Recombinant DNA Recombinant DNA TechnologyTechnology

Bacterial Transformation & Bacterial Transformation & GFPGFP

Frederick Griffith - 1928Frederick Griffith - 1928

PlasmidPlasmid:: small, usually small, usually ring-shaped molecule of ring-shaped molecule of deoxyribonucleic acid deoxyribonucleic acid (DNA), (DNA), Plasmids are present in Plasmids are present in almost all bacteria and almost all bacteria and may also be found in may also be found in some yeasts and other some yeasts and other fungi, protozoa, and even fungi, protozoa, and even some plants and animals. some plants and animals. They are separate from They are separate from chromosomes.chromosomes.

Plasmids generally carry Plasmids generally carry fewer genes than do fewer genes than do chromosomes, and the chromosomes, and the genes that they carry are genes that they carry are useful, but not essential, useful, but not essential, to the survival of the cell.to the survival of the cell.Most bacteria have only Most bacteria have only one chromosome under one chromosome under normal circumstances, normal circumstances, but may contain 1 to 100 but may contain 1 to 100 or more copies of a given or more copies of a given plasmid.plasmid.

Plasmid Replication Site

Example: pAmp

Recombinant DNA, like most other Recombinant DNA, like most other DNA technologies, relies on DNA technologies, relies on restriction enzymesrestriction enzymes, specialized , specialized enzymes that act like molecular enzymes that act like molecular scissors to cut DNA strands at certain scissors to cut DNA strands at certain points in a base sequence, which may points in a base sequence, which may be four to eight bases long. This be four to eight bases long. This creates “sticky ends.” creates “sticky ends.” Another enzyme, called Another enzyme, called DNA ligaseDNA ligase, , glues an isolated gene to a glues an isolated gene to a vectorvector—a —a fragment of DNA that is able to transfer fragment of DNA that is able to transfer from one organism to another from one organism to another (plasmids). (plasmids). In this way, scientists cut and paste In this way, scientists cut and paste pieces of DNA from different sources pieces of DNA from different sources together to create molecules that will together to create molecules that will then be known as then be known as recombinant DNArecombinant DNA..

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The ability to combine human and bacterial DNA The ability to combine human and bacterial DNA has given rise to a number of medical advances. has given rise to a number of medical advances.

The first commercial use of a recombinant The first commercial use of a recombinant plasmids came in 1982, when scientists created plasmids came in 1982, when scientists created a genetically engineered bacterium able to a genetically engineered bacterium able to produce human insulin. produce human insulin.

Insulin is a hormone that helps regulate blood Insulin is a hormone that helps regulate blood sugar and is needed by many people with sugar and is needed by many people with diabetes. diabetes.

Plasmid Map (Simple)Plasmid Map (Simple)

*ORI is the origin.

Plasmid MapsPlasmid Maps

Growth and Purification of PlasmidsGrowth and Purification of Plasmids

Plasmid growthin bacteria Purification of

plasmid

ExtractionTransfection

Recombinant DNA Technology Recombinant DNA Technology and Vaccine Developmentand Vaccine Development

CHO, yeast,or insect cells

In vitro antigenproduction

In vivoantigen

production

Vaccine vector