Genetic Transformation

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 (Ampr) and target gene on bacterial plasmid

Bacterial clones

Cell division

Transformation mixture is platedon to agar plate containing Ampicillin

Only E. coli containing plasmidsurvive on Ampicillin plates

Individual colony is selected andcultured to amplify recombinant DNA

Plasmid enters somebacteria

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.

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 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

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