Genetic Engineering

• “In Greek mythology, the chimera was part lion, part goat, part serpent, which was slain by the hero Bellerephon. In modern day biology, a chimera is a genetically engineered creature created from the DNA of different species. What once was fiction has now become fact; through the process known as DNA recombinant research, scientists are able to splice genes together from different species that would never be able to mate under normal, non-laboratory circumstances. “

• Linda MacDonald Glenn

What is Genetic Engineering?

• Involves the manipulation of DNA in order to alter the trait(s) of an organism.

• Used in:

-genetically modified foods

-make crops pest resistant

-make crops more frost/drought resistant

-mass production of human proteins such as growth hormone and insulin

- more examples

Bacteria are very useful in genetic engineering fortheir ability to pick up free DNA in their environment. Thisprocess is known as TRANSFORMATION.

Transformation requires the presence of PLASMIDS – circular pieces of DNA that exist in some bacteria cells.

Plasmids exist outside the main bacterial chromosomeand carry their own genes – often genes that conferresistance to antibiotics and are capable of self replication.

How are Plasmids Used in Transformation?

Steps in Transformation Experiments

1. A restriction enzyme is used to cut the genes of interest (a section of DNA) from the foreign DNA

2. The same RE is used to cut the plasmid at the specific recognition site for the RE – the plasmid is now linear

3. The RE used will generate sticky ends in both the foreign and plasmid DNA. Placing the cut foreign DNA in the same solution as the cut plasmid will cause the sticky ends of the foreign and plasmid DNA to anneal (join) - creating a recombinant DNA molecule

4. The resulting piece of recombinant DNA is then introduced into a bacterium. A bacterium that has taken up foreign DNA is referred to as being transformed.

How do bacteria cells take in genetically

engineered plasmids? The bacteria cells need to be made “competent” – need toreadily take up foreign DNA.Cells are made competent by placing them in a cooledsolution of CaCl2. The Ca2+ ions interact with negativelycharged phosphate group on phospholipid head stabilizingthe cell membrane. The cold temperature makes themembrane less fluid.The cells are then heat shocked (at ~ 40°C) for a few seconds.This temperature difference (between inside/outside of thecells) creates a draft which sweeps the engineered plasmidinto the bacteria cell.Engineered Plasmid DNA is copied whenever bacterial cellsreproduce.

• Animation of plamid cloning http://www.sumanasinc.com/webcontent/animations/content/plasmidcloning.html

• Animation of plasmid uptake http://www.dnalc.org/view/15918-Transformation.html

How do you know if the bacteria cells were transformed?

If you grow the bacteria on a nutrient agar plate that contains an antibiotic, only the transformed bacteria will survive and express the gene of interest (such as the “glow in the dark” gene). All other bacteria that do not contain the plasmid (and the antibiotic resistance gene) will not survive.