DNA Transformations

This is part of a series of posts by recipients of the 2021 Career Services Summer Funding Grant. We’ve asked funding recipients to reflect on their summer experiences and talk about the industries in which they spent their summer. You can read the entire series here.

This entry is by Sean Hilgendorf, COL ’24

During the summer I conducted research under the mentorship of Wil Prall in the Brian Gregory Lab. Our lab focuses on the RNA structures, functions, and interactions within the cell of the Arabidopsis thaliana plant. Overall I was able to learn so much, especially in molecular biology, that I have not learned at Penn thus far. My journey over the summer can be broken down into three important sections. Firstly, I was conducting experiments to insert 6 separate DNA sections into bacterial DNA code. Secondly, I was conducting bacterial DNA transformations for 13 separate DNA lines. Then lastly, using the 13 bacterial DNA lines, I infected the Arabidopsis plants in order to insert the coding into their DNA.

For the most part my work dealt with DNA transformations, so I will expand a bit about what those are. A DNA transformation is when you insert exogenous DNA into a competent cell, also known as a damaged cell that is open to taking in external material. The standard DNA transformation protocol requires a heat shock treatment. To do this one must obtain competent cells. I worked with XL Blue Cells, Zymo Competent cells, or NEB DH5a Competent Cells. (The XL blue cells needed to be prepared with 2-Mercaptoethanol right before use. But the rest of the steps for the protocol were done the same no matter what cells were used.) Next, I had to add the exogenous DNA that I wanted to insert into my cells into the cell solution and let it sit on ice for 30 minutes. Then I had to shock the cells with warm water for 30 seconds and let them sit on ice for another 2 minutes, before immediately saving them with a bacteria medium. I then let these newly transformed cells incubate for an hour before plating them on an antibiotic plate. The reason for this antibiotic plate was that our transformed cells had a gene that gave them resistance to the antibiotic. Therefore, any untransformed cells would die with exposure to the antibody. I then let these plates incubate overnight. The next day I would obtain a single bacteria colony with a pipette tip and grow it in a liquid culture overnight. Using the liquid culture, I would then use a Thermo Fisher MiniPrep kit to isolate the bacteria DNA. This DNA can then be used for the next levels of transformation.

After I reached the final level of transformation with my 13 bacteria DNA lines, I had to transform them into Agrobacterium Cells (GV3101 cells), in order to infect my plants. These cells needed to be prepared differently. After adding exogenous DNA to the cells, they needed to be electroporated, and immediately saved with the bacterial medium. They also need to be incubated at a lower temperature for a longer time. Similarly, they are plated on an antibiotic plate, but it contains 3 different antibiotics and need to be incubated at a lower temperature for a longer time. I finally used single colonies to create larger liquid cultures to dip our plants into.

Sean running an experiment in the lab.

At the end of summer my plants grew and I collected their seeds and under a microscope I had to obtain the seeds that glowed under a certain light, since our DNA inserted caused it to. This confirmed that the specific seeds had the DNA we inserted.

Overall, I really loved getting to work with Wil, Dr. Gregory, and my other lab coworkers (Geethu Annamalai, Bishwas Sharma, Rong Guo, and Xia Hua). I also enjoyed getting to meet other undergraduate students in our building throughout our weekly journal clubs. In these journal clubs we discussed biological research papers from our labs, and got to learn more than just the material our individual labs focused on. Additionally, outside of the lab I was able to live more on my own, without having a dining hall or residential perks given by a normal school semester in the college dorms.

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