PGLO Transformation LAB AP LAB 7 Lancaster High 改造实验室7实验室pGLO AP兰开斯特高.pptVIP

* * * * * * * * * * operator promoter Inducible operon: lactose DNA TATA repressor ACTIVE repressor protein repressor gene1 gene2 gene3 gene4 Digestive pathway model GLUCOSE is food of choice Don’t need lactose digesting enzymes Gene is turned off RNA polymerase Slide from Kim Foglia mRNA enzyme1 enzyme2 enzyme3 enzyme4 operator promoter Inducible operon: lactose DNA TATA RNA polymerase repressor repressor protein repressor lactose – repressor protein complex lactose lac repressor gene1 gene2 gene3 gene4 Digestive pathway model When lactose is present, binds to lac repressor protein triggers repressor to release DNA induces transcription 1 2 3 4 lac lac lac lac lac lac lac conformational change in repressor protein makes it INACTIVE! lac lac Slide from Kim Foglia Lactose operon What happens when lactose is present? Need to make lactose-digesting enzymes Lactose is allosteric regulator of repressor protein Slide from Kim Foglia ARABINOSE OPERON REGULATION = INDUCIBLE OPERON PRESENCE OF ARABINOSE TURNS ON GENES THAT MAKE ENZYMESTO DIGEST ARABINOSE (along with pGLO gene) Adding ARABINOSE to media makes bacteria GLOW Reasons for Each Transformation Step CaCl2 treatment Positive charge of Ca+2 ions neutralizes: negative charge of DNA phosphates negative charge of membrane phospholipids Ca++ Ca++ O CH2 O P O O O Base CH2 O P O O O Base OH Sugar Sugar O Ca++ Incubation on ice slows fluidity cell membranes Heat-shock increases permeability of cell membrane Nutrient broth incubation allows beta lactamase expression Reasons for Each Transformation Step Selection for plasmid uptake Antibiotic becomes a selecting agent only bacteria with the plasmid will grow on antibiotic (ampicillin) plate LB/amp plate LB plate all bacteria grow only transformed bacteria grow a a a a a a a a a a a a a a a cloning a a Bacterial Transformation Plasmids Chromosomal DNA Bacterial Cell The uptake of DNA pGLO LAB SUPPLIES FOAM tube holder/float 4 - flip top m