90- How can we make more insulin? V.1.2.2
How can we make more insulin? By Transforming Bacteria V.1.2.2
“transformed cell” – cell has acquired new characteristics “characteristics” – due to the expression of incorporated foreign genetic material
AP Biology Green with envy?? Jelly fish “GFP” Transformed vertebrates
AP Biology Genetic engineering… grow bacteria harvest (purify) protein transformed bacteria plasmid gene from other organism + recombinant plasmid vector
Plasmids Small circles of DNA –carry extra genes 2-30 genes genes for antibiotic resistance Plasmids
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
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
The Transformation Process
preparing competent cells for transformation Lipid bilayer (inner) Lipid bilayer (outer) Peptidoglycan layer Adhesion zone Calcium ions
transforming Escherichia coli with pGLO Recombinant Plasmids Competent Cells pGLO
Calcium ions pGLO transforming Escherichia coli with pGLO Adhesion zone Lipid bilayer (inner) Lipid bilayer (outer) Peptidoglycan layer
GFP expression araC gene GFP Gene P BAD Transcription mRNA Translation araC protein
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
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
Work on pGLO Reading