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pGLO Transformation LAB AP BIO LAB 6

araC. ori. pGLO. GFP. bla. pGLO Transformation LAB AP BIO LAB 6. BIO-RAD lab book. http://www.mshri.on.ca/nagy/GFP%20mice.jpg. Big Idea 3: Living systems store, retrieve, transmit and respond to information essential to life processes.

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pGLO Transformation LAB AP BIO LAB 6

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  1. araC ori pGLO GFP bla pGLO Transformation LABAP BIO LAB 6 BIO-RAD lab book http://www.mshri.on.ca/nagy/GFP%20mice.jpg

  2. Big Idea 3: Living systems store, retrieve, transmit and respond to information essential to life processes. Enduring understanding 3.A: Heritable information provides for continuity of life. Essential knowledge 3.A.1: DNA, and in some cases RNA, is the primary source of heritable information e. Genetic engineering techniques can manipulate the heritable information of DNA and, in special cases, RNA.  To foster student understanding of this concept, instructors can choose an illustrative example such as:  • Electrophoresis  • Plasmid-based transformation • Restriction enzyme analysis of DNA  • Polymerase Chain Reaction (PCR) f. Illustrative examples of products of genetic engineering include:  • Genetically modified foods  • Transgenic animals  • Cloned animals  • Pharmaceuticals, such as human insulin or factor

  3. PLASMID Extrachromosomal DNA Often carry genes for antibiotic resistance Can 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

  4. Image from: BIO-RAD lab book - GFP = Green fluorescent protein taken from jellyfish (Aequorea victoria) glows green in UV- light - ori = Origin of replication allows plasmid to replicate itself - araC = codes for arabinase = enzyme to break down arabinose sugar Turns on when arabinose sugar is present (INDUCIBLE) - bla On all time makes enzyme beta-lactamase that breaks down ampicillin provides antibiotic resistance (=ampR) pGLO plasmidContains genes for:

  5. Image from: BIO-RAD lab book • genetically engineered plasmid • used in biotechnology as a vector creating genetically modified organisms. • contains several “reporter“ genes green fluorescent protein (GFP) – glows under UV light ampicillin resistance gene (ampR) – allows growth on media containing ampicillin • produces an observable phenotype to help identify cells that contain the plasmid (AND ANY OTHER GENES THAT SCIENTISTS ATTACH TO THE PLASMID!) pGLO plasmid

  6. Aequorea victoria: Source of “glowing gene” for this experiment

  7. Jellyfish Gene put into Other Critters http://www.lafuga.de/GFP_pig.jpg http://www.technologyreview.com/files/21291/monkey_x600.jpg

  8. Bacterial Transformation Uptake of DNA from environmentChanges phenotype Image modified from BIORAD pGLO lab manual

  9. pGLO LAB SUPPLIES • FOAM tube holder/float • 1- colored eraser (to ID your tubes in water bath) • 4 - flip top microtubes Blue- Transforming solution (CaCl2) Yellow- LB nutrient broth Pink- label - Purple- label + • 1-pkg yellow innoculating loops • 2- Sterile pipettes • 4 poured agar plates 1 - LB 2 - LB/amp 1- LB/amp/ara • PERMANENT MARKER • Tri-pour beaker with crushed ice

  10. PLATE ABBREVIAIONS LB (LURIA & BERTANI) BROTH • contains nutrients for bacterial growth AMPICILLIN (amp) • Antibiotic that kills bacteria ARABINOSE (ara) = sugar used for food if glucose is unavailable

  11. LABEL TUBES purple = +pGLO pink = -pGLO Images from BIORAD pGLO lab manual

  12. Use sterile pipette to add 250µL transformation solution to pGLO + and – tubes Transformationsolution (CaCl2) Images from BIORAD pGLO lab manual

  13. Get your TUBES on ICE! Images from BIORAD pGLO lab manual

  14. Images from BIORAD pGLO lab manual INNOCULATE TUBES WITH E. coli BACTERIA Using more colonies canDECREASE transformationefficiency! Gently pick up 2-4 “fat” colonies with inoculating loop Twirl loop in + pGLO tube Use a new loop and repeat.Twirl loop in – pGLO tube. USE SPECIAL GARBAGE BAG FORDISPOSAL OF USED SUPPLIES FOR THIS LAB

  15. EXAMINE pGLO plasmid DNA • Use UV light to examine pGLO plasmid vialDOES IT GLOW? • UV light can be harmful to your eyes! • GFP =Green Fluorescent Protein isolated from jellyfish USED AS A GENETIC TOOL http://www.mshri.on.ca/nagy/GFP%20mice.jpg

  16. 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 + TUBE DO NOT ADD PLASMID TO - TUBE Images from BIORAD pGLO lab manual

