2. 90- How can we make more insulin? V.1.2.2

3. How can we make more insulin? By Transforming Bacteria V.1.2.2

4. “transformed cell” – cell has acquired new characteristics “characteristics” – due to the expression of incorporated foreign genetic material

5. AP Biology Green with envy?? Jelly fish “GFP” Transformed vertebrates

6. AP Biology Genetic engineering… grow bacteria harvest (purify) protein transformed bacteria plasmid gene from other organism + recombinant plasmid vector

7. Plasmids Small circles of DNA –carry extra genes 2-30 genes genes for antibiotic resistance Plasmids

8. AP Biology How can plasmids help us?  A way to get genes into bacteria easily  insert new gene into plasmid  insert plasmid into bacteria  bacteria now expresses new gene  bacteria make new protein + transformed bacteria gene from other organism plasmid cut DNA recombinant plasmid vector glue DNA

9. Plasmid of Interest Plasmid of interest Genes in Plasmid: Amp r- Gene for ampicillin (an antibiotic) resistance araC- Gene for araC protein which regulates GFP transcription GFP- Gene for green fluorescent protein pGLO ori Amp r GFP araC

10. The Transformation Process

11. preparing competent cells for transformation Lipid bilayer (inner) Lipid bilayer (outer) Peptidoglycan layer Adhesion zone Calcium ions

12. transforming Escherichia coli with pGLO Recombinant Plasmids Competent Cells pGLO

13. Calcium ions pGLO transforming Escherichia coli with pGLO Adhesion zone Lipid bilayer (inner) Lipid bilayer (outer) Peptidoglycan layer

14. GFP expression araC gene GFP Gene P BAD Transcription mRNA Translation araC protein

15. GFP expression P BAD araC protein araC gene araC protein prevents GFP transcription by causing a loop to form in the region of the gfp gene GFP Gene

16. GFP expression araC protein arabinose araC geneP BAD arabinose – araC protein complex RNA polymerase Arabinose – araC protein complex prevents DNA looping and helps to align RNA polymerase on the promoter site (P BAD ). mRNA Transcription Translation GFP (green fluorescent protein) GFP Gene

17. Work on pGLO Reading