GMO LAB1,) GENETIC MODIFICATIONSSelection- Selection of phenotypes alters genomes- Both natural and artificial selection are in this categoryDeliberate alterations- Mutagenesis- Polyploidy/Aneuploidy- HybridsEngineering- Direct introduction or alteration of DNA sequences2.) BACTERIAL TRANSFORMATION- Uptake and expression of foreign DNA- Usually a plasmid in bacteriaStarting materialsE.coli cells - Inhibited by antibiotics- Can’t grow and DO NOT GLOW- Competent: able to be transformedPlasmid- Has ampicillin resistance gene (always expressed)- Arabinose promoter (turned on in the presence of arabinose)GFP - Green fluorescent protein- Glows under UV light3.) PLASMID- Has AmpR, Ara and GFP- Recombinant bacteria - Can grown in the presence of antibiotic- And can glow in the presence of UV light- AmpR: makes all transformed bacteria to ampicillin. Is the BLA (beta lactamase enzyme gene)- GFP: codes for fluorescent protein- Ara: promoter that controls GFP gene expression, only turned on in the presence of arabinose4.) METHODS OF TRANSFORMATION- Electroporationo Electrical shock makes cell membranes permeable to DNA- Calcium Chloride/Heat Shocko Chemically competent cells uptake DNA after heat shock5.) TRANSFORMATION PROCEDURE- Suspend bacterial colonies in Ca2+ transformation solution- Add pGLO plasmid DNA- Place tubes on ice- Heat shock at 42C and place on ice- Incubate with nutrient broth- Streak plates6.) REASONS FOR EACH STEPTransformation solution is CaCl2- Positive charge of Ca2+ ions shields negative charges of DNA phosphates- Charged molecules are very hard to get across a membraneIncubate on ice- Slows fluid cell membraneHeat Shock- Increases permeability by altering membrane potential- By placing it on ice afterward, increases permeability of membranes through shedding of lipids and proteins (makes pores)Nutrient Broth Incubation- Allows time for beta lactamase expression before exposure to antibiotic7.) WHY GM CROPSGrowing population, loss of farm land, soil remediation, enrich nutrient food value, decrease losses due to pests, herbicide tolerance, viral resistance, drought resistance, increased nutritional value, altered ripening, pest resistance8.) POSSIBLE RISKS OF GM FOODS- Insects might develop resistance to pesticide producing GM crops- Herbicide tolerant crops may cross pollinate weeds, resulting in super weeds- Gene products may be allergens- Unintended effects on wildlife and beneficial insects- Promotion of monoculture increases vulnerability9.) PLANT GENETIC ENGINEERINGBuilding transgenes  plant transformation -> screening and selection  plant breeding  seed production and marketing10.) LAB SAMPLESSample 1: Control “Non-GMO” foodSample 2: “Test” foodSample 3: Control “GMO-positive” DNA11.) DUPLEX PCR- Tests for the presence of two different GMO associated sequences- 35S promoter of the cauliflower mosaic virus (CaMV 35S)- Terminator of the nopaline synthase (NOS) gene of Agrobacterium tumefaciens-If present, PCR amplifies these sequences12.) PRIMERSFirst Set of Primers- GMO Primerso Detects GMO-specific sequenceso Amplifies a 203 bp fragment of the cauliflower MV 35S promoter and A 225 bp fragment of the NOS terminator used in most GMO plantsSecond Set of Primers- Plant Primerso Detects plant-specific sequenceso Amplifies a 455 bp region of the PSII chloroplast gene found in most plants13.) GMO POSITIVE CONTROLS- Controls against false negatives- Indicates problems with PCR- Provides reference bands for test samples14.) GMO NEGATIVE CONTROLS- Controls against false positives (if non-GMO test is positive, you cannot trust the result of a positive result for unknown sample)Lane NumberContains1 Non GMO food with PLANT PRIMERS2 Non GMO food with GMO PRIMERS3 Test food with PLANT PRIMERS4 Test food with GMO PRIMERS5 GMO positive control with PLANT PRIMERS6 GMO positive control with GMO PRIMERS7 PCR MW rulerIf a food is GMO + it will contain the band for GMO primers seen in lane 6 (control) in lane 4 (test food with GMO)15.) 5 STEPS TO GENETICALLY MODIFY1. Identify protein of interest2. Isolate the gene that codes for the protein3. Engineer the gene so plant/bacterial cells can transcribe and translate the protein4. Introducing the gene into a plant cell/bacterium5. Grow the crop/bacteria with recombinant DNAPROTEIN PURIFICATION LAB1.) TRANSFORMATION EFFICIENCYDetermines how well the GFP transformation workedExample:- pGLO DNA solution = 0.08 ug/ul- 10 ul spread on LB/amp/ara agar plates with 0.8 ug/plate- Number of colonies = number of transformed cells- Transformation efficiency = total number of cells growing/ug pGLO DNA- If number of colonies = number of transformed cells = 500- Then 500/0.8 ug pGLO DNA = 6.25E2 transformants/ug2.) IMPORTANCE OF KNOWING THE EXTENT OF GENETIC TRANSFORMATIONThe more cells that are transformed to produce a desired protein, