1. Genetic Engineering BSC 1010L Transformation of E. coli with Jellyfish GFP

2. What is transformation? Transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation, and expression of exogenous DNA taken up from the cell’s surroundings Plasmid BacterialchromosomalDNA Cell wall

3. Escherichia coli It has become an important research organism for molecular biology Growth requirements are well characterized E coli is the most common bacterium in the human gut A single microscopic cell can divide to form a visible colony with millions of cells overnight Molecular biology of E coli is well understood Has been extensively studied Genome has been sequenced Many strains commercially available Reproduces very rapidly

4. Bioluminescence – the production and emission of light by a living organism This phenomenon found in fungi and many animals including fireflies and other insects, marine invertebrates and vertebrates Blackdragon fish Mushrooms Firefly Dinoflagellates

5. In the jellyfish, Aequorea victoria, a greenish biolumunescence results from the activity of Green Fluorescent Protein (GFP) GFP is composed of 238 amino acid residues that exhibits a bright green fluorescence (509 nm) when exposed to blue light (395nm)

6. The Transformation Plasmid The GFP gene has already been inserted into the pGLO plasmid. A restriction enzyme was used to cut out the GFP gene in jellyfish DNA The same restriction enzyme was used to cut open the pGLO plasmid The GFP gene fragment and pGLO plasmid were incubated with DNA ligase The recombinant pGLO plasmid is now ready for use

7. The pGlo plasmid –Beta Lactamase Provides ampicillin resistanceProvides ampicillin resistance –araC regulator protein Regulates GFP transcriptionRegulates GFP transcription –Green Fluorescent Protein Aequorea victoria jellyfish geneAequorea victoria jellyfish gene pGLO ori bla GFP araC

8. How does it work? GFP Beta lactamase (ampicillin resistance) pGlo Plasmids BacterialchromosomalDNA Transform bacteria with the pGlo plasmid and grow under various conditions

9. Ampicillin Action and Resistance Antibiotics have various methods of interfering with bacterial growth: inhibiting cell wall biosynthesis or blocking protein synthesis Ampicillin inhibits peptidoglycan synthesis: the cell wall polymer consisting of sugars and amino acids The ampicillin resistance protein, β-lactamase, cleaves the β- lactam ring of ampicillin molecules, which leaves them unable to interfere with peptidoglycan synthesis

10. Transformation Procedure: Overview Suspend bacterial colonies in Transformation SolutionSuspend bacterial colonies in Transformation Solution Add pGLO plasmid DNAAdd pGLO plasmid DNA Place tubes on icePlace tubes on ice Heat shock at 42 o C and place back on iceHeat shock at 42 o C and place back on ice Incubate with LB nutrient brothIncubate with LB nutrient broth Streak platesStreak plates

11. Why perform each step?  CaCl 2 treatment on ice crystallizes fluid membranes and stabilizes distribution of charged molecules  CaCl 2 Transformation solution provides Ca ++ cations that neutralize the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane, allowing the DNA to enter the cells Ca ++ O CH 2 O P O O O Base CH 2 O P O O O Base OH Sugar O Ca ++

12. Why perform each step?  Heat-shock increases permeability of cell membrane  Luria-Bertani Nutrient broth incubation allows beta lactamase expression Beta lactamase (ampicillin resistance) pGlo Plasmids Bacterialchromosome DNA Cell wall

13. Selection for Transformants Grow transformed bacteria under various conditionsGrow transformed bacteria under various conditions On which plates will colonies grow?On which plates will colonies grow? On which plates will colonies glow?On which plates will colonies glow?

14. The pGlo System Areas of Special Attention Timing is important…be efficient!! Mix contents before pipetting!!! A film of plasmid must be on the loop!

15. 1 – LB/AMP/Ara plate Supplies (each lab group): 2 – micro test tubes Micro tube rack Sterile transfer pipette Sterile inoculation loop 2 – LB/AMP plates 1 – LB plate

16. Step 1: Add CaCl 2 solution to tube:

17. Step 2: Add competent E. coli to tube:

18. Step 3: Add pGLO plasmid to +pGLO tube: Be sure to label the bottom of the petri dish.

19. Step 4: Heat Shock Bacteria: It is very important to directly transfer tubes from the ice to the water bath and then directly back to the ice after 50 seconds have elapsed.

20. Step 5: Plate Bacteria: