## Module 2 pGlo Transformation

In your first genetic engineering experiment, you will give E. coli bacteria a gene from jellyfish. The jellyfish gene encodes Green Fluorescent Protein (GFP). When GFP it is hit by ultraviolet (UV) light, it releases green light. (For the jellyfish Aequorea victoria, the ability to glow is useful because it reduces the shadow that the jellyfish casts. Lack of a shadow makes predators less likely to spot the jellyfish.) E. coli don’t naturally ma ke the GFP protein, but by giving them the gene, we provide the bacteria with the instructions to make GFP.

E. coli normally lives in the human intestines but is frequently used in biotechnology labs for research and production since it is a very well understood organism. Like all other cells, bacteria like E. coli carry their genetic information encoded in a chromosome that has DNA in it. However, in addition to one large chromosome, bacteria often contain one or more small circular pieces of DNA called plasmids. You can think of them as mini-chromosomes that are independent from the main chromosome. The main chromosome carries thousands of genes but plasmid DNA usually contains genes for only a few traits.

Giving a cell new DNA is called transformation. In this lab you will be transforming bacteria with a plasmid containing the gene for Green Fluorescent Protein (GFP). The plasmid DNA that has the GFP gene is named pGLO. Plasmids are almost always given a name that begins with a lowercase “p” to let you know that it is a plasmid. In addition to the GFP gene, the pGLO plasmid also contains the gene for an enzyme called beta-lactamase (bla) that can break down a chemical called “ampicillin”. Bacterial cells that do not get the pGLO plasmid cannot grow in the presence of ampicillin since it is toxic to them. In other words, ampicillin is an antibiotic. Antibiotics prevent the growth of certain kinds of cells. So, it is pretty easy to selectively grow the cells that have been transformed with the pGLO plasmid since they will be able to grow on nutrient plates that contain ampicillin but untransformed cells will not.

DNA has lots of information other than the information in genes, including regulatory information. That is information that controls when and where a gene is activated or expressed. pGLO includes information like this in the form of a positive gene regulation system. This regulatory system is used to control expression of the green fluorescent protein in transformed cells. In this system, the presence of a special kind of sugar called arabinose causes the GFP gene to begin making RNA. Since the gene is being copied into RNA in the presence of arabinose, it doesn’t take long for that RNA to be translated into protein. As the cell continues to grow in the presence of arabinose, the GFP protein will accumulate inside the cell and the cells will glow green…just like the jellyfish from which the gene originally came.Download and read the pGLO Student Manual to learn more about the experiment, and how you will carry it out. It is not required that you answer all the questions in the manual, but you are invited to consider them. The video below shows a technician carrying out the steps in the protocol.

Once you have read the protocol and watched the video, take the pGLO transformation quiz to verify that you’re prepared to complete the lab.