Genetic Transformation

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

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

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

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

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

Key Steps for Transformation Bacterial cell suspension is placed in CaCl 2 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.

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

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)

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

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

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

Preparing Competent Bacteria Heat Shock: –Drives DNA into cells –Hold cells on ice in presence of CaCl 2 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

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

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)

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

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

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

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?

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

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

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

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

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

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