AAAS Convention

Last weekend, a couple of the other interns and I had the opportunity to go to the AAAS (American Association for the Advancement of Science) Convention in Boston!

We were able to choose what lectures we wanted to go to, and we spent all day listening to different scientists talk about their work! I attended lectures including:

• Language as an Adaptation to the Cognitive Niche - Steven Pinker
• More Than Pretty Pictures: How the Process of Making Science Images and Graphics Clarifies Understanding - Felice Frankel 
• The Robotic Moment: What Do We Forget When We Talk to Machines? - Sherry Turkle 
• Resurrected Ancestral Proteins: Fundamentals and Applications - Joseph Thornton 
• Stroke Research: New Concepts and Innovative Solutions - Moll Shoichet, Constantino Iadecola, and Stephen Meairs 
• The Connectome: - William Seeley, Jeff Lichtman, Mark Bear, and Steve Petersen 

I really enjoyed the lecture about resurrecting ancestral proteins because it used some of the same technology that I am learning to use in my internship! I also really enjoyed Felice Frankel's lecture because it combined science and art in a really interesting way! We were also able to visit different booths that were set up, including one that focused on enantiomers. An enantiomers are a pair of molecules that are mirror images of each other. They have identical chemical properties, but they can react very different in biology! For example, +Limonene smells like oranges and -Limonene smells like pine! We were able to build these molecules (below). 

Overall, it was a great experience to be immersed in the world of science! It definitely inspired me to pursue my next steps in becoming a scientist! 

Concentration Experiment!

Today (February 19th), I went over to RPI to continue my work in the lab. Since last week, slides of peptide arrays were printed using the amino acid solutions I made. Today, I helped use these printed slides to set up and run an experiment. In this experiment, the goal is to determine what part of loop 1 of the Claudin-5 protein in the endothelial cell tight junctions reacts with loop 2 on the opposite side of the junction. These loops can be seen in the diagram below. 

In this experiment, we are using a solution of 90% unlabeled peptide and 10% TAMRA labeled peptide. The TAMRA binds to terminal alkynes via a copper-catalyzed click reaction, and it is fluorescent red. The fluorescent tagging is useful in determining where the peptide binds to the printed 56 peptide array via intensity analysis. In this experiment, There are 10 slides containing 56 printed peptides each. Each of the slides will be exposed to different concentrations of TAMRA tagged peptide, the highest being 500 mM and the lowest being 100 mM. On the highest concentration slide, we expect to see all of the dots tagged at a high intensity, and on the lowest concentration slide, we expect the TAMRA tagged peptides to bind only to the highest binding proteins on the slide. To analyze the intensity of each peptide binding spot, it will be scanned into a computer program, and the intensity will be evaluated using pixels. This data will then be plotted on an intensity vs. concentration graph. We expect the data to resemble the graph shown below, with the intensity increasing as the concentration increases and eventually plateauing. This graph will then be used to find 1/2 of the plateau intensity, and identify the concentration of that intensity. This value will be used to find the Kd, or the affinity of the peptide towards the substrate. 

In my work today, I helped to set up this experiment. Overnight, the slides were placed in individual petri dishes and coated with BSA (Bovine Serum Albumin) to prevent the peptide from binding with the blank space on the slide where no peptide dots are printed. My first job was to wash the excess BSA off of the slide with PBS. The PBS solution I used today was actually the PBS solution I made on one of my first visits to the lab! To wash a slide, I first poured the excess BSA out of the petri dish. Then I added 10 mL of PBS to the petri dish, making sure to not add it directly onto the slide to avoid forcefully removing BSA from the slide. I then repeated this process for the other nine slides, and then put on them on a rotator for 10 minutes. This process was repeated three different times to ensure that all of the excess BSA was removed. 

While the slides were on the rotator, JP and I did calculations to determine the volume of unlabeled peptide, TAMRA peptide, and PBS solution to put on each slide. The amounts were different for each slide because the concentration of TAMRA peptide was different. To add the peptide solution, we used the properties of surface tension, as in adding water droplets on a penny. However, the slide had to remain wet, so it made the process more difficult. First, I poured the PBS solution off of the slide. Then, JP dried the sides and bottom of the slide to allow us to use the properties of surface tension. While JP was drying the slide, I dried the petri dish with Kimwipes. The slide was then put back in its labeled petri dish. We then added the specified concentration peptide solution to the slide, making sure it was evenly dispersed and that none went over the edge of the slide. We then repeated this for each of the slides, using the different concentration peptide solutions. After all of the slides were coated with their specified solutions, we covered them with black paper because the TAMRA is light sensitive. The slides are going to sit and be slowly rotated for three hours. 

I can't wait to see the results from this experiment next week! It should give us useful information going forward in our research. It was very interesting today to see different activities I have done in the lab come together. The amino acid solutions I made last week were used in the printing of the peptide slides, and the PBS solution I used today was the solution I made! I look forward to continuing my research and learning more new procedures!

Amino Acid Solutions

This Tuesday (February 12th), I went to RPI for my weekly internship meeting. This week, I was in charge of making amino acid solutions to be used in the peptide printer. First, I had to calculate the volume of the solution that needed to be added to each amino acid powder. I was given each of the 20 amino acids in their powder form in separate tubes. The mg of powder in the tube was labeled on the side, along with the three letter abbreviation of the amino acid. I was also given a sheet of the dilution concentrations (mg and mL) for each amino acid. Using these numbers, I had to calculate the unknown, which was the volume of solution that needed to be added to the amino acid powder. For example, if the given concentration was 2 mg / 10 mL, and the tube contained 0.5 mg of powder, I would need to add 2.5 mL of solution. The concentrations for each amino acid were different, so I had to do that calculation 20 different times. After my calculations were finished, I went to the lab next door to use the fume hood to create the solutions. To do this, I used an Eppendorf that measured in microliters. The maximum volume that the Eppendorf could hold was 1000 microliters, or 1 milliliter, and the average volume I needed to add to the tubes was 4 mL, so I had to add the solution in smaller amounts. After each amino acid, I had to change the tip of the pipette to prevent contamination between amino acids. After adding the specified volume of solution to each of the 20 amino acids, I took the amino acid solutions back to our lab. There, I used a vortexer like the one shown below to dissolve the amino acid powder in the solution. Some were much easier to dissolve due to their polarity. These solutions will be used in the peptide printer to print arrays of peptides for future experiments. 

I'm looking forward to finding out what I will do next week!

Modeling Peptides

This Tuesday (February 5th), I went to RPI to continue my project from last week. Last week, I identified possible α helix binding sites on the protein. SInce last week, JP has worked on using the color coded model to come up with amino acid sequences that would form an α helix structure and bind to one of the sites. My job was to test the possible structure of the amino acid sequences by entering them into a program called PEP-FOLD. This program uses the amino acid sequence to identify possible  reactions between the amino acids that would make the protein's secondary structure. Ideally, the amino acid sequence we are looking for should have an almost perfect α helix secondary structure in order to bind well with the binding site on the protein. The figure below was the most successful α helix model we made. 

We also built the amino acid sequence using the MOE software. We set the program to make the sequence into the ideal α helix shape to determine where the bases would be located on the α helix. This allowed us to align our α helix sequence with its binding site on the protein model to identify the possible reaction sites and see how we could change the amino acid sequence to improve the binding. 

Overall, my work today really increased my knowledge about amino acid sequences and protein structure. I look forward to continuing to work on this project!