Bacterial Transformation

Transformation is the process by which exogenous, or foreign, DNA is taken into a cell. The plasmids used in transformation can be designed by researchers to confer different traits onto a cell. This simple yet powerful technique is often used:

• To make many copies of DNA (“DNA cloning”) 
  
• To make large amounts of specific proteins such as insulin.
  
• To genetically modify a bacterium or cell
  

In this lab, we will be transforming Escherichia coli (E.coli) so that the bacteria will express a particular gene of interest located on the pFLO plasmid. This gene codes for a fluorescent protein found in marine coral. To help identify and isolate transformed cells, the plasmid also carries the gene for ampicillin resistance.

Click Through Animation - How scientists genetically modify organisms

The Plasmid

Plasmids are circular pieces of DNA. A plasmid can be modified by scientists to contain specific genes of interest. Scientists can then insert the plasmid into a bacteria and the bacteria will transcribe and translate the genes on the plasmid. As a result, the proteins coded for by the genes will be made inside the bacteria. 

An example of this is in the formation of insulin. Insulin is a protein that allows humans to regulate blood sugar. Diabetics aren't able to produce their own insulin. Scientists put the gene for human insulin into a plasmid and then put the plasmid into bacteria. The bacteria then make the insulin protein. Scientists can extract the insulin protein and diabetics can use injections of the protein to help them manage their disease. 

In this experiment the plasmid you will be using has two genes. One of the genes is for a fluorescent protein. This is the gene that you want your bacteria to produce. The other gene is for ampicillin resistance. Ampicillin is an antibiotic that kills e.coli bacteria. The gene for ampicillin resistance allows the bacteria to make an enzyme that breaks down the ampicillin so it can no longer kill the bacteria. 

​When you mix the plasmid with the bacteria only some of the bacteria will actually absorb the plasmid. Bacteria that do not absorb the plasmid will not make the fluorescent protein and they will be killed by ampicillin. Only the bacteria that absorb the plasmid will be able to survive in an environment with ampicillin. Adding the ampicillin resistance gene to the plasmid allows you to kill all the bacteria that do not absorb the plasmid. This allows you to select for just the bacteria that have the plasmid. 

Ampicillin is a molecule that binds to the bacterial cell wall, preventing the bacteria from making more cell wall. As a result, the bacteria have holes in their cell wall and they are killed.

The gene for ampicillin resistance codes for an enzyme that breaks down the ampicillin molecule. Bacteria with the ampicillin resistance gene secrete the enzyme into their environment. The enzyme breaks apart the ampicillin so it can no longer bind to the cell wall of the bacteria. 

Video on bacterial transformation - PBS

Transformation vocabulary

Transformation - Altering an organisms traits by inserting extra DNA into the organisms cells
Plasmid - piece of circular DNA 
e.coli - the bacteria that will be transformed 
pFLO - the particular plasmid we will be using. It contains a gene for fluorescence and a gene for ampicillin resistance.
LB agar - "Luria broth" nutrient agar that is used to grow e.coli
AMP - ampicillin - an antibiotic that kills e.coli
Resistance - the ability to survive in the presence of an antibiotic