  17. Put rack on ICE for 10 MIN! Images from BIORAD pGLO lab manual

  18. WHILE TUBES COOL CHECK LABELS ON PLATES LB (Luria and Bertani) – broth & agar provides nutrients for bacterial growth LB/amp Nutrients + ampicillin (antibiotic) LB/amp/ara Nutrients + ampicillin + arabinose sugar + plasmid will be added - NO plasmid added Images from BIORAD pGLO lab manual

  19. MAKING CELLS COMPETENT“COMPETENT” cells have the ability to pick up plasmids TRANSFORMATION SOLUTION (CaCl2) • Positive charge of Ca++ ions neutralizes: ~ negative charge of DNA phosphates ~ negative charge of membrane phospholipids HEAT SHOCK • Increases permeability of cell membranes so plasmid can enter Image modified from BIORAD pGLO lab manual

  20. SHOCKING INCREASES UPTAKE OF FOREIGN DNA (PLASMID) • OSMOTIC SHOCK = Transformation solution CaCl2 • HEAT SHOCK increases the permeability of the cell membrane to DNA RAPID TEMPERATURE CHANGE is the key 2 MINUTES 50 SECONDS Images from BIORAD pGLO lab manual

  21. NOT ALL BACTERIA WILL PICK UP THE PLASMID Bacteria that pick up the plasmid are said to be “COMPETENT”

  22. Images from BIORAD pGLO lab manual • Place foam rack with + and – tubes on desktop • Use new sterile pipette to add 250 µL LB broth to + tube • Use new sterile pipette to add 250 µL LB broth to – tube • Incubate at ROOM TEMPERATURE for 10 min

  23. TAP TUBE WITH FINGER TO MIX! Use NEW STERILEpipette for each vial to add 100 uL bacterial suspension to CORRECT DISH (CHECK LABELS!) Use a NEW STERILELOOP FOR EACH PLATE to spread suspension evenly on surface of plate DON’T DIG INTO AGAR! QUICKLY REPLACE LIDS Images from BIORAD pGLO lab manual

  24. HOW TO INNOCULATE A PLATE Images from BIORAD pGLO lab manual http://www.cdn.sciencebuddies.org/Files/620/7/MicroBio_img_006.jpg

  25. FLIP PLATES UPSIDE DOWNSTACK AND TAPE LABEL WITH YOUR GROUP NAMEPLACE IN INCUBATOR Images from BIORAD pGLO lab manual

  26. MAKE A PREDICTION WHAT WILL HAPPEN?

  27. Slide from Kim Fogliahttp://explorebiology.com Selection for plasmid uptake • Antibiotic becomes a selecting agent • only bacteria with the plasmid will grow on antibiotic (ampicillin) plate only transformedbacteria grow all bacteria grow a a a a a a a a a a a a a a a a a LB plate LB/amp plate cloning

  28. +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

  29. Transformation Results LB PLATELuria Broth + - PGLO = NO Plasmid → All cells grow since there is no antibiotic on the plate

  30. Transformation Results LB/AMP PLATELuria Broth with antibiotic + - PGLO = NO plasmid → NO GROWTHCells without plasmid don’t have antibiotic resistance. Can’t grow on media with antibiotic added.

  31. Transformation Results LB/AMP PLATELuria Broth with antibiotic + + PGLO = Plasmid added → LAWNCells with plasmid have antibiotic resistance gene so can grow on media with antibiotic

  32. Transformation Results Cells with pGLO plasmid GROW & GLOW-can grow on media with antibiotic ONLY GLOW on media with arabinose (turns on GFP gene) LB/AMP/ARA PLATELuria Broth+ antibiotic|+ arabinose + + PGLO = Plasmid added →

  33. Slide from Kim Fogliahttp://explorebiology.com RNA polymerase repressor repressor promoter ACTIVE repressor protein operator Inducible operon: lactose Digestive pathway model GLUCOSE is food of choice Don’t need lactose digesting enzymes Gene is turned off gene1 gene2 gene3 gene4 TATA DNA Slide from Kim Foglia http://explorebiology.com

  34. RNA polymerase repressor repressor repressor enzyme1 1 enzyme2 2 enzyme3 3 enzyme4 4 promoter repressor protein operator lactose lac lac lac lac lac lac lac lactose – repressor protein complex Slide from Kim Fogliahttp://explorebiology.com Inducible operon: lactose Digestive pathway model When lactose is present, binds to lac repressor protein & triggers repressor to release DNA • induces transcription lac gene1 gene2 gene3 gene4 TATA DNA mRNA lac conformational change in repressor protein makes it INACTIVE! lac

  35. Slide from Kim Fogliahttp://explorebiology.com 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

  36. Slide from Kim Fogliahttp://explorebiology.com RNA polymerase promoter operator gene1 gene2 gene3 gene4 TATA DNA

  37. Slide from Kim Fogliahttp://explorebiology.com ARABINOSE OPERON REGULATION = INDUCIBLE OPERON PRESENCE OF ARABINOSE TURNS ON GENES WHICH MAKE ENZYMESTO DIGEST ARABINOSE (along with pGLO gene) Adding ARABINOSE to media makes bacteria GLOW

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