Genetic engineering Recombinant DNA technology
Questions: • Name 3 things you know about bacteria. • What are some characteristics that make bacteria a good choice for genetic engineering? • Why are we using bacteria for biotechnology rather than some other organism?
Bacteria are useful hosts. • They are easily grown • They are cheap to grow • They grow fast • They are easily manipulated in the laboratory • DNA can be inserted - transformation • DNA can be easily isolated • Bacteria contain natural plasmids and viruses which are useful vectors for recombinant DNA
Bacterial plasmids • Most bacterial DNA is on a single large chromosome, but some DNA is in a small circle called a plasmid.
Bacterial Plasmids in Nature • Occur naturally in bacteria and usually carry genes that are useful but not essential to survival: e.g. genes which make bacteria resistant to antibiotics. • Plasmids are released by dead bacteria and absorbed by those still living thus genetic information is exchanged (sexual reproduction?).
Bacterial Plasmids in Nature • Some plasmids even contain genes that build a transfer tube between bacteria. • There can be as many as several hundred copies of a single plasmid in each bacteria.
Plasmid Structure • Plasmids only need an “origin or replication” and a “useful” gene to be considered complete. • Molecular biologists have been able to “insert” custom built restriction sites into many plasmids so they can be used to “insert” DNA fragments from other genes into them and thus have a way to propagate those DNA pieces.
Plasmids can be manipulated easily in the laboratory • Plasmids can be collected from bacteria. • Restriction enzymes can be purified and used to identify and cut out specific sequences of DNA along with the plasmid vector. • Ligase (enzyme) can be purified and used to “glue” pieces of DNA together. • Bacteria can be transformed by taking in plasmids given to them.
Transforming Bacteria • When a bacterial cell takes in a plasmid from the environment, it has new DNA (and therefore, new traits). • Scientists say that the bacteria has been transformed.
How to produce human insulin from bacteria and become a multimillionaire • Isolate plasmid DNA from bacteria and insulin gene from human. • Cut both DNAs with the same restriction enzyme. • Mix the DNA together with ligase. • Insert the new DNA into bacteria (transformation). • Use antibiotics to kill any bacteria without the plasmid. • Grow bacteria and harvest the insulin. Human DNA Insulin gene Insulin Purification
Some Links • Basic construction of recombinant DNA http://www.dnai.org/b/index.html • Click on “techniques” then “cutting and pasting” • An excuse to study on Youtube!! http://www.youtube.com/watch?v=x2jUMG2E-ic
Your Turn • Plasmid Maps • Restriction Enzyme Challenge • Activity I and II • Recombinant DNA • Paper plasmid Activity