7/20/07

7/19/07

Today, we did chromatography with various plants today- native AZ plants, yellow plants, purple plants in order to ascertain their pigments, absorption, and cholrophyll. Interestingly, we found out that raddichio was treated rather harshly and thus, has its white- bleached like color has no more chlorophyll. It was at this point that I started making connections with my previous studies on folivory and howler monkeys behavioral adaptations to a diet rich in mature leaves. It was a conversation that sparked and renewed my previous interest in my undergraduate honor's thesis that had me perform a comparative study on three primate folivores. We also found out that we could each take back 2 vials of GFP each!

7/18/07

Today, we earned the PSI crystals in the cold room yet again and found out that we had one vial of crystals from the seeding crystals that was large enough for x-ray diffraction. Dr. Fromme was quite impressed. We were also informed that our phyocyanin crystals were being measured and analyzed at Berkeley due to their clarity. Additionally, we placed our isolating GFP through a nickel column and allowed it to drip out as the truly isolated GFP. This took several hours even beyond our time there but in the end, we will be bringing back the GFP that will glow with a black light- a sure way of grabbing students attention! In Wachter's more bio-type lab, there is a lot of waiting which allowed us to go back and forth.

7/17/07

Again, worked on GFP and worked with copper sulfate and potassium chloride crystals. Also, took pictures and focused upon our powerpoint presentation as we are all anxious to make this very professional. We will be taking GFP home- relatively stable and will keep in fridge. Am very pleased with the group I am in- both the teachers and the researchers. GFP is not classroom transferrable but is applicable to future lectures in class as it applies protease inhibitors and various bio jargon I have used but never seen in use.

Two labs were going on in parallel this morning. Di worked with Jennifer and myself in creating GFP. Di had put modified e-coli bacteria in a test tube to be multiplied. Then we added it to a flask with a liter of broth. Every hour we checked the bacteria growth by seeing the 600nm absorbtion of a sample. The bacteia had been modified with a plasmid which contained DNA for GFP and an antibiotic resistance protein. The antibiotic resistance was used because an antibiotic was added to the broth to kill any bacteria that was not resistant. After 3 hours there was enough bacteria. GTDP was added, and the temperature reduced. This stopped the bacteria multipling and instead in started to produce GFP.

Meanwhile prof Fromme was running a inorganic crystal lab using copper sulphate.

Today's pics 7/16/07

7/16/07

Today I divided my time between Fromme's lab and Wachter's lab. In Wachter's lab, we are isolating GFP through the use of primers and a broth in E. coli with an antibiotic that the plasmid is resistant to. In Fromme's lab, we were trying to replicate experiments creating inorganic crystals using the materials that would be typical of a high school lab and Dr. Fromme commented on how much more difficult simple experiments then become. She showed us a particular catalog that would allow us to purchase better equipment- not lab quality- but certainly above rudimentary for our work creating copper sulfate crystals. We found out how to apply the terms saturated and supersaturated in the lab setting as well and will be meeting once again with Dr. Fromme at 2:30 PM to review our powerpoint thus far.

7/13/07

Today was rather stressful and we were trying to figure out our presentation. We listened to a grad student present his current data very succinctly- since there were no questions and he was a physics person amidst chemists. We also ate more cake and then tried to figure out what would be best to do for next week in light of our powerpoint and poster session. As such, there was a bit of disagreement between our research groups so we decided to split up. Fortunately, Petra Fromme came to see us and talk with us about how best to do our powerpoint so we had a much better idea and it will be started over the weekend as we have divided the tasks.

7/12/07

We finished working in the cold room to perform macroseeding for PSI crystals which required us to have the hands and steadiness and concentration of a surgeon. I loved b eing in a "different" environment at 4 degrees Celsius and I found it unusual and exciting. Additionally, I got more time to speak with Petra even more on a personal level and find out about some of her amazing "science" stories. We then took her out to lunch and continued to talk about the possibility of having some of our students working with her- networking at its best. Additionally, I believe the professors are very impressed and grateful for the work we have done and the feeling is very much mutual.

Pics

Our DNA! GFP!

7/11/07

Today, we continued work on PCR and gel electrophoresis in Dr. Wachter's lab. We learned that it is absolutely imperative to have a steady hand when placing the mix of buffer, dye, and polymerase into a gel well due to its small size ( at least it was for me...since I had to redo it!). Fortunately, we actually had the dye. The researcher informed us that sometimes this has to be done with colorless solution so the level of difficulty is increased. We waited for an hour, added another dye, and mixed it for 15 minutes and then saw the sample DNA along with the segment we had replicated underneath UV-C light- picture forthcoming here.

We also listened to the research of Dr. Wachter and I learned the real-world applications of her work. Essentially, if you can input GFP- green flourescent protein- into a nematode (it is originally from a jellyfish) and target nerve cells to flouresce specifically when they are firing, you can use this particular protein as a "biosensor." Since, CFTR malfunction is the main cause of Cystic Fibrosis, we could then use a GFP clone to follow the misfunction via flourescence lighting up a specific piece of information such as chloride transport in this case.

Lastly, GFP takes aboput an hour to turn green but an hour may be too long with live cells so Nam- a researcher- is currently working on the development of GFP variants that mature- "turn green" - more rapidly and this is done by creating a DNA library to look for the ones that flouresce quicker- similar to artificial selection in dogs- i.e. breeding selectively for specific morphological or behavioral traits.

