Charged on 3 Counts of AsSalt

In the Andresen lab we research biophysics, specifically involving DNA. Fun Fact: DNA needs a trivalent cation to condense (hence the title). Here is some in depth information about our projects:

My name is Savannah Miller and I am a rising senior with a BMB major and a Physics minor. I worked in Professor Andresen’s lab last year on a similar project and enjoyed it so much that I decided to work here again this summer. I decided to work in Professor Andresen’s lab because I was attracted to the interdisciplinary nature of the biophysics research. I felt that this lab would give me the chance to apply my background in both biochemistry and physics and learn how to approach research questions from a wide variety of angles. My project this summer is an exploratory investigation into how cationic gold nanoparticles interact with DNA. I am collaborating with Professor Thompson’s lab who are providing me with gold nanoparticles and their expertise. Gold nanoparticles are nanoscale gold particles that are stabilized by charged organic molecules like CTAB and citrate. We have been using CTAB stabilized gold nanoparticles that are positively charged and thus will electrostatically attract the negatively charged phosphate backbone of the DNA.

IMG_5141

Flow Reactor used to make large quantities of CTAB AuNPs

The DNA we have been using is calf thymus DNA which ranges from 8-15 kb in length. We expect the nanoparticles to act as a condensing agent for the DNA and the DNA to maybe even form histone-like structures around the nanoparticles. Unfortunately the addition of the long DNA caused instability and aggregation of the nanoparticles so we are now planning on using smaller DNA that will be less likely to incite aggregation. To obtain smaller DNA we have decided to shear down the DNA we have using a sonication protocol mentioned in a paper. The protocol uses a probe sonicator (see below) to instigate cavitation, the formation and collapse of microbubbles that together spread damaging shockwaves through a solution, in order to shear calf thymus DNA down to at least 800 bp.

Using an old probe sonicator from the biochemistry lab, we were able to run the protocol listed in the paper. Gel electrophoresis shows that the sheared DNA solution contains a variety of fragment ranging from 1kb to far below 0.5 kb. This is promising because it means we have successfully sheared the DNA and obtained fragments that may be able to interact with the nanoparticles without stimulating aggregation.

We have run out of CTAB gold nanoparticles, which are more difficult to make than citrate gold nanoparticles, so I am going to start using the citrate ones. Citrate is naturally negatively charged which would repel the negatively charged phosphate backbone. Instead, I plan to layer lysine onto the nanoparticles on top of citrate. Lysine is positively charged at physiological pH so it will create the correctly charged surface for DNA electrostatic attraction.

My name is Sarah Hansen and I am a rising senior, majoring in physics and minoring in chemistry. For the past two summers, I have worked with Professor Andresen, whose research involves biophysics. Last summer, my project focused on ion-counting studies around arrays of DNA strands, but this summer my project involves nucleosomes.

Because DNA strands are negatively charged (due to the phosphate groups), they need to wrap around histone proteins in cells to condense. Several histone proteins will form a “bead on a string” type conformation, eventually forming chromosomes. Though, the exact structure and electrostatics of these interactions is unknown. The goal of my current project is to investigate the electrostatics of DNA-wrapped histone proteins, perhaps using ion-counting techniques like last summer.

Before I study the properties of DNA-histone complexes though, I must first make a sample of them. I began my summer with 50 mL of whole chicken blood (see image below), which contains plasma, white blood cells, and red blood cells. Unlike humans, the red blood cells in birds actually carry DNA, so the goal was to isolate the red blood cells, and then further isolate the nuclei.

Components of Whole Blood-1

 

Through a series of biological and chemical techniques, I have been working on isolating the nucleosomes from the whole chicken blood (see image below for an example of spinning down my samples to achieve nuclei). Although I have not reached my end goal yet, I am close to making my sample. Throughout the rest of the summer, I will work on isolating the nucleosomes, as well as running electrostatic experiments on them.

Sample image

In summary this is what research is like in the Andresen lab:

 

 

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