene protein crystals. They were created under different conditions. The one on the right had higher amount of PEG, which absorbs water, This meant is crystallised quicker and is the reason why the crystals are smaller.

This morning, Raymund Fromme, led us through the process of taking X-ray diffraction pictures of crystals. Firstly we selected a crystal. It turns out that a nice looking crystal may not have good internal crystal structure.
I was amazed how quickly Raymund was able to fish out a crystal with a small loop and set up the crystal correctly in front of the x-ray beam. The two pictures at 0 and 90 degress took 1 minute each to take and 2 minutes for the computor to read. We got good diffraction patterns with the second crystal and the computor was able to calculate the cell size of 38,74,79 A. This compares well with the accepted values of 36,78,78 Angstroms.
We also took digital photos of our philocyonene crytals which came out very well.

The morning was first spent with Raymund Fromme( the husband of Petra Fromme). We were preparing vapor diffusion plates for another protein. It was comforting that I knew what, why and how we were doing the technique.

I then spent an hour with Gabbi. She showed how software is used to analyse the data. First the X-ray diffaction data(1 exposure 8Mb, 500 exposures) is used to create the electron density map. Using more software the electron density map s then used to create an atom structure model. This can be very time consuming. One example took 1 1/2 years as it needed so many iterations.
Finally the atom model can be transfromed into a ribbon model.

Here are some vapor diffusion crystals of lysozyme