PGLO Transformation LAB AP LAB 7Brookings …：pGLO转化实验室AP实验室7 布鲁金斯… .ppt

pGLO Transformation LABAP LAB 6,http:/www.mshri.on.ca/nagy/GFP%20mice.jpg,BIO-RAD lab book,Aequorea victoria:Source of“glowing gene”for this experiment,Jellyfish Gene put into Other Critters,http:/,http:/www.lafuga.de/GFP_pig.jpg,PLASMIDExtrachromosomal DNAOften carry genes for antibiotic resistanceCan be passed from one bacterium to another,http:/www.agen.ufl.edu/owens/age2062/OnLineBiology/OLBB/www.emc.maricopa.edu/faculty/farabee/BIOBK/14_1.jpg,Bacterial Transformation,The uptake of DNA,pGLO LAB SUPPLIES,FOAM tube holder/float4-flip top microtubes Blue-Transforming solution(CaCl2)Yellow-LB nutrient broth Pink-label+Purple-label-1-colored eraser(to ID your tubes in water bath)1-pkg yellow innoculating loops2-Sterile pipettes4 poured agar plates 1-LB 2-LB/amp 1-LB/amp/araPERMANENT MARKERCup with crushed ice,LABEL TUBES,Purple=+pGLO pink=-pGLO,Use sterile pipette toadd 250L transformationsolution to pGLO+and tubes,Transformationsolution(CaCl2),Get your rack on ICE!,INNOCULATE TUBES WITH E.coli BACTERIA,Pick one colonyTwirl loop in+pGLO tubeGet new loopPick one colonyTwirl loop in pGLO tube,USE SPECIAL GARBAGE BAG FORDISPOSAL OF USED LOOPS,EXAMINE pGLO plasmid DNA,Use UV light to examine pGLO plasmid vialUV light can be harmful to your eyes!Wear your goggles.Do not shine in eyes.GFP=Green Fluorescent Protein isolated from jellyfishUSED AS A GENETIC TOOL,http:/www.mshri.on.ca/nagy/GFP%20mice.jpg,PLASMID DNA TRANSFER,THIS STEP IS CRUCIAL!Look closely to make sure you have a film of solution across the ring.(Similar to soapy film when you blow bubbles),ADD PLASMID TO+TUBEDO NOT ADD PLASMID TO-TUBE,Put rack on ICE for 10 MIN!,WHILE YOUR TUBES COOLLABEL YOUR PLATES,FLIP UPSIDE DOWN AND WRITE LABELS ON BOTTOM NOT ON TOP!,LB(Luria and Bertani)broth&agar provides nutrients for bacterial growthLB/amp Luria agar+ampicillin(antibiotic)LB/amp/ara Luria agar+ampicillin+arabinose sugar,SHOCKING INCREASES UPTAKE OF FOREIGN DNA(PLASMID)OSMOTIC SHOCK=Transforming solutionCaCl2HEAT SHOCK RAPID TEMPERATURE CHANGE is the key,50 SECONDS,2 MINUTES,Place foam rack with+and tubes on desktopUse new sterile pipette to add 250 L Luria broth to+tubeUse new sterile pipette to add 250 L Luria broth to tube Incubate a ROOM TEMPERATURE 10 min,TAP WITH FINGER TO MIX!Use NEW STERILEpipette for each vial to add 100 uL bacterial suspensionto CORRECT DISH(CHECK LABELS!)Use a NEW STERILELOOP FOR EACH PLATEto spread suspension evenly on surface of plateDO NOT DIG INTO AGAR!QUICKLY REPLACE LIDS,FLIP PLATES UPSIDE DOWNSTACK AND TAPE LABEL WITH YOUR GROUP NAMEPLACE IN INCUBATOR,pGLO plasmid,bla(beta-lactamase)-On all time-Makes protein that breaks down ampicillin-Provides ampicillin resistance,GFP-Green Fluorescent Protein-Glows green in fluorescent light,ARABINOSE OPERON(INDUCIBLE)Turns on when arabinose sugar is presentAllows bacteria to digest this sugar,Ori-PlasmidReplicationgenes,Inducible operon:lactose,DNA,TATA,gene1,gene2,gene3,gene4,Digestive pathway model GLUCOSE is food of choiceDont need lactose digesting enzymesGene is turned off,Slide from Kim Foglia,mRNA,Inducible operon:lactose,DNA,TATA,lac,gene1,gene2,gene3,gene4,Digestive pathway model When lactose is present,binds to lac repressor protein&triggers repressor to release DNAinduces transcription,conformational change in repressor protein makes itINACTIVE!,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,DNA,TATA,gene1,gene2,gene3,gene4,Slide from Kim Foglia,ARABINOSE OPERON REGULATION,=INDUCIBLE OPERONPRESENCE 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,Incubation on ice slows fluidity cell membranesHeat-shock increases permeability of cell membraneNutrient broth incubation allows beta lactamase expression,Reasons for Each Transformation Step,Selection for plasmid uptake,Antibiotic becomes a selecting agentonly 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,Transformation Results,All cells grow since there is no antibiotic on the plate,LB PLATELuria Broth+-PGLO=NO Plasmid,Transformation Results,NO GROWTHCells without plasmid dont have antibiotic resistance.Cant grow on media with antibiotic added.,LB/AMP PLATELuria Broth with antibiotic+-PGLO=NO plasmid,Transformation Results,LAWNCells with plasmid have antibiotic resistance gene so can grow on media with antibiotic,LB/AMP PLATELuria Broth with antibiotic+PGLO=Plasmid added,Transformation Results,Cells with pGLO plasmid GROW&GLOW-can grow on media with antibioticGLOW on media with arabinose(turns on GFP gene),LB/AMP/ARA PLATELuria Broth+antibiotic|+arabinose+PGLO=Plasmid added,+pGLOLB/amp,+pGLOLB/amp/ara,-pGLOLB/amp,-pGLO LB,http:/faculty.clintoncc.suny.edu/faculty/michael.gregory/files/Bio%20101/Bio%20101%20Laboratory/Bacterial%20Transformation/results.htm,