Genetic Transformation

1. Genetic Transformation

2. Historical Perspective • Frederick Griffith 1928 London • First controlled demonstration of genetic transformation • Griffith made the observation that nonpathogenic bacteria (Streptococcus pneumoniae)became pathogenic when mixed with a virulent strain of heat-killed S. pneumoniae (i.e. injected mixture killed mice) • The mechanism of transforming nonpathogenic bacteria to deadly bacteria was not known • In 1944 Oswald Avery demonstrated that DNA is responsible for conferring pathogenic properties

3. What is Genetic Transformation? • Genetically modification of a cell • Involves uptake of foreign DNA • Replication within organism • Gene expression DNA RNA Protein • Introduction of foreign DNA: Terms to know • By viruses: Transduction • Between bacteria: Conjugation • In mammalian cells: Transfection

4. Gene Cloning • Amplification and isolation of a particular gene sequence • Requires the generation of recombinant DNA (rDNA) i.e. combining DNA that does not naturally occur • Insertion of the gene into a plasmid (circular DNA) • Transformation of bacteria for replication • Select for cells that have received the recombinant DNA • Select individual colony for scale-up culture and replication of cloned DNA

5. BamH1 sites Ampicillin-resistance gene PCR-generated target gene with BamH1 sites Plasmid BamH1 digest Sticky ends DNA ligase Plasmid containing Ampicillin resistance gene and target gene Cloning a Gene into a Plasmid

6. Genetic Transformation into E.coli Ampicillin resistance gene (Ampr) and target gene on bacterial plasmid Individual colony is selected and cultured to amplify recombinant DNA Plasmid enters some bacteria Only E. coli containing plasmid survive on Ampicillin plates Cell division Transformation mixture is plated on to agar plate containing Ampicillin Bacterial clones

7. Key Steps for Transformation • Bacterial cell suspension is placed in CaCl2 solution • Cells must be in log phase of growth. • Cells are kept on ice until heat shock treatment • Heat shock at 42 ˚C for one minute • Recover period in LB broth • Cells are spread on appropriate selection plates Protein of interest Protein for antibiotic resistance Plasmid DNA enters the bacterial cell and the genes are expressed.

8. Components of Gene Cloning • Plasmid (to carry rDNA into cell) • Enzymes: • Restriction enzymes for cutting vector and insert • DNA ligase for joining DNA fragments • Selection process

9. Plasmids • Small circular dsDNA separate from bacterial DNA • Plasmids exist in bacteria, yeast, organelles • Single or multiple plasmid copies per cell • Easy to isolate and manipulate • Used as vector for transforming bacteria with foreign DNA • Foreign DNA is inserted after cutting with restriction enzymes • Plasmids contain certain genes which offer a competitive advantage for bacteria (i.e. antibiotic resistance) • Positive Selection: confers growth advantage i.e. able to grow in presence of antibiotic • Insert gene for expression (<10kb insertion)

10. Arabinose Operon • Gene induction • Arabinose operon • Three structural genes: araB, araA, and araD encode enzymes for arabinose metabolism • Initiator region, araI contains both the operator and promoter • The araC gene encodes an activator protein, AraC, which binds to initiator region

11. Arabinose Operon Regulation • Activation • Arabinose binds the activator protein • AraC/arabinose complex facilitates binding of RNA polymerase to the promoter which turns on the ara operon. • Activation also depends on cyclic AMP • Repression • Without arabinose, AraC protein binds araI and araO regions forming a loop and preventing transcription of the ara operon • Inducible promoter is used to control gene expression

12. Competent Cells • Competence is the ability of cells to take up exogenous DNA from the environment • Two types of competence: • Natural competence: Bacteria have cellular machinery to take up DNA from environment • Artificial competence: Cells are made competent in the laboratory allowing them to take up DNA

13. Preparing Competent Bacteria • Heat Shock: • Drives DNA into cells • Hold cells on ice in presence of CaCl2 to promote permeability of cells to plasmid DNA • Cells are heat shocked at 42 ºC for 50 – 60 seconds to allow circular plasmid DNA to enter cells • Electroporation: • Subject cells to electric shock to perforate membrane • Plasmid DNA enters cells through temporary holes • Efficient transformation of large plasmids

14. Plant Transformation • What is plant transformation • Objective: To transform the entire organism not individual cells • Systemic infection of Arabidopsis by transformation of female gametes

15. Genetic Engineering • Involves: • Isolating genes • Modifying genes for improved function • Packaging gene for insertion into new organism • Developing transgenes • Development of organisms that express new traits not found in nature • Extended shelf-life (produce) • Herbicide resistance (Roundup Ready) • Faster growth rate, larger • Terms: • Transgene is a genetically engineered gene added to a species • Transgenic refers to an organism containing an artificially introduced transgene (i.e. not through breeding)

16. Agrobacterium tumefaciens • Natural tool for plant transformation • How it works – tumor induction • Transfer of DNA to plant

17. Methods of Plant Transformation • Agrobacterium • Easiest and most simple • Cut plant tissue in small pieces, soak in Agrobacterium suspension • Some cells will be transformed by the bacterium • Grow on selection medium (rooting or shooting) • Some plants will not transform with the method • Particle Bombardment • DNA is coated onto gold or tungsten particles • Particles are shot into young plant cells • Low efficiency • Most plants can be transformed • Electroporation • Electric shock induces transient holes in cell membranes • DNA enters cells • Viral transformation • Use plant virus as vector to introduce DNA • Not always integrated into plant genome

18. Applications and Potential • Genetically Modified Organisms • Agriculture • Health and Medicine • Biotechnology • Scientific Research • Industry and Environment • Gene therapy

19. Genetically Modified Organisms (GMOs) • GMOs • Express traits not normally found in nature • Result of introducing foreign DNA • Highly controversial • Safety concerns • Environmental implications • Can we blindly trust profit-driven industry?

20. Agriculture • Herbicide resistant crops • Soybean, corn canola, lettuce, strawberry, potato, wheat • Virus resistance • Papaya resistance to papaya ringspot virus • Golden rice • Engineering rice to produce Vitamin A • Edible vaccines in development • Plant containing pathogen protein is ingested • Body produces antibodies against protein • Conferring resistance (ex diarrhea, hepatitis B, measles) • Bananas, potato, tomato

21. Health and Medicine • Biotherapeutics • Antibodies • Hormone • Enzymes • Disease Indications • Liver disease • Genetic diseases • Kidney disorders • Digestive disorders • Cancer • Infectious disease

22. Biotechnology • Chymosin: • Genetically engineered enzyme • Used for curdling milk productsin cheese production • Revolutionized cheese production • Previously rennin was isolated from newborn calf intestine (expensive, inhumane) • Inexpensive, readily available • Bovine somatotropin (bST): • increased milk production in cows • Other examples: • Insulin • Interleukin • Human growth hormone • Interferon

23. Scientific Research • Protein production using genetic transformation • Objectives: • Generate antibodies • Assay development • Structure determination • Protein-protein interaction

24. Industry and Environment • Bioremediation: Using bioengineered microbes to clean up pollution and contaminated sites • Indicator bacteria: Detecting pollution and contamination in the environment • Waste management • Sewage • Petroleum products

25. Gene Therapy Overview • Viral vector is used to deliver genetic material to target cells (ex. liver, lung) • The viral vector then injects the gene for a defective or missing protein • The cell then produces the functional protein and restores the target cell to a normal state • Viruses used for gene therapy • Retroviruses • Adenoviruses • Adeno-associated viruses • Herpes simplex viruses • Gene therapy is experimental with poor success in clinical trials • There are no FDA-approved gene therapy products on the market