71 Bacterial Transformation Lab
Dr. Lisa Bartee and Jack Brook
Genetically Modified Organisms
Dictionary.com defines a genetically modified organism (or GMO) as any organism whose genetic material has been altered using genetic engineering so that its DNA contains one or more genes not normally found there. GMOs are created by manipulating the genes of an organism to cause a change in the organism’s traits. GMOs are currently a hotly debated topic. Opponents of the technology claim that GMOs pose a health risk to humans as well as potential environmental risks. Supporters of GMOs believe that the benefits such as increasing the available food supply, increasing the nutritional content of foods, and the production of medicine outweigh the risks.
The glowing mice seen in Figure 1 are one example of a GMO. These mice contain the gene for green fluorescent protein, which was originally isolated from jellyfish. During this lab, you will be creating your own genetically modified organism by adding the green fluorescent protein gene to E. coli bacteria.
Bacterial Transformation
Transformation is the genetic alteration of a cell by the update of DNA from the environment. This process can occur naturally in some types of bacteria, but is typically rare. In a lab, we can subject bacteria to conditions that will cause them to take up DNA from the environment (to become “transformed”). There are several ways to transform bacteria in a lab setting, but one of the most common involves changing the concentration of ions in the bacteria’s surroundings and then heating the cells in a specific way. Bacteria that are able to easily take up DNA from the environment are called “competent”. Making cells competent renders their cell membrane more permeable to DNA. After the new DNA has entered the bacteria, it is used by the cell to make RNA and then protein. The new proteins produced from this DNA are what cause the change in the traits of the cells.
Plasmids
In addition to their DNA genome (which is circular), bacteria can also contain additional smaller circles of DNA called plasmids. A plasmid is a small, circular piece of double-stranded DNA that can be copied by bacterial cells. Plasmids occur naturally in bacteria and they are widely used by scientists as a method of for introducing foreign DNA into these cells because the sequence of DNA within the plasmid can be modified in the lab. Once a plasmid has entered a cell, it is copied by the cell’s DNA replication machinery. When the bacterial cell divides, each new daughter cell receives copies of the plasmid. One original transformed bacteria will divide to form a visible colony made up of one million or more transformed bacteria, which each contain a copy of the plasmid (Figure 3).
Selecting for transformed bacteria
In order to transform bacteria using plasmid DNA, biotechnologists must overcome two problems. First, cells that contain plasmid DNA have a disadvantage since cellular resources (such as energy) are