1. Biotechnology & DNA Technology Genetic Engineering Chapter 12 12.1-12.2Pgs. 231-234 Objective: I can describe several different types of biotechnology, such as plasmid cloning and transformation, in order to best determine how to cut a gene of interest out and insert it into a plasmid. http://www.youtube.com/watch?v=ebSWG1QmxnY

2. Prokaryotic DNA Packaging 1 circular bacterial chromosome ▫ NOT plasmid… Plasmid can refer to small(er) circular molecules of DNA besides “main” chromosome ▫ Self-Replicating, so will pass on ▫ Confusing, because plasmid can be incorporated into “main” Prokaryotic Cell Division is called ▫ Binary Fission ▫ Asexual Reproduction

3. Prokaryotic Sexual “Reproduction” Conjugation ▫ Uses pili (singular pilus) ▫ Transfers DNA  Not reproduction - recombination Can pass plasmid(s) or part of “main” ▫ Can either remain as plasmid in receiver ▫ Can be incorporated into main bacterial chromosome

5. Conjugation: A Natural Transformation Scientists realized that bacteria naturally transfer genetic info (in certain conditions)  helps survivial (adaptation + evolution) Similar to… Griffith’s Experiment

6. Can we artificially transfer genes? If bacteria can naturally take in foreign DNA (undergo transformation), can we make them take DNA we want them to? Yes: Biotechnology (DNA Technology) Genetic Engineering  Recombinant DNA What’s the point? Allow for gene cloning (just gene) Allows for GMO (Genetically Modified Organisms) How? (specifically)?

7. Bacteria of Choice E.coli Escherichia coli Use bacteria as “factory” Can clone a gene (DNA) by letting E.coli reproduce (divide)

8. E. coli bacterium Bacterial chromosome A plasmid is isolated. Gene of interest Plasmid The cell’s DNA is isolated. DNA A cell with DNA containing the gene of interest 1 2

9. Figure 12.1B_s2 E. coli bacterium Bacterial chromosome A plasmid is isolated. Gene of interest Plasmid The cell’s DNA is isolated. DNA A cell with DNA containing the gene of interest 1 3 2 4 The plasmid is cut with an enzyme. The cell’s DNA is cut with the same enzyme. Gene of interest

10. Figure 12.1B_s3 E. coli bacterium Bacterial chromosome A plasmid is isolated. Gene of interest Plasmid The cell’s DNA is isolated. DNA A cell with DNA containing the gene of interest 1 3 2 4 5 The plasmid is cut with an enzyme. The cell’s DNA is cut with the same enzyme. Gene of interest The targeted fragment and plasmid DNA are combined.

11. Figure 12.1B_s4 E. coli bacterium Bacterial chromosome A plasmid is isolated. Gene of interest Plasmid The cell’s DNA is isolated. DNA A cell with DNA containing the gene of interest 1 3 2 4 5 6 The plasmid is cut with an enzyme. The cell’s DNA is cut with the same enzyme. Gene of interest The targeted fragment and plasmid DNA are combined. DNA ligase is added, which joins the two DNA molecules. Gene of interest Recombinant DNA plasmid

12. Figure 12.1B_s5 Gene of interest The recombinant plasmid is taken up by a bacterium through transformation. Recombinant bacterium Recombinant DNA plasmid 7

13. Figure 12.1B_s6 Gene of interest The recombinant plasmid is taken up by a bacterium through transformation. The bacterium reproduces. Clone of cells Recombinant bacterium Recombinant DNA plasmid 7 8

14. Figure 12.1B_s7 Gene of interest The recombinant plasmid is taken up by a bacterium through transformation. Harvested proteins may be used directly. The bacterium reproduces. Clone of cells Recombinant bacterium Recombinant DNA plasmid Genes may be inserted into other organisms. 9 7 8

15. animation file

16. How can cut gene of interest out? How can cut plasmid to insert? Restriction Enzymes: natural defense (a.k.a. restriction endonucleases) Enzymes that recognize a particular sequence in DNA and cut there ▫ Sequence typically symmetrical (complementary palindrome) ▫ Area that is cut = restriction site animation file

17. Restriction Enzymes Details Usually cut in a staggered format ▫ Results in “sticky ends” ▫ Allows to hydrogen bond to other restriction fragments  Fragments of DNA cut by restriction enzymes Connections made permanent by DNA ligase