W3D1

Today I prepared a new calibration series, and some more DNA samples to run. We ran these samples under the supervision of a PerkinElmer tech, and took away some important lessons. We learned that altering the current through the plasma coil can give us higher counts for elements that require ionization. We also learned that slowing the flow of sample through the nebulizer will increase this effect. I prepared a new calibration series to run new PEG samples tomorrow.

Week 3 Day 1

After a memorial weekend of reading up on the theoretical models of the binding of cations to DNA, the lab took a day to tidy and read more on what the future holds. A lab tech came in to assess the OES and answer any questions we had. Hopefully getting our hands dirty in the lab tomorrow.

Week 3- Day 1

We had a technician show us better ways of using the spectrometer today. I also started reading another article named Electrostatic mechanism of chromatin folding which was written by David J. Clark and Takeshi Kimura.The article talked about the behavior of the nucleosomes in a salt solution of NaCl with the cations also present in the solution. It investigated the behavior with low and high concentrations of the salt solution and the DNA. With kurt back, we will soon kick into a higher gear in the research.

W2D5

Today I spent more time looking into papers on osmotic pressure. The first one I read was by Hansen et al, and it described a new way of looking at ions around DNA called the cell model formulation. This model is more accurate for determining osmotic pressure at low salt concentrations. It says that this "cell" acts as a neutralization volume for the counterions, and the electric field vanishes at the cell wall. Within the cell, the ions follow the PB equation. I also read another paper by Evilevitch (EVIL!) about DNA ejection from viruses. This paper suggested that PEG can be used to prevent the ejection of DNA from viruses. The use of smaller osmotic pressure changing molecules such as Glycol does not prevent the ejection of the DNA.

W2D4

Today I ran another competition between Ben and Fash, aiming to hone their pipette and calibrating skills. I also read a paper on DNA counterions. This paper suggested that counterions can contribute to the macroscopic properties of DNA solutions, such as osmotic pressure. It said that at High DNA concentrations, the counterion contribution to osmotic pressure prevails. Also, this paper suggested that there was a certain DNA/SAlt concentration range where osmotic pressure was proportional to the DNA concentrations, and independent of the salts.

Week 2- Day 5

Today, with Ben away in Phili,I went over the articles we read last week to further strengthen my knowledge in the biology of the research.

Week 2- Day 4

Today, Ben and I wanted to become more familiar with pipetting,calibrating and operating the spectrometer in general. So we decided to compete in making solutions of Mg,Co and P of 0.05,2,4,6,and 8ppms.

We then ran random samples which John provided with our respective calibrations to see which ones matched better. Well, to cut the story short, I won!! Although I have to admit that my Mg calibrations were a bit off. We will be working more on our pipetting skills over the next few days.

W2D3

Today we began with some plans for aesthetic work to be done to masters hall. This involves a lot of poster renovation and updating. I then got out some leftover DNA samples from my work last summer for Ben and Fash to analyze. They used diluted versions of their calibration standards from yesterday to calibrate the spectrometer, and then attempted to determine the DNA and ion concentrations. We found that there was actually a few errors in their calibration series, but they were still able to obtain decent results. I also spent some time today educating myself on osmotic pressure, because many of the series I will be soon looking at deal with PEG and osmotic pressure. I found one article that suggested that divalent cations could cause attractive forces between DNA molecules under proper solution conditions. No such attractive forces were found with +1 ions.

W2D2

Today I prepared mystery solutions for Ben and Fash to identify using the spectrometer. These solutions had random amounts of Mg, Co and P that I had predetermined. I gave them some rough guidelines on what range my concentrations were in, which is around what we have when testing actual DNA samples. They made an accurate calibration series, and were able to identify the mystery solutions with very high accuracy. I also spent some time today looking over a lot of my results from last year, getting ready to start running actual DNA samples again.

Week 2- Day 3

Today, we ran solutions similar to the ones we did yesterday. The only was change was that we diluted the 1,2,4,6,8,10(ppm) solutions to 1,20,40,60,80,100(parts per billion(ppb)) respectively and then calibrated the spectrometer with them. Also , the samples we used this time were some of the DNA samples John used last year.

We also learned more about analyzing the results from the spectrometer. We noticed one point( the 80ppb) on the graphs were out of place. We probably added a little too much of Cobalt in the original 8ppm solutions we made yesterday. Otherwise, every other thing seemed pretty okay.

Week 2 - Day 3

Today the veteran put the foreign powers to the test by pulling out an old batch of DNA and left us alone to test the ions present in the solution. The foregin fact passed fairly sucessfully with most of the data corresponding to Johns previous tests. One of the calibrating solutions (80ppb) had a out lying concentration of Cobalt, which threw the calibration and results off a little. But all and all a good day, and feeling more comfortable using the equipment.

Week 2 - Day 2

Today,John "the veteran" made solutions of magnesium, cobalt and phosphorus of unknown concentrations and our task was to use results from the spectrometer to make good estimates of their concentrations. We started off by making calibrations of the same solution varying from 1ppm(parts per million) to 9ppm.

