Review of MSTTF

Blogging is difficult because our school district does not allow access to blogging from school computers where all my lesson plans and materials are so I will be doing this at home.

I did complete the lesson plans that I had originally planned and they went reasonably well with my 9th grade chem/physics class

Day1: Introduction to measurement of volumes
Day2: Mixing different volumes of colored water to increase measuring skills
Day3: Limestone rocks and vinegar crstalling growing. This illustrated that different strengths of acetic acid got different results
Day4: Growing copper sulphate crystals using different slution concentrations. Done on a Friday so we could see they results on Monday
Day5: The students wrote an essay recording there thoughts about the experiments

I was disappointed that at present no other teachrs have shown any interest in using these lesson plans.

But I thought that the lessons were a great way of showing the importance of precise measurements and led into discussion of atomic property of mattter as we discussed the crystal structure showed an underlying symmetry to matter

Third week of school

The school year is now 2 1/2 weeks old. I missed the first week as my wife was thrown from a horse while on vacation in Utah. Fortunately there was no nerve damage but she had to have C5-C6 vertebrae fused. In the first days of school I have concntrated on measuring length ( in imperial system and the metric system) and mass in grams. We have spent a day discussing the difference between variables, vlaues and relations between these variables. I have used the program "Understanding Science". This will all lead well into our inorganic crystal section.

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.

6/29
Today we studied the crtstals that we had made withthe three different techniques. We found that all three methods gave us crystals which was very exciting. We took photos of the crystals using a digital camera.

We also sat in a seminar were a grad student talked about his research project. In his project he was taking a piece of DNA which generates the c-complex( part of the ATPsynthase molecule) and implanted in an ecoli bacteria with the idea of generating the C-complex protein. He was having problems detecting the C-complex. This may have been to the problems with dtection methods or because the bacteria was not generate the required protein.

I also found that all my wiki entries have disappeared

6/28

6/28

Today we created two more ways to crysrallize lysozome protein.
Free interface diffusion. Capillaries of protein solution are place in vials of salt solution of various concentrations. By adding solute we will create crystals
Vapor diffusion. We put drops of protein solution over a strong concentration resevoir. The drop will slowly evaporate leaving crystals

We also looked at yesterday’s experiment and we have crystals.

We also looked at an old plate(1 year) which was crystallizing an HIV protein,the source was a tobacco plant. Prof Fromme excited because there were good crystals where previously there were none.

Xenon and kryton gasses are used to create a reference point in the crystal structure. Also mercury is used.
To be a good scientist you need
Knowledge in chemistry, biology, physics, patience, sense of adventure, good team work, sense of humor.

Wed 27th June

Today we created crystallisation capillaries tubes of lysozome at different salt concentrations.

What did we learn
Measurements must be done very carefully. It took us 3 attempts to get the correct mixtures( 20 in all).
To get protein crystallographers passionate mention
- Rosalind Franklin(The female scientist who was the first to crystallize and use x-rays to study the structure of DNA)
- Stochiometry and dimensional analysis
- 9/11 and how this means that crystals can no longer be taken on airplanes because the compulsory x-ray machines degrade the crystals.

Copy of diary

MSTF Diary
Barry Siegwart
7/25
Prof Rebecca Watcher, Prof Petra Fromme
Lab installation
Green bubbler. Produces large amounts of cells (16 gm). Many liters. Gives cells correct heat (54C) and light and air to reproduce. Requires 2.3 days to run completely. Symbiotic cells needed for growth.
Cell walk breaker. Uses compressed nitrogen gas. Better that physical breakers (French press). Does less damage. Does not need protease inhibitors.
Centrifuge. Like spin dryers. Large high revs.
Small scale separator Uses a column of beads to attract ionized parts of the proteins. Then different concentrations of salt solution are passed through the column. Different proteins release from column at different salt concentrations. Equipment also has a frequency monitor (Not sure point/use of this equipment)
Large scale separator. Same principal as small scale. Used in green room because green light does not activate the photosynthesis proteins. The outputs at different salt concentrations are collected in a set of test tubes.

Prof Fromme first to find the structure of P1 and P2 which are the parts of the photosynthesis

Looking to find the structure of complete ATP protein which is used to create ATP (energy source) in both plants and animals. Use plants because the switch off at night, animal do not. Two Nobel prizes have been given for work in ATP.



7/26
We worked in the lab to create a protein crystals.(Lysozyme). Creation of correct molar solutions.
P2 has 150,000 atoms/molecule. Requires 3.4A.
Crystallization is an art/magic
Needs clean material
Needs the correct concentration of solution
Zero gravity. Use space shuttle. (4mm crystals)
Special detergent removes the P2 without destroying it.

Group is now trying to crystallize ATPsynthase. X-rays are high energy, need syncotron to generate. Brookhaven labs. X-rays generate an electron density map. This is used to generate a 3-D model.
Use cynobacteria instead of plant material. Plant material is not very stable.
Cynobacteria came into existence 2.5 billion years ago. Created oxygen. Cynobacteria is prokyatic not eukroactic.

There is a high degree of cooperation between researchers.

Humans have 23 pairs of chromosomes containing about 20,000 genes. DNA is the molecule that carries

Science observes and measures the natural world. From those data it infers laws that govern physical and biological processes. Explanations of large classes of phenomena must make testable predictions and be falsifiable. That is, there must be a way to make an observation that could disprove the explanation. The requirement of falsifiabilty rules out supernatural explanation. You cannot disprove, for instance , the claim that God scattered fossils throughout rock strata to make it look as if species had evolved over millions of years. God may have done that, but we may never know and there is no way to disprove it. I that way, faith is fundamentally different from science.
Good science distinguishes correlation from causation. If kids who play violent video games commit more violence, before you blame the game you’d better be sure that violence-prone kids are not more drawn to violent games than other kids. If so, then violent behavior causes the playing of violent videogames, and not the other way around.

6/26

We were impressed by how open Prof Petra Fromme was to us.