Question to ponder:

Can we improve upon nature?

Certainly depends upon specific conditions.

Today, we met with Dr. Wachter who re-introduced me to PCR analysis. I was very excited to be performing this relatively simple procedure since I have been teaching it for many years but have never had the opportunity to actually perform such procedure. It was just as simple as I thought, except for the minute quantities we were dealing with. Apparently, many of the materials were simply purchased from larger lab corporations. Then, we had the opportunity to see how agarose gel was mixed. I have to admit that I felt much more comfortable in this type of lab. Later, we changed concentrations several times to get the correct amount of crystals for seeding for PS I. Then, we had to work with a dialysis tube and capillary with a very small "loop" and we had to place it in a miniscule tube without touching the side in a cold room at 4 degrees Celsius! Fun, adventurous activity!

7/9/07

Today, we tried a dialysis technique to crystallize PS I which required the use of many different equations as well as a series of rather complicated steps such as preparation of probes,detremination of chrolophyll concentration, reactor assembly. We were able to see "fast and immediate crystallization," use the spectrometer again, and deliberately denature the protein. We learned the secret of crystallization of PSI- sometimes you need to use a lower salt concentration. We are "recrystallizing" essentially for PS I or will be microseeding tomorrow and working with Dr. Wachter for PCR.

This work was painstaking and complex to me. I am excited to see what will happen due to our groups' work.

07/06/2007

Today was exciting. Our x-ray diffraction pattern was used on a cryo lysozyme crystal and input into HKL 2000. This program was used to get a 3- D data set of the crystal. With the information, we created an electron dot configuration and further created our "protein picture" from work we had all done. Additionally, all our crystals have been doing well. The researchers are all relatively impressed with us and willing to share a multitude of techniques. Next week, we will be working with PCR, and Western blot analysis and learning more about DNA shuffling so we can share our FIRST-HAND knowledge with our students regarding these currciulum topics. I know that when I convey learning to my students based upon my personal field experience, they always do well because I have examples, activities and enthusiasm for the subject. I only wish this could go on longer since there is so much I would like to learn!

7/5/07

We checked for phycocyanin crystals. All wells had great bluish crystals that resembled hairs or nails depending upon your perspective. We decided to wait before putting those through the x-ray machine in the hopes that they would grow even larger. As such, we used our lysozyme crystals for x-ray diffration and watched the x-ray machine work as observers since that is a more dangerous piece of equipment. The first crystal gave a relatively poor diffraction pattern despite its look on the outside. I guess- in crystals- as well as in people- it's whats in the inside that counts. We then waited for the next pattern and while we did that, we took more pictures of these new crystals using the microscope camera once again, only this time we took pictures of all wells. Tomorrow, we will be diffracting the crystals under cryo conditions- i.e. liquid nitrogen or propane- in the hopes that this will help the crystals retain more moisture. However, too much humidity is also a problem so it is like Goldilock's theorem where you have to get it "just right." My question is this: how do you get this beautiful colorful picture of the structure of these membrane proteins showing the symmetry and ribbon-like structure of the amino acids? That is the question that I will ask next and possibly do some independent research upon myself as well.

7/3/07

Today, we did vapor diffusion technique with the real thing in the hopes of making large crystals for x-ray diffraction. We also used the spectrometer to determine the purity of the proteins being used based upon different dilutions. More importantly, we got to chatting about our own personal experiences. I am starting to feel more comfortable and confident with this process.

7/2/07

Lots of lecture today and I was very excited to find out the implications that this research has. With the splitting of water comes the vast possibility of endless energy and power for our future growing population. Humans are so cool. I look at this from an ecological perspective indeed which is why humans have not yet reached carrying capacity and I discuss this with my students as part of our standards all the time. Additionally, this has implications for drug interactions since humans are composed of 60% membrane proteins and drugs target these in particular. Plants are even more complicated- again another twist on the order and complexity of our classification system. It would indeed indicate that plants are much more complex and have to be especially in their defense mechanisms since they cannot move.

Additionally, we worked an ultra-centrifuge today and will be working with x-ray diffraction soon. The patterns are truly beautiful... hauntingly so- since they are so complex. Revisting vocabulary from my MS. Am very excited about the ecological standpoint of this!

Crystals are beautiful and my students would love to create them and I could then extrapolate upon solubility curves, titration, salt concentrations etc.

Techniques

Vapor diffusion technique- nice colorful crystals, batch technique- lots of crystals, and interface technique- more difficult to use with High School?

7/1/07

Just trying to add pix to my blog and the wiki. Having trouble with that since they often require a link or a URL and am not used to doing that. Will consult with my group tomorrow.

6/29/07

Found crystals usable for x-rays at A3, A4, D3 from vapor crystallation technique. Will be making photos of the crystals. Sat in on grad seminar. Dr. Fromme served cake to grad students for their birthday. The atmoshere was one of comraderie and collaboration. Student Rob Lawrence was having some trouble with his data- overexpressing a C subunit of spinach in E. coli. Several suggestions were given and an effort was sincerely made to explain this to us and we were not treated as if we were clueless either. Very nice.

Later, we took pictures of our crystals with a computer. Very sensitive and not as easy as it looked and I broke one of the capillaries so it had to be resealed. Liked the decision theatre but will write more on that in the wiki.