pGLO Transformation Lab

In this lab you will perform a procedure known as genetic transformation. This term means change caused by genes; it involves inserting a gene into an organism in order to change one or more characteristics in that organism. We will use E. coli K-12 as our host organism; ie the organism that will receive the gene. The gene we will transfer to E. coli K-12 codes for green fluorescent protein (GFP). The source of this gene is the bioluminescent jellyfish Aequorea Victoria. If all goes well, our bacterial host cells will incorporate the gene for GFP into their genome and will transcribe and translate the gene to produce GFP. We will know the procedure has worked if our bacteria glow green when UV light is shined on them.

There are several methods that can be used to transfer a gene from one organism to another; we will be using a plasmid as our vector. Bacterial cells have one circular chromosome which carries the genes needed for the cells to continue to thrive. In addition to this circular chromosome, most bacteria naturally contain one or more plasmids: smaller circular pieces of DNA that may contain only a few genes. Plasmids are naturally transferred between bacteria cells. The genes carried on the plasmid can then be transcribed and translated by the cell; this can lead to a cell having new traits that may lead to a higher rate of survival, especially if the conditions are changing. The transmission on plasmids is exactly how many bacterial strains have recently become resistant to the antibiotics that we commonly use to treat patients who are suffering from bacterial infections.

Plasmids are a great example of a naturally occurring situation which we can harness to manipulate genes in order to bring about a desired outcome. (Restriction enzymes are another example.) If we can insert a gene into a plasmid, the bacteria will take up and begin to express the gene naturally. Most of the work is done for us when the cell naturally follows its normal behavior.

In our lab, the plasmids are engineered and sent to us in freeze dried form. When we rehydrate the plasmids, the gene necessary to produce glowing bacteria, along with several genes that are part of the control for the experiment will be contained in a neat package that the bacteria will take up on its own.

One of the difficulties when working with bacteria is the fact that the individual cells are very small, so at the end of the procedure, it is almost impossible to pick out which cells have the desired trait. Even if we can pick out the cells which successfully transformed, it would be very hard to isolate them from the cells which did not. We can solve this problem by ‘killing’ all the cells that did not transform, leaving only those that did transform in our plates. We can easily kill the cells by adding an antibiotic (ampicillin in this particular case) to the medium upon which we culture our cells. The complication here is that we need a way to allow the transformed cells to survive in the antibiotic laced medium. This is easy: included in the plasmid is the gene for beta-lactamase. Beta-lactamase protein is produced by the transformed bacteria and secreted into the medium; it inactivates the antibiotic allowing the bacteria that produced it to survive. So the transformed cells will be antibiotic resistant and the non-transformed cells will be killed by the antibiotic in the medium.

The gene for GFP will be contained within an operon—a group of genes that are regulated together so that when the appropriate signal is present, all of the genes will be turned on or off simultaneously. Operons are a very common strategy used by bacterial cells to regulate gene activity; in eukaryotic cells, other regulating strategies are more common. (See ch 18.1 in Campbell’s 10th for information on how operons work.) In this particular situation, the operon that is used is related to metabolism of the sugar arabinose.