Transformation and the production of human insulin

Transformation is a genetic engineering technique where DNA is introduced into cells, allowing them to produce new proteins. This method is crucial for producing  proteins with medical and industrial uses. Human insulin is the foremost example of this technique, where the insulin gene is inserted into bacteria, enabling its efficient manufacture. This biotechnological process provides a safe and reliable source of insulin for treating individuals with insulin-dependent diabetes.

Use this page to revise the following concepts of transformation and the production of human insulin:

Creating a Recombinant Plasmid


Creating a recombinant plasmid involves combining a DNA fragment of interest with an existing plasmid to form a new genetic tool.

The following describes the steps that must be carried out in order to create a recombinant plasmid.

  1. Choose the DNA of Interest:
    Select the gene or DNA segment you want to study or work with. For example, the human insulin gene.
  2. Select a Plasmid Vector:
    Choose a plasmid that will carry the human insulin gene. The plasmid should contain:
    • An origin of replication (so it can copy itself in a host ).
    • A selectable marker (e.g., an antibiotic resistance gene to identify transformed cells containing the plasmids).
    • Restriction enzyme sites that match those found in the gene of interest.
  3. Cut the DNA and Plasmid:
    Use restriction enzymes to cut both the human insulin gene DNA and the plasmid at specific sequences, creating sticky ends, so they can be joined together.
    Note: If the gene of interest (insulin gene) and the plasmid do not have recognition sequences for the same restriction enzyme that creates sticky ends, a restriction enzyme that creates blunt ends can be used. However, it is preferable to use a restriction enzyme that creates complementary sticky ends.
  4. Mix DNA and Plasmid:
    Combine the cut DNA fragment with the cut plasmid in a test tube.
  5. Join the DNA and Plasmid:
    DNA ligase is added to the mixture to join the plasmid DNA to the insulin gene. This forms strong covalent bonds in the DNA backbone, known as phosphodiester bonds and a recombinant plasmid is produced. The overhanging sticky ends form hydrogen bonds between complementary base pairs, and DNA ligase completes the phosphodiester backbone of the DNA strands
    This process is shown in the diagram below.

Transformation Process

The recombinant plasmid needs to be inserted into the bacteria. This process is called transformation. 

This can be done in a number of ways, some of which are:

  1. Heat Shock:
    Bacteria are briefly exposed to a sudden increase in temperature after being mixed with the DNA. This creates small openings in their cell membranes, allowing the recombinant plasmid to enter.
  2. Electroporation:
    Bacteria are exposed to a short burst of electricity, which temporarily disrupts the cell membrane, making it easier for the recombinant plasmid to enter.
  3. Chemical Treatment:
    Cells are treated with chemicals, such as calcium chloride, to make their membranes more permeable, allowing the recombinant plasmid to pass through.
    The diagram below shows a summary of the process.

Selecting for transformed cells

Not all bacteria will take up the recombinant plasmid. Some will end up without plasmids. Others may end up with a plasmid that does not contain the insulin gene.  After the bacteria are transformed, scientists need to confirm that the process was successful and find the bacteria that have taken up the recombinant plasmid.

This can be done in a number of ways, often used in combination.

  1. Using antibiotics
    The plasmid used for transformation typically contains a gene for antibiotic resistance, such as the ampicillin resistance gene. Transformed bacteria are grown on agar plates containing the antibiotic.
    If the transformation was successful, only bacteria that took up the plasmid (and thus the resistance gene) will survive and grow into colonies.
  2. Blue-white screening
    The plasmid also contains the lacZ gene for the enzyme β-galactosidase that can turns the substrate X-gal blue. The blue colour is used to differentiate colonies of bacteria that took up recombinant vs non-recombinant plasmids. This is because the DNA of interest is inserted within the lacZ gene, disrupting it and causing transformed bacteria to lack β-galactosidase.

As detailed in the diagram below, only transformed bacteria will be able to grow into colonies on the antibiotic-infused medium. If the transformation was successful, the colonies will be white because they contain the inserted DNA, disrupting the lacZ gene. If the transformation was not successful, the colonies will be blue because they do not contain the inserted DNA, and lacZ remains active.
Diagram showing 3 steps to identifying transformed bacteria. Three bacteria along the top - A transformed cell with a plasmid that does not contain the insulin gene, a transformed cell with a recombinant plasmid that contains the insulin gene and a non-transformed cell. An arrow points to a Petrie dish that cantains nutrient medium+ampicillin+X-gal to indicate all three bacteria types are introduced to the dish and growth medium. The next arrow is labelled overnight growth and leads to a Petrie dish that shows small blue or white circles. This is labelled "Blue colonies: transformed cell with plasmid only" and "White colonies: transformed cell with recombinant plasmids

These testing methods ensure that only bacteria carrying the correct recombinant plasmid are used in further experiments or applications.

Let’s assume our plasmid contains the insulin gene.

Production of Human Insulin

The transformed bacteria are grown in large bioreactors under optimal conditions, allowing them to grow and multiply while producing the insulin protein.

The insulin protein is extracted from the bacterial cells and  the final product is purified to ensure it is safe and effective for medical use.