Genetic Engineering Aulanni’am Biochemistry Laboratory Chemistry Department Brawijaya University

What is genetic engineering? The transfer of genes (segment of DNA) from one species to another. This is impossible in naturally breeding.

How to get the gene from species A to species B? vector A vector is used to carry the gene into the host nucleus.

What are vectors commonly used in GE? Plasmid (of bacteria) Plasmid (of bacteria) Viruses Viruses Shotgun technique (blindly shoots tiny particles coated with the gene into the host cells) Shotgun technique (blindly shoots tiny particles coated with the gene into the host cells)

What is a plasmid?

Natural role of a plasmid in bacteria? Plasmids usually contain one or two gene that confer a selective advantage on the bacterium e.g. antibiotic resistance gene. Plasmids usually contain one or two gene that confer a selective advantage on the bacterium e.g. antibiotic resistance gene. Every plasmid possess a replicator gene can duplicated independently from the chromosomal DNA. Every plasmid possess a replicator gene can duplicated independently from the chromosomal DNA.

1&2 : antibiotic resistance genes 3: replicator gene

Recombinant DNA technology Cut insulin production gene from a human DNA Cut insulin production gene from a human DNA Paste it into the DNA of Escherichia coli Paste it into the DNA of Escherichia coli The E. coli reproduces rapidly to produce large numbers: gene cloning The E. coli reproduces rapidly to produce large numbers: gene cloning

restriction enzyme  In bacteria, restriction enzymes are found naturally. They cut DNA at specific sites.

DNA double helixes are cut at the axis of symmetry:

Cut plasmid (by adding restriction enzyme) Cut insulin gene from human cell (by adding restriction enzyme) adding ligase enzyme Insulin gene has been inserted into the plasmid

Place the recombinant plasmids back into the bacteria Leave the bacteria to grow and multiply

How do I know which bacteria will have the plasmid? Add specific antibiotics to the agar culture Add specific antibiotics to the agar culture All those bacteria which do not have the plasmid will die All those bacteria which do not have the plasmid will die Only those with the recombinant plasmids can grow and multiply Only those with the recombinant plasmids can grow and multiply

Application of GE: Medical Medical GM Food Industrial production Environmental protection

Medical Production of pharmaceuticals for treatment of diseases e.g. human insulin, interferons Production of pharmaceuticals for treatment of diseases e.g. human insulin, interferons Production of pharmaceuticals for disease prevention e.g. vaccine (hepatitis B vaccine)

Medical Gene therapy: Gene therapy: Artificially replace the disease-causing gene with a normal allele. The normal allele can be carried by a virus vector to the target tissues. e.g. treatment of cystic fibrosis

Medical Clonal propagation: Clonal propagation: a source of tissue or organ for transplantation avoid all problems of immunoincompatibility.

Agricultural Transgenic plants and farm animals Transgenic plants and farm animals pest-resistant (reduce use of pesticides), increase yield Increase storage time e.g. green tomato tomato with beef genes

Industrial: Use of GM microorganisms to make stone-wash jeans Use of GM microorganisms to make stone-wash jeans Use of GM microorganisms to produce enzymes e.g. detergents

Environmental protection GM E. coli possesses gene to break down cellulose, speeding up recycling of the most abundant biomass on earth GM E. coli possesses gene to break down cellulose, speeding up recycling of the most abundant biomass on earth GM microorganisms with enhanced ability to break down environmental pollutants

Dispute in the development of GM Dangerous pathogens formed in the course of rDNA Dangerous pathogens formed in the course of rDNA New tools for militarists and terrorists New tools for militarists and terrorists Triggering of catastrophic ecological imbalance Triggering of catastrophic ecological imbalance Moral problems in the use of GM techniques in man e.g. germ cell gene therapy Moral problems in the use of GM techniques in man e.g. germ cell gene therapy Unknown effect of GM food on men Unknown effect of GM food on men