Bacterial Transformation

1. Bacterial Transformation AP Biology Transformation Lab

2. What is Transformation? • Changing the genes and phenotype of a bacteria by uptake of foreign/new DNA • a natural process that bacteria have evolved in order to obtain DNA from their environment. • enables scientists to insert genes by recombinant techniques and place the plasmid into a bacteria for expression

3. What is Transformation? • In order to transform bacteria we need to overcome two problems • Disadvantage –cells that contain plasmids grow more slowly • There is pressure on cells to get rid of their plasmids • Needs to be an Advantage to keep plasmids • Antibiotic resistance • How do we tell which cells have the plasmid? • We use a marker • Grow the bacteria on plates that contain the antibiotic • Use a color pigment marker that is present when a particular enzyme is present Let’s review bacterial DNA first…

4. Let’s Review: Bacterial genome • Bacteria are prokaryotes—no nucleus. • The area where DNA is located is called the nucleoid • DNA is organized in one double stranded circular molecule

5. Bacterial DNA Bacterial cell Plasmid DNA Genomic DNA

6. Size

7. Which bacteria will we be using? • Escherichia coliis the most common bacterium in the human gut. It has been extensively studied in the laboratory and is an important research organism for molecular biology. • E. colireproduce very rapidly; a single microscopic cell can divide to form a visible colony with millions of cells overnight. • Like all bacteria, E. coli has no nuclear envelope surrounding the bacterial chromosome and thus no true nucleus. • All of the genes required for basic survival and reproduction are found in the single chromosome.

8. A circular piece of autonomously replicating DNA exists outside the main bacterial chromosome Originally evolved by bacteria Carries separate genes for specialized functions. In genetic engineering, plasmids are one means used to introduce foreign genes into a bacterial cell. What is a Plasmid? Scanning electron micrograph

9. What is carried on the Plasmid? • The plasmid contains genes necessary for survival and can be passed from one bacteria to another • ampR gene • confers resistance to the antibiotic ampicillin. • E. coli cells containing this plasmid, can survive and form colonies on LB agar that has been supplemented with ampicillin. • Cells lacking the ampR plasmid are sensitive to the antibiotic, which kills them. • An ampicillin-sensitive cell can be transformed to an ampicillin-resistant cell by its uptake of a foreign plasmid containing the ampR gene. • The same can be said for the lac gene, which codes for lactose. I • If this gene is taken in, the organism can break down lactose.

10. Transformation has 4 main steps • Prepare cells • Make them competent • Incubate with plasmid • Plasmid associates with membrane of cells • Shock the cells • To initiate plasmid uptake • Allow cells to recover and plate on agar • Allow cells to recover in rich medium for 0.5 – 1 h (without antibiotic to avoid stressing the cells) • Plate on agar with antibiotic for selection • Next day can pick single colonies or clones

11. The Transformation Lab… • Our plasmid: pBlu plasmid • Into E. coli (scary?…no!) • Our plasmid contains genes for: • AMP= ampicillin (an antibiotic) resistance • Beta-galactosidase-an enzyme that converts X-Gal  Indo Blu RNA Protein that allows for antibiotic resistance RNA Enzyme that breaks down X-Gal to make Indo Blu

12. How do we get the plasmid inside of the bacteria? • To transform cells, you first need to make them competent to take up extracellular DNA. • Obtain E. Coli bacteria cells + Add to ice cold CaCl2 • Helps plasmid attach to bacteria • Makes the cell competent • Add plasmid to same microtube 1. E. Coli 2. pBlu plasmid

13. Transformation solution CaCI2 Positive charge of Ca++ ions shields negative charge of DNA phosphates DNA becomes “neutral” and can pass through the cell membrane How It Works Ca++ O Ca++ O P O Base O O CH2 Sugar O Ca++ O O P Base O O CH2 Sugar OH

14. How do we get the plasmid inside the bacteria? • Wait…and then • Heat shock! This temporarily opens pores to allow the plasmid to enter the bacteria…timing is critical!!!

15. Chemical transformation Bacterial chromosomal DNA • Ice-cold CaCl2 • To make competent • Slows the fluid cell membrane • Heat shock • Increases permeability of membranes by opening pores • Plasmid DNA is taken up • Nutrient broth incubation • Allows beta-lactamase expression Cell wall GFP Beta lactamase (ampicillin resistance) pBLU plasmids

16. What is Nutrient Broth? • Luria-Bertani (LB) broth • Medium that contains nutrients for bacterial growth and gene expression • Carbohydrates • Amino acids • Nucleotides • Salts • Vitamins

17. How will we know if the bacteria actually got into the plasmid? • Any ideas? • We can grow the bacteria on a plate: • That contains ampicillin and X-Gal • Regular bacterial medium • What do you predict will happen in each?

18. Predict What will we observe??? Amp X-Gal pBlu pBlu Control Control Regular Amp X-Gal Regular

19. Growing the bacteria • After they have received the plasmid… • Place on a growth media and allowed to grow. spread evenly discrete colonies (~106 cells) ~ 100 µl overnight 37 ºC