4/10-4/16: Week 9 – Entering the Wet Lab

                             

This week I finally entered the Mills Lab's wet lab and began working on research alongside a student pursuing his master's degree, Anthony (Tony) Meza.

Tony's research focuses on designing a functional metalloprotein which incorporates the non-canonical amino acid (not occurring in nature) containing bipyridine as its side chain. This research may lead to the development of protein assemblies with photochemical and photophysical properties that would be difficult to achieve with standard amino acids.

To accomplish this goal, several plates of mutated and wild type (non-mutated) E. coli bacteria were grown and thereafter harvested. Using the harvested cells, I used a polymerase chain reaction (PCR) kit to rupture the cells and thereafter induce an artificial, exponential DNA replication process.

Nano drop I used to measure concentrations of E. Coli DNA. By understanding the concentration and size patterns, I was able to identify the best samples to use for the PCR.

Tony's rack of primers and E. coli genetic material from various cultures awaiting replication.

While working on the project, I also got to learn about many tangential topics, such as the dangerous nature of ethidium bromide and how it can kill cells. I also learned that the LD50 (the dosage of a substance that kills 50% of a test population) for orally ingested caffeine is between 150 and 200 mg/kg for an average adult, or roughly 80 to 100 cups of coffee.

The molecular structure of ethidium bromide (EtBr), which is utilized as a nucleic acid stain in gel electrophoresis. Just like all other polycyclic aromatic hydrocarbons (hydrocarbons with several aromatic rings in their structures), EtBr is thought to be carcinogenic because of its ability to be incorporated into DNA. However, Tony explained to me that EtBr actually prevents DNA replication and therefore is actually cytotoxic rather than carcinogenic. There has also been no significant evidence for EtBr's supposedly carcinogenic nature. This isn't important to my research in the lab as EtBr is still dangerous even if it isn't carcinogenic, but I thought that this was an interesting tidbit. 

Loaded agarose gel in a buffer solution. The pink well holds the "ladder" (metric) and the green wells hold what I hope to be concentrated E. coli DNA. Running an electric current through the gel and surrounding buffer will cause the DNA to migrate through the porous gel. This is due to DNA's intrinsic negative charge, which attracts it to the positive end of the plate while also repelling it from the negative charge created by the current.

As seen in the image, the green food dye has decayed into its components: blue and yellow. This is due to the two dyes' different interactions with the current. These dyes do not matter to the end result of the gel electrophoresis as they were simple visual markers that don't bind to nucleic acid. The EtBr, on the other hand, bound to the nucleic acids in the wells will create patterns parallel to the ladder, thus identifying the molecules by their sizes, which determine how far each molecule migrates. I'll post the result in my next update this coming week!

See you next week!