Hello! I'll be taking an extended break from blogging and temporarily putting a greater focus
A quintessential skill that's often utilized for industry application is bacterial transformation. The idea behind transformation is to give a strain of bacteria a gene to express, allowing us to have, in essence, protein factories. This is seen all around us, from the insulin many diabetics use to the various petri dish art pieces on the internet. So for today's post, I'll explain how to conduct a basic transformation.
First and foremost, you would want to have a plasmid with your gene and an antibiotic resistance gene. While it may seem unnecessary initially, the resistance gene serves 2 roles. For one, we want to ensure we keep the bacterial colonies that take up the plasmid and kill off the ones that don't. By having the plasmid contain a gene that prevents them from dying to an antibiotic, we can select for transformed cells. The other reason is that the bacteria need a reason to pick up the plasmid. Bacteria will have to spend more energy to express the plasmid, so bacteria won't normally take in the plasmid if it has no reason to. By introducing an antibiotic resistance gene and placing the bacteria in a harsh environment, the bacteria are presented with a reason to pick up and keep the plasmid.
So once you have your plasmid, we need 4 plates (LB, 2 LB/Antibiotic, 1 LB/Antibiotic/Sugar). The initial LB plate (aka your traditional nutrient plate) is to make sure your bacteria are alive. We want to see our bacteria grow! The next two plates are to check whether or not the bacteria cells are competent or have a natural resistance gene. If the untransformed bacteria can survive the antibiotic plate, that means the strain is unsuitable for transformation since it's already resistant. If the transformed bacteria don't survive the antibiotic plate, that means the bacteria isn't competent or didn't pick up the plasmid. The final plate, then, is to actually have the bacteria express the intended gene added via the plasmid vector.
So once your prep is done, you first must make your cells competent. This is done by typically mixing calcium chloride with your cells, creating pores in the cells. Then, you'll add your plasmids and either heat shock or electric shock your cells. This makes the cells stressed and causes them to pick up the plasmids in their environment. From here, you'll put the cells into a water bath at an appropriate temperature to allow them to reacclimate and replicate. This step is essential to ensuring the bacteria don't die from harsh environments. After all, that, plate the bacteria and watch the magic happen!
I decided to write about transformations after so long due to AP Biology conducting one. As Ms. Bliss's teaching aid, I got the opportunity to help in the process of conducting the lab (I made the plates and felt very proud of it)! All in all, this is one of the more remarkable labs, however, the lab is often failed. Due to the delicate nature of bacteria, students would often mistime various steps in the process of transformation. Despite this, the opportunity to learn is priceless!