1. BRIDGES 2014

2.  DNA ➔ RNA ➔ PROTEIN ➔ TRAIT Genotype Phenotype

3.  Adds new information to DNA  Cell can take up (take inside) and expresses a new piece of DNA ◦ Transformation  New genetic information often provides organism with new trait-identifiable after transformation  Genes can be cut out of human, animal, or plant DNA and placed inside bacteria via transformation

5.  Agriculture ◦ Frost, pest, or drought resistance  Bioremediation ◦ Bacteria to digest oil spills  Medicine ◦ Transforming a sick person’s cells with healthy copies of the defective gene ◦ Creating insulin for diabetes

6.  Cells that take up DNA  Cells in log phase ◦ Growing cells take up DNA better  Transformation solution-CaCl 2 ◦ Ca 2 + cation neutralizes the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane  Heat shock ◦ Increases the permeability of the cell membrane ◦ Duration of the heat shock is critical

7.  Vehicles for foreign DNA  Plasmid DNA usually contains genes for one or more traits that may be beneficial to bacterial survival  Bacteria can transfer plasmids back and forth, allowing them to share beneficial genes  Allows for adaptation to new environments  Recent occurrence of bacterial antibiotic resistance due to transmission of plasmids

10.  Competent cells take up DNA  DNA is brought in on plasmids  Process is called transformation

11.  GFP gene from jellyfish- Aequorea victoria ◦ Causes cells to glow green under ultraviolet light  Mutations to GFP enhance fluorescence  Modified GFP gene in pGLO plasmid  After transformation bacteria express the jellyfish gene and glow bright green

12.  Gene for GFP ◦ Switched on by adding the sugar arabinose to the cell’s nutrient medium ◦ Transformed cells are white on plates not containing arabinose ◦ Transformed cells glow green when arabinose is included in the nutrient agar  Gene for resistance to the antibiotic ampicillin

13.  ori- origin  bla- ampicilin resistance  araC- activated by arabinose, allows down stream transcription  GFP- green fluorescent protein

14.  Add E. coli grown overnight to transformation solution ◦ Should be in log phase  Add pGLO plasmid to +pGLO sample  Heat ◦ Should make cells competent (take up plasmid)  Add to LB plates with ampicillin and arabinose  Grow overnight

15.  Label tubes ◦ Initials ◦ Group number ◦ +pGLO ◦ -pGLO

16.  Add 250µL transformation solution

17.  Put on ice

18.  Add E. coli grown overnight to both tubes

19.  Add pGLO plasmid to +pGLO tube  DO NOT ADD TO –pGLO TUBE

20.  Add pGLO plasmid to +pGLO tube  DO NOT ADD TO –pGLO TUBE  Use pipet to transfer 10µL of plasmid

21.  Put back on ice for 10 mins

22.  Label plates

23.  Heat Shock ◦ Time this! ◦ Then ice for 2 mins

24.  Add broth

25.  Use broth YOU made  Using sterile technique pour some into beaker and transfer from there

26.  Add broth  Longer room temperature incubation will improve results

27.  Add to plates

28.  Spread plates ◦ Use different spreaders for each plate

29.  Incubate overnight ◦ Label with initials ◦ At 37˚C