Exercise 16: Transformation of Bacteria

• How are cells made competent to be transformed with bacteria?

1. Growing cells to mid-log phase (typically around three hours after inoculation).

2. Incubating cells on ice with divalent cations (Ca²⁺) and with naked DNA.

3. Heat-shocking the cells and DNA at 42°C.

• What are the sequences/genes on the pGREEN plasmid and what is their purpose?

During this laboratory exercise, you and your lab partner will perform a genetic transformation. You will be using a plasmid called pGREEN (see Figure 16.1). Plasmids are small, circular DNA molecules. They are not chromosomes, but are capable of autoreplication

. This particular plasmid carries an antibiotic resistance gene that confers resistance to ampicillin.

The AMP^R gene carried on the plasmid DNA allows the bacterium to make the beta-lactamase enzyme= SELECTIVE GENE

The plasmid contains a short DNA sequence known as the origin of replication, or ORI. The ORI is needed for the cells to make copies of the plasmid DNA. If a plasmid does not have an ORI, it will not be copied and is not passed down to the daughter cells

the plasmid DNA also contains a gene that codes for a green fluorescent protein (GFP) - REPORTER GENE, which is a bioluminescent gene from a jellyfish. In the deep ocean, the jellyfish is able to glow in the dark. After the transformation procedure, the bacteria will express the jellyfish gene and produce the fluorescent protein, which causes them to glow a brilliant green color under ultraviolet light

How do you know if your transformation was successful based on the observation of your results

Place the plates under a UV lamp to see which plate contains transformed cells expressing the pGREEN gene. Make sure to note any changes in the appearance of the colonies growing on different plates.

ok!

Ok!