1. Bacterial Transformation

2. Chromosome? A long piece of DNA with many pieces of information on it, each piece is a set of directions for making a protein or regulating the production of a protein.

3. Gene? A piece of DNA that codes for the production of one protein.

4. Plasmid? A small circle of DNA with a few pieces of information on it, each piece a set of directions for making a protein or regulating the production of a protein.

5. Ampicillin? An antibiotic; a chemical that inhibits reproduction of bacteria.

6. bla (amp)? One of the genes on our plasmid codes for beta- lactamase, we sometimes call this gene amp because it provides the bacteria with the code for producing a protein (beta-lactamase) that inactivates ampicilin (AMP) pGLO ori bla GFP araC

7. GFP? The gene that codes for the production of the green fluorescent protein. pGLO ori bla GFP araC

8. araC? The inducible promotor that tells the bacterial cell to start using the GFP gene to make green fluorescent protein. pGLO ori bla GFP araC

9. pGLO The plasmid containing the GFP, araC and bla genes. pGLO ori bla GFP araC

10. Luria Broth (LB) Nutrient solution/food. Like chicken soup for bacteria.

11. Agar? A gel made with seaweed protein.  Not unlike Jello (which is made with gelatin - made from dissolved connective tissue from horse and cow and pig skeletons)

12. Transformation? The process of getting a bacteria to take in a new piece of DNA (a plasmid).  If successful the bacteria will be “transformed” into something totally new - a bacteria that is genetically different from the original and now is partly bacteria and partly jellyfish. Or is it?

13. Transport Nucleus Cytoplasm Protein gene DNA mRNA The Cell:

14. Circular piece of autonomously replicating DNA What is a plasmid? Originally evolved by bacteria May express antibiotic resistance or be modified to express proteins of interest ori bla

15. pGLO ori bla GFP araC pGLO Plasmid  Beta Lactamase Ampicillin resistance  Green Fluorescent Protein Aequorea victoria jellyfish gene  araC regulator protein Regulates GFP transcription

16. Uptake of foreign DNA often a circular plasmid What is transformation? GFP Amp Resistance

17. pGLO Transformation GFP pGLO plasmids Beta lactamase (ampicillin resistance) Bacterial chromosomal DNA Cell wall

18. p GLO Regulation araC RNA Polymerase Effector (Arabinose) B B B A A A D D D araC ara Operon RNA Polymerase araC ara GFP Operon GFP Gene araC GFP Gene araC GFP Gene Effector (Arabinose)

19. Transformation Procedure Suspend colonies in Transformation Solution Add pGLO plasmid DNA to “+” tube Incubate tubes 10 minutes on ice Heat shock 50 seconds at 42 o C Incubate 2 minutes on ice Incubate 10 minutes with LB broth Plate

20. Why perform each step? 1. Incubation on ice slows fluid cell membranes 2. Heat-shock increases permeability of cell membrane 3. LB recovery allows beta lactamase expression GFP Cell wall Beta lactamase (ampicillin resistance)