Biotechnology: Bacterial Transformation Lab

1. Biotechnology: Bacterial Transformation Lab How can we manipulate inheritable information?

2. Genetic information passed from parent to offspring via DNA provides for continuity of life. • In order for DNA to direct cellular activities it must first be transcribed. • Some of the RNA’s are used immediately for ribosomes or to control other cellular processes. • Other RNA’s are translated into proteins that have important roles in determining metabolism & development.

3. When the DNA of a cell changes, the RNA’s and proteins they produce often change, which in turn changes how the cell functions. • DNA can change in different ways: • Mutated (spontaneously, environmental effects or DNA replication error) • Biotechnologists can cause an intentional change

4. Biotechnologists have a most powerful tool: • They have the ability to transfer the DNA of one organism into another & make it function in the new organism. • With this ability they can make cells produce novel protein products that the cells did not make previously.

5. Fredrick Griffith Experiment CONCLUSION: The living R bacteria had been transformed into pathogenic S bacteria by an unknown, heritable substance from the dead S cells that allowed the R cells to make capsules.

6. What they do today… • Biotechnologists have succeeded in inserting a gene (Bt) from the bacterium: • Bacillus thuringiensis into the corn genome. • When expressed the Bt gene produces a toxin that kills caterpillars & controls earworms that damage corn

7. Applications for genetic engineering

8. Human manipulation of DNA has raised several ethical, social, and medical issues

9. Working with bacteria

10. Natural Transformation Cells have surface proteins that bind to DNA and bring it into the cell. Then the orig. DNA and the new DNA are compared. If there is enough similarities the new DNA will be welcomed. If there isn’t then the new DNA is digested by the cellular enzymes.

11. Artificial Transformation • It is rare that bacteria uptake DNA naturally from the environment. • Subjecting bacteria to artificial conditions can enable bacteria to take in DNA. • Referred to as competent. • So…how is this done?

12. Rendering bacteria competent • Changing the ionic strength of the medium and heating the cells in the presence of positive ions (usually calcium). • This treatment renders the cell membrane permeable to DNA. • Recently high voltage has also been used to render cells permeable to DNA • electroporation

13. Once the DNA has been taken in by the cell, the use of that DNA by the cell is referred to as expression • The expression of the new DNA depends on its integration with the host DNA. • Normally scientists want to introduce DNA that is not similar to the host DNA & therefore the cell would destroy the introduced DNA. • Scientists have found a way around this by introducing the new DNA as plasmid DNA. • Does not have to be similar to the host DNA

14. What’s a plasmid? A plasmid is a small circular piece of double-stranded DNA that has an origin of replication.

15. Selecting for transformed bacteria • 2 problems to overcome: • Cells containing plasmids reproduce at a slower rate & the pressure is great to rid themselves of the plasmid. • To overcome this there needs to be an advantage • We have to be able to determine which bacteria have received the plasmid.

16. Solutions to the problems • Scientists use a system involving antibiotics and genes for resistance of antibiotics. • In a typical transformation, billions of bacteria are treated and exposed to plasmid DNA. • Only a fraction (less than 1 in 1000) will acquire the plasmid. • Antibiotic resistance genes provide a means of finding the bacteria that took in the plasmid.

17. The treated bacteria are then exposed to the antibiotic. • The bacteria that took in the plasmid will grow on the antibiotic plate. • Resistance to an antibiotic is known as a selectable marker; we can select for cells that contain it. • Other marker genes are color marker genes, which change the color of a bacterial colony.

18. Green Fluorescent Protein (GFP) This is an example of a color marker scientists use.

19. Color markers used as reporter molecules • If you wanted to know what a certain protein did or where it went you could attach the color marker. • Let’s say you wanted to know if a particular protein had anything to do with making blood vessels. If the blood vessels started glowing you would have your answer.

20. Suppose you have a plasmid that contains both the gene for GFP and a gene for resistance to ampicillin (an antibiotic), how will you be able to determine if the bacterial cells have been transformed?

21. What’s this lab about?

22. What are you going to do? • Some bacteria are naturally resistant to antibiotics, but others are not. • How could you use LB/agar plates, some E. coli, and some ampicillin (an antibiotic) to determine how E. coli cells are affected by ampicillin? • What would you expect your experimental results to indicate about the effect of ampicillin on the E. coli cells?