We then ran these calibrations in the machine along sides johns samples so we could use our calibrations to deduct the concentrations of his solutions. This was another opportunity for us to improve our pipetting skills. Ben taught me how to pipette properly after he noticed that I was unknowingly drawing out to much solution each time.

We ended up getting the right answers to the concentrations of johns mysterious solutions.We are looking forward to the next task.

W2D1

Today we began with a lesson on chemical safety. After that, I gave a crash course of the basic chemistry that is required for our lab work. We then each prepared a set of samples, spanning from 1 to 10 ppm of Mg, Co and P. Using my set as the calibration, we tested the other sets for accuracy, and they were both very close, especially for first tries.

W1D5

Today I spent the morning brushing up on some reading, looking at a review article done by Bai, et al. This article described research very similar to my own, because they are also using spectroscopy to look at the ion atmosphere around Nucleic Acids. They did not use precipitated DNA though, rather they use a buffer equilibrium technique to preserve the atmosphere around the DNA. They found that the total ions around the DNA almost always was within error of exactly compensating for the DNAs negative charge. They also studied the competition between different molecules with similar charges, finding that bulkier polyions were often less competitive than the alkali and alkaline earth ions. I then spent the afternoon brushing up on my OES skills, preparing and running a test series and calibrations set.

Week 2 Day 1 - Experiments

As promised, learning the experimental ropes began today. It began with a crash course in stoichiometry, led by John, the student veteran of the Andresen lab. Once we had recalled the number crunching of high school chemistry, we were sent to the pipettes to create solutions of varying concentrations of Mg, P, and Co. We created these samples so we could be trained on the main machine of the lab, the Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). To quickly summaries what this machine does, it pumps solutions through a high temperature plasma, which blasts the molecules in solution apart and detects the resulting ions by analyzing the discrete wave lengths detected at different regions throughout the machine.
Using the ICP, we can send DNA on NCP molecules that have been bathed in different ionic solutions, we can detect the ions that attach themselves to molecules. It's important to note that the main error in the experiments comes from pipetting, so the Foreign Factor will be getting lots of practice on that, competitions galore.

Week 2-Day 1

Kurt was not around today so John was in charge. We had a session on lab safety early on, to start the day. Jeremy Kumar gave us essential information on how to handle situations gone wrong in the lab and he gave us helpful information about appropriate lab behavior.

Next, John showed us how to prepare solutions of Cobalt, Magnesium and Phosphorus. We learned how to use the pipettes and brushed up our old school chemistry skills in stoichiometry as we calculated concentrations of solutions.

The most exciting part of the day was learning how to actually run the Spectrometer as John lead the way!!

Day 4- Last day of reading

We rounded up reading the articles we had to cover today. We read the "Physics of Chromatin" article by Helmut Schiessel. This article was like an all round summary of all the other articles we read during the week. First , it talked about the structure of the Nucleosomes, then proceeded to say that DNA is packed in a way that the information would be easy to access even though it is packed in a complex way.

Next, we read about how an increase in ionic strength makes the DNA thicker and hydrogen bonding being responsible for the linking between the DNA- backbones and the histones surrounded by the DNA.

The paper also confirmed once more that counter ion release was responsible for overcharging. The paper ended by using a sphere ad chain model to explain the DNA and histone interaction in three different cases.
1) weakly charged
2) strongly charged and
3) physiological condition

Day 4 - Last day of reading

With today being the last day of reading and next week the beginning of learning experimentation procedures, it seems appropriate to discus a few of the experiments we have read about during these past few reading days.

With certain aspects of these nucleosome structures fairly ambiguous, such as how they are packed along the genome, investigation into these uncertain aspects of nucleosome function is currently being looked into. The main work we have looked at is the paper, Structure and Phase Diagram of Nucleosome Core Particles Aggregated by Multivalent Cations, by Aurélie Bertin, Stéphanie Mangenot, Madalena Renouard, Dominique Durand, and Françoise Livolant, where they examined the behavior of NCP in various polyvalent ion solutions. The general trend was that when a solution of NCP had polyvalent ions added, the NCP rapidly fell out of solution. However, as more solution was added the NCPs went back into solution until it reached its original concentration, an unusual trend that that could suggest certain processes and structures of chromatin and NCP.

Day 3

Ben has basically said all we did today at the lab and has given a little lecture on the Necleosome core particles(NCP),but i will add a little to that. I will explain a little about the experiment that was done on the NCP he talked about. As he said already, the NCP has a net negative charge and so the experiment involved adding two different cations ,namely a magnesium ion and a spermidine ion to a solution containing the NCP.These ions were added separately in two different experiments. The behavior of the NCP was observed as they precipitated when the ions were added but also came out in re-dissolution when the number of ions added reached a certain threshold. They used different concentrations of the NCP to see the different behaviors it will have. We learned that, the lower the concentration of the NCP was, the greater the interaction it had with the ions. We will keep you posted, but for now the second half of the international factor is out till tomorrow.

Day 3 - Nucleosome Core Particles (NCLs)

Today the foreign factor was churning through papers again, this time looking at prior experimentation done with Nucleosome Core Particles (NCLs). These publications are along the lines of the work us internationals will be doing around the lab, so as we know what its like to be in a strange land, for those of you who are foreign to NCLs, we'll turn this post into a lesson on NCLs and how they act in certain environments.

If you can imagine wrapping a thick string around a cylinder, that is a basic picture of an NCL, with DNA strands being the string and a histone octamer, or protein being the cylinder. As seen in the image to the right, the 'nucleosome' refers to this whole string and ball structure of a 146-147bp (base pair) DNA strand wrapped around the histone. This tiny structure (10nm) has a lesser negative charge than isolated DNA, due to the positive core, with an overall charge of -150. The nucleosomes can be thought of as the building blocks of chromosomes, as multiple nucleosomes can be wound together to form chromatin fibers, which be further arranged to form chromosomes. The reason we are interested in these nucleosomes is that due to the small, dense and complex structures, not much is known about the processes that compacts nucleosomes into chromatin fibers.

We'll let that information be absorbed and talk about experimentation tomorrow. Foreign factor gone.

Day 2 - Studying continues

We moved on to different article that also tries to explain why the liked charged macroions/polyelectrolytes (DNA in particular) will want to stay attached together,in a condensed form ,when counterions are added to a solution in which they are in. We also learned ,from kurt, about the role that enthalpy and entropy play in this procedure. We learned about why the system as a whole chooses to preserve its entropy at the expense of its enthalpy by using its highest mutli-valent ion in the bonding process. I have learned a lot in just two days. It is going to be a great summer!!

first day of "freshmen year" once again!! But for research this time

Today, we basically had to catch up with the readings for research. We have a lot of fun stuff to read and get acquainted with, because the research involves a decent measure of biology. So I decided to get going with it right away and discover why two molecules(DNA) of the same charge will want to sit beside one another holding hands(be attracted to one another). They should be enemies according to the physics we all know. So to get to the bottom of this, Ben and I read our first article and then bombarded Kurt with all our uncertainties(there most always be uncertainties in labs right?)and he made them all clear. Tomorrow,we will be on to the next one.

p.s make sure you laugh at the uncertainty joke. Thanks

Day 1 - Foreigners hit the books

With a summer theme of Bio-Physics, it would seem important for Fash and Benjamin (the Foreign factor of the lab), the young Nigerian and New Zealand aspiring physicist to brush up on the prefix (Bio). Currently, although we are well versed in physical ideas, such as electrostatic repulsion, Fash's and my biological knowledge doesn't extend further then a semester of bio 112; leaving us about as lost as we felt line dancing at the local rodeo last weekend (although Fash did suit his pink rodeo hat).

Today -- and most likely the remainder of the week -- will be dedicated to becoming acclimated with the biological and chemical content of the Andressen lab, and also getting a good grasp on prior work that has been conducted on the unusual properties of DNA.

From the literature we covered today (DNA-Inspired Electrostatics - W. Gilbert, Electrostatics of Strongly Charged Biological Polymers: Ion-Mediated Interactions and Self-Organization in Nucleic Acids and Proteins - G. Wong, L. Pollack) the interesting behavior of DNA came to the forefront of Fash and my bilingual discussions. Switching effortlessly between New Zealandish, English and American, we conversed about the theme of electrostatics. We agreed that one of the most striking aspects of DNA was its charge to length ratio. The long thin DNA coil contians one unit of negative fundamental charge every 0.17nm of length. Even more remarkable than this fact, are the experimental observations that despite the repulsive effects one might expect from such dense, like charge, the DNA attracts itself under a range of solution conditions. This was a recurring theme in the literature studied, with numerous theories including Poisson-Boltzmann Mean-Field Theory, presented in an attempt to explain this unintuitive phenomena.

Also applicable to the work we will be doing was the descriptions of how DNA acts in varying ionic solutions. When placed in "physiological conditions," (1MolL-1 NaCl) the DNA follows the shape of a coil, however, when placed in a highly dilute solution, the DNA forms into a torus (donut) shape with an average radius of 50nm. This resembles the experiments the foreign factor will most likely be looking into. However, instead of dealing with isolated DNA, we will be looking at how DNA, with nucleosomes still present within the structure, will act in varying solutions.

That seems like enough of an introduction, apologies for the accents, and until tomorrow, the Foreign half of team Andressen is out.

The Summer Begins Anew

What an exciting time! This summer we have one returning researcher, John Giannini, and two new researchers, Olayinka O. Fasawe (Fash) (below) and Ben Constable (right). These two will be taking the reins on the nucleosome work that I just got funding for through the Research Corporation Cottrell College Science awards. They will be looking at all of the interesting ways that ions interact with nucleosomes and how these ions sometimes manage to make the nucleosomes behave in peculiar ways. John will be continuing his research on DNA condensation.

In addition to helping them all with this work, I'll be working on a project that uses x-ray scattering to study these same nucleosomes. It should be a busy summer!

But with this talent and the wonderful resources available to us, we're ready for it!