Week 1 (9/5/22 – 9/9/22): An Intro to the World of Transformation

Welcome to the first of many blogs that will provide you insight into the world of transformation (my current research endeavor), where the team I am on will be working on importing “superpowers” to an organism whose natural abilities would likely already put Superman’s to shame. Now, I’m not here to debate about which species would win in a match (Although doesn’t one by the name Deinococcus sound much more tenacious, sophisticated, and enigmatic than a Kryptonian species that is just so 20th century)? But I am hoping that, by incorporating both illustrative and text-based pedagogy, you will join me in my journey of undergraduate research. 

To provide a brief background, I joined Dr. James Tuohy’s Independent Research Lab at GCC just three months ago, and was excited to learn that I recently received the TRAIN Grant to help fund my educational expenses (thank you, Dr. Tuohy and Co!). Since then, I’ve learned quite a bit about what it means to truly put the Scientific Method to work, with many an exciting moment of learning and growth (and a few failures – or shall I rather say “areas of opportunity” – along the way). My group, specifically, is focused “transformation,” a process by which bacteria integrate foreign DNA (DNA that it already doesn’t have) into its own genome, such that it will express the characteristics provided by the new DNA. In our case, we are working on transforming the bacterial species Deinococcus radiodurans (also referred to as D. rad). 

First, let me tell you something you may find interesting. Can you believe that even though this organismal genus is one of the most radiation-resistant ones in existence (that we currently know of at least), a mere concentration of 3 micrograms per milliliter (3 ug/mL) of the antibiotic Chloramphenicol will prevent it from growing in an otherwise perfectly suitable environment? This extremely tiny “fatal dosage” is known as the minimum inhibitory concentration (the “mic”). To put into perspective just how minute of a dose is needed to keep D. rad from growing, let’s start with that a single grain of salt is just 0.065 grams. Since a concentration of 3 micrograms per milliliter is the same (after using conversion factors) as a concentration of 0.003 grams per liter, you’d actually need to cut that single grain of salt into approximately 22 pieces and drop just one piece (1/22nd of a grain of salt) into a liter of water to visualize an equivalent concentration of Chloramphenicol that would keep D. rad from growing. 

This might seem small, but you also might be thinking that this is a teeny tiny bacterial species we’re talking about, one that we can’t even see without a microscope – Of course D. rad might not survive at such a low concentration! But let’s put this into perspective even further. If we stood around 20-25 cm (approximately 8-10 inches) away from an object, our eye’s visual acuity would generally allow us to see something that was just 60 micrometers (60 um) wide. Now, the bacterium Deinococcus radiodurans is only about 2 micrometers (2um) wide, which is way too small for us to see with the naked eye. But, if we took our three main components (1 liter of water, 1/22nd of a grain of salt, and 1 single D. rad bacterium) and scaled them to 9,000 times their size, we would have a single D. rad bacterium the size of a dime immersed in a 10-foot round aboveground pool (only 4 feet in height) that has had just 4.5 teaspoons of salt added to it. Just 4.5 teaspoons of salt in a 10-foot round pool would be an equivalent concentration of all that is needed to keep D. rad from growing in the antibiotic Chloramphenicol. 

Hopefully this 2-paragraph visual of D. rad’s “mic” helps you to understand just how difficult it is for D. rad to survive in Chloramphenicol. However, what if there were a way for us to provide this resistance? There is, and this is where our team comes in. Now, transformation of Deinococcus radiodurans to make it Chloramphenicol-resistant has already been done, both by scientists who have published their work on this specific type of transformation and even by our own lab colleague and esteemed mentor, Jonathan Hill. However, our team’s current job is to make sure we can not only perform the same type of transformation ourselves, but to also make sure that we can perfect our techniques of the entire process (from plasmid extraction to transformation itself), with the next steps being to transform Deinococcus aquaticus, a feat that has yet to be performed (at least according to what has been verifiably published thus-far). 

With this, you are entering this blog at a time when we are in the middle of preparation for transformation, so I will provide a brief synopsis of what has occurred this week. Our goal is for D. rad to uptake the pRADZ1 plasmid (and in a separate transformation, the plasmid pRADZ3) extracted from E. coli and integrate it into its own genome, ultimately expressing the Chloramphenicol-resistance trait provided by this foreign gene that D. rad otherwise does not have naturally. For transformation to happen, we needed to create the environments that would allow it to grow and that would prove that our D. rad were transformed. To do this, we needed to create two sets of TGY agar plates (one set would have Chloramphenicol and one set would not). Since we had already created the TGY agar media last week and had it autoclaved to prevent contamination, we needed to heat the now-solidified TGY agar flasks so that they became liquified. While waiting for our TGY agar to heat up, we verified and re-checked our calculations that told us how much of our stock solution of Chloramphenicol (at a concentration of 34 milligrams per milliliter (34 mg/mL)) we would need to add to one of our two 400 mL flasks to have a working / ending Chloramphenicol concentration of 3 micrograms per milliliter (3 ug/mL), which is also D. rad’s mic for Chloramphenicol. We figured out that, before pouring our plates that would have Chloramphenicol (“+Cm”), we would need to add 35.3 microliters of Chloramphenicol to the 400 mL of agar (the other 400 mL flask of TGY agar would not need to have anything added, as the plates that were poured from this flask would be without Chloramphenicol, “-Cm”).

We ended up making our plates that week and were able to begin the transformation process, but we were unable to continue with it as there was one key step we missed (An “area of opportunity”): Our plan was to originally plate our competent D. rad cells (ones which had been immersed in a solution that allowed the cells to become permeable and thus transformable) the same day that we plated our transformed cells. However, we had an important piece of information pointed out to us: If we plated our transformed cells and ended up seeing no growth, how would we know that the original competent cells used for transformation were even viable (able to stay alive just as they were in an otherwise healthy environment) before transforming them? In other words, how do we know they were even still alive before we tried transforming them? Proving the viability of our competent cells before starting transformation is a key to helping make sure we have as many controls as possible (and are thus covering our bases). Because of this, we will have to go back to the drawing board and delineate each part of the transformation process to ensure that we are taking the best steps toward “quality assurance” of our research. Onward and upward!