1. Transforming E. coli with a Recombinant Plasmid

2. What is biotechnology? Employs use of living organisms in technology and medicine Modifying living organisms for our use Mass production Recombinant DNA Recombine DNA fragments from various sources

3. Introduction Restriction enzymes Cut DNA molecules from various organisms and recombine pieces Recombinant DNA Restrict the growth of viruses in bacteria Digest the DNA molecule at specific nucleotide sequences Restriction fragments DNA fragments Sticky ends Allow annealing and recombination of DNA fragments from different sources

4. Engineering the Plasmid: ligation of rfp gene into p-ARA sticky end BamH I sticky end Hind III sticky end BamH I sticky end Hind III Bruce Wallace

5. Introduction Bacterial plasmids Circular pieces nonessential DNA found in bacteria Can be engineered to carry genes Express proteins encoded by these genes

6. pGLO Plasmid Contains: Green Fluorescent Protein gene Codes for green fluorescent protein Ampicillin resistance gene Great example of : DNA RNA PROTEIN TRAIT

7. Introduction Purpose: Get the pGLO plasmid containing the gfp gene into bacterial cells Get those cells to express the gfp gene and make the mutant fluorescent protein Transformation Process of taking up foreign pieces of DNA In this case, a plasmid Not very common in mature! Can occur under experimental conditions (1 cell in a thousand!)

8. Plasmid DNA Insertion

9. Transgenic Colony Allowed to Grow

10. Introduction What are competent cells?!?! Cells ready to receive plasmids through lab procedures Soaked in calcium chloride PM and DNA negatively charged Calcium ions (Ca++) neutralize charges to allow for plasmid to pass through PM Need to “heat shock” cells Creates pressure differences to allow cell membrane to be permeable to DNA Cold then hot Then feed and recover!!

11. Media Spread cells on various plates (3) LB plate Only bacterial food “control” LB/amp plate Contains ampicillin Antibiotic prevents bacteria from forming CW Cells that contain amp r produces protein that destroys ampicillin “-” and “+” LB/amp/ara plate Contains arabinose Needed to express the gfp gene If take up plasmid, this helps with transcription of gene

12. Predictions…… LB LB/ampLB/amp/ara -pGLO +pGLO LB/amp

13. Predictions…… LB LB/ampLB/amp/ara -pGLO +pGLO + + - + LB/amp