Genetic Transformation

What is transformation? When a cell takes up and expresses a new piece of genetic material—DNA— in order to change the organism’s traits A gene is a piece of DNA which provides the instructions for making (coding for) a particular protein When genes from one organism are transferred into the DNA of another organism In this lab, we will transform bacteria, giving it the ability to make fluorescent proteins. These are proteins that absorb light at one wavelength (color) and re-emit the light at a different wavelength, or color. Some things will glow with a new color when you shine the "right light" on them. Most people are accustomed to seeing fluorescence produced by ultraviolet light, often called "black light." DNA Protein Trait

History of Transformation 1928 – Fredrick Griffth first described transformation Pneumococcus bacteria (pneumonia) and mice 1944 – Oswald Avery, Colin MacLeod, Maclyn McCarty identified the “transforming principle” as DNA

History of Transformation 1952 – Alfred Hershey and Martha Chase provided final proof that DNA was the molecule of heredity 1970 – Morton Mandel and Akiko Higa developed a protocol for transforming E. coli bacteria

What is transformation used for? Agricultural Genes coding for traits such as frost, pest or drought resistance can be genetically transformed into plants

What is transformation used for? Environmental Bacteria can be genetically transformed with genes enabling them to digest oil spills or remove pollutants from the environment

What is transformation used for? Medical Production of human proteins to treat genetic diseases Protein Disease/Disorder Human insulin Diabetes mellitus Human Growth Hormone Deficiency in children Erythropoietin Anemia DNase I Cystic fibrosis Human antibody blocker Asthma Genes can be cut out of human, animal or plant DNA and placed inside bacteria and the bacteria will produce the “foreign” protein coded for by the gene. For example, a healthy human gene for the hormone insulin can be put into bacteria. Under the right conditions, these bacteria can make authentic human insulin just as they would make their own proteins.

What are we doing? We will transform bacteria (E. coli), giving it the ability to make green fluorescent proteins In this lab, we will transform bacteria, giving it the ability to make fluorescent proteins

Green Fluorescent Protein Used in science as a visual marker… Biological processes (protein production) Localization and regulation of gene expression Cell movement Cell fate during development Formation of different organs Marker to identify transgenic organisms

How does it work? Cell membrane Bacterial chromosomal DNA *plasmids* In addition to one large circular chromosome which contains all of the genes a bacterium needs for its normal existence, bacteria naturally contain one or more tiny circular pieces of DNA called plasmids. Plasmid DNA contains genes for traits that may be beneficial to bacterial survival under certain environmental conditions. In nature, bacteria can transfer plasmids back and forth, allowing them to share these beneficial genes. This mechanism allows bacteria to adapt to new environments. The recent occurrence of bacterial resistance to antibiotics is due to transmission of plasmids.

= + Making Plasmids Plasmid Gene A circular piece of autonomously replicating DNA Originally evolved by bacteria May express antibiotic resistance gene or be modified to express proteins of interest

pGLO Plasmid To make green fluorescent proteins GFP araC To survive with Ampicillin (antibiotic)