This morning we continued with the gfp extraction. We put a resin/nickel mixture in a syringe. Then we put the gfp sample on top of the nuckel solution. The gfp should bind to the nickel. Tis is a time consuming process.

We aso took photos of our ps1 crystals. We had three sizes, micro, small and large. The large are big enough to be used in x-ray diffraction.
We also found that that our phlocyanin crystals are in Berkley and may be used on the syncchotron.

Jenniferand I worked with with Di on the GFP extraction. Di had previously centrifued the broth to extract the e-coli bacteria which had been kept an -80 C overnight. The cell walls of the baacteria was then broken using a sonicizer, sort of like a high tech sonic toothbrush. The mixture was then centrifued again. Tomorrow we will purify using a nickel column.

Also we carried on with our inorganic cystallisation. Our crystals of copper sulphate were excellent. We aslo created crystals of potassium chloride.

Jodie, Jannette and Petra were here unitl 9pm last night working on the power point. It looks excellent.

At this morning's seminar Craig gave an excellent power point presentation on the physics of photosynthesis and fluoresence. Craig was mostly concerned with the emitted light after PS1 have absorbed light. He studied this process with a smear camera which allows the amplitude and wavelength of a light pulse to be studied over a short period of time. Craig had found that here were four different light emissions with different time constants.

Next we talked with Petra about some possible inorganic crystal growing projects we could do next week which might be suitable for our classrooms. Also , next week, we will work on isolating the GFP protein.

This morning we took pictures of our lysozyme crystals. The we spent time in the cold room working n our PS1 crystals. WE had already planted small crystals inn a protein mix. There dialysis vessels had now created medium size crystals which were in the form of rods. These rod crystals were planted into another dialysis vessel with the correct buffer/salt concentration with the hope of creating large crystals

Today we continued our PCR lab. Our cultures had spent an hour in the PCR machine and had hopefully multiplied. Now we had to use a gel and electric voltage to see if that was true. We had created the gel yesterday. A sample of each PCR run with a loading dye was put into a small well at the top of the gell. We had 5 samples plus a standard. A volatge of 100V was placed across the gel and left for an hour. To see the DNA we had to add ethidium bromide which binds to the DNA. We then looked at the gel under a UV lamp. DNA had been multplied, the PCR had worked.

We also spent an hour with Professor Wachter discussing green fluorescent proteins(GFP). GFP are an immense area of interest. Nam is creating mutants of GFP to see if the can react in a faster amount, at present 1/2 hour

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

Today, I spent the first hour with Ptra Fromme. She carried on with her tutorialabout PS2 and PS1.Show highlights
PS1 and PS2 evolved 2.5 billion years ago. PS1 in cynobacteria now have the abilties to absorb green light using phycobilisones anttennae. Plants can only absorb blue and red light.
It crstallization you add sucrose to act as a cryogenic protector(Stops ice crystals forming)
PS1 has 96 chlorophylls apparantly randomly placed. But there is a reason for there positioning. They act as a net so there are multiple pathways. Aslo the different orientations allow light from any direction to be absorbed.
Carotene is in the PS1 molecule and acts as sunscreen as an antioxidant.

PS2 is a water splitter molecule. Understanding it may win a Nobel prize. The core of the PS2 is 4 manganese atoms. Understanding this structure is very difficult.

I then spent time with Han a researcher. He is looking at GFP(green flourescent protein). He has a mutant which works. He istrying to crstallize it to see how different its structure is.
We also went to the X-ray diffraction lab.