A Lab Filled with DNA

Hi! We are the Golden Trio under Professor Andresen and we are interested in learning about DNA reconstitution. We have a diverse group of chemistry and physics majors. Our lab is located in Master’s hall, however we are often in our collaborator, Professor Buettner’s lab in the science center. 

We all have our independent projects, however, we are all studying the effects a variety of salt will have on the structure of nucleosome arrays.

From left to right: Aisha, Sofia, & Tam

The Golden Trio:

  • Hi, My name is Sofia, and I am a Chemistry major and a Math minor from Nicaragua, class of 2025. In my free time I love to read, watch movies and solve Rubik’s cubes.
  • Hello! My name is Aisha. I am a rising sophomore, majoring in Physics and minoring in Mathematics. I will possibly also be minoring in Sociology. In my free time, I enjoy going to the gym and cooking. In lab, my favorite thing to do is make DNA stocks. 
  • Hi! I’m Tam and I’m majoring in Physics. I also take some Maths and Econ classes (still don’t know if I want to minor in those fields or not). I like to play video games and watch movies in my free time.

Our Projects:

Sofia: 

My project is based on measuring the effects of Zinc binding to DNA. To understand DNA, we have to think about whether certain ions bind to DNA, which is the main focus of my project.

How do I go about this? First of all, thanks to the work of previous students, we now know how DNA is supposed to look when out in water, and salts like sodium chloride and magnesium chloride. My job is to do these experiments again, in order to see if we get the same results, and to collect a good amount of data.

I spend my days making samples of DNA in different solutions, but mostly zinc acetate. I do this so the zinc ions will bind to the DNA and hopefully we’ll be able to see a change in the DNA structure. This is where everybody’s favorite instrument comes in, the Circular Dichroism Spectrometer, or CD, for short. This instrument sends polarized light through molecules, and in our case, mosnitor structural changes to the DNA caused by the ion binding. We have found that as we increase the zinc concentrations of our solutions, the DNA structure changes more.

After I had firmly established that zinc definitely did something to the structure of DNA, it was time to see if this change could be reversed. The next step was to try to wash good ol’ zinc from the samples that we had been making. How do we do this? This is where our good friend, the centrifuge, comes in. We put our samples in the centrifuge, but we put them through a filter that lets the DNA stay in place, but lets out other liquids. I assign each sample a liquid to wash them with, so each sample gets washed with either Tris buffer, sodium chloride, or magnesium chloride, five times, and then in the centrifuge they go. 

After this thorough process, I run the samples through the CD again, to see if any of the changes have reversed.

This is our latest graph, and we can see that DNA that has been washed with salts and a buffer looks very different from DNA in zinc solution that has not been washed. We can interpret this as the zinc unbinding from the DNA, which makes the peak at 275 nm revert back to how it was before zinc was added. However, we can still see some differences between the washed and unwashed DNA.

The next step for me is to repeat this experiment a couple more times to gain some insight and see if the data keeps being consistent with what has been done in the past.

Aisha:

My project has to do with using time-resolved approaches to investigate the structure and kinetics of DNA surrounded by zinc ions.. I am using the stopped flow attachment of the circular dichroism instrument under the supervision of Professor Andresen. Zinc is a significant metal that is known to disrupt the structure of DNA so we will be comparing zinc samples to non-zinc samples to carry out our experiment. The Stopped Flow is an instrument  that rapidly mixes two or more solutions to monitor the reaction in the range of one millisecond to hundred of milliseconds. 

With the stopped flow mixer, we will be investigating the structural changes of the DNA caused by the zinc. So far, we are doing this by injecting a zinc sample in one syringe and the DNA sample in another. We then compare this to a run of a non-zinc sample. With these different sample runs, we should be expecting to see the changes of the DNA structure when it is in contact with zinc. While the Circular Dichroism Spectrometer(CD) investigates the kinetics of protein folding, the stopped flow mixer investigates the time-dependence of the DNA structural change. 

The procedure of the stopped-flow consists of two different samples, or “reactants”, loaded in separate syringes which are fitted in a valve. The procedure continues by transferring the sample into the mixer by using our software to control the amount of sample that’s being absorbed by the mixer. We monitor the triggering reaction by treating the CD signal as a time function instead of a wavelength function as it appears on our software. 

Unlike my lab partners, I don’t have a specific poster to look back to for reference. When I’m not in Professor Buettner’s lab using the Stopped-Flow mixer, I spend most of my days in Master’s hall, reading articles to gain more knowledge on the dynamics of compact nucleosome packaging. 

This is our first graph plotted for the stopped-flow so far. We can see that there is a difference between the Zinc and the Tris solutions. There is a strong decrease in the zinc compared to that of Tris as they both remain the same time frame. Although we can’t come to conclusions solely on this graph, we can infer that the difference in structure might be caused by the presence of zinc ions. I will continue to graph the rest of our data and hopefully the results of the data remain consistent. 

Tam: 

Hi! I’m Tam and I am working with Professor Andresen in his Biophysics lab. I’m currently focusing on the thermodynamics of DNA binding utilizing the Isothermal Titration Calorimeter (ITC). My daily work includes preparing DNA and Cobalt Hexamine  stock, running the ITC and analyzing the result from the ITC.

Basically, DNA is negatively charged. Therefore, if we put positive ions into it, they would bind into each other. This binding phenomenon can either release or absorb heat. In my project, I am trying to perform the experiment from the previous students to get more data in order to confirm or to see if I can explore something new. The experiment is to put Cobalt Hexamine (or CoHex for short) into DNA samples because Professor Andresen is working on the binding of +3 ion and the DNA . To get the data, I need the help from the ITC at the Physical lab of the Chemistry department. The ITC is a machine that can identify a very small change of energy of the solution (I am working with the change around 1 µJ/sec.)  

For the first 4 weeks, the ITC was very unpredictable. The data was very noisy and it looked nothing like what me and Professor Andresen expected. We tried rinsing the machine with a lot of water, detergent and even acid and still cannot get the right data. 

Then came the savior, Professor Frey from the Chemistry department. She brought a new syringe (an important part of the ITC) and the data looks a lot cleaner and now everything is on the right track. 

For the remaining weeks, me and Professor Andresen will bring polyethylene glycol to explore more about the energy under the osmotic pressure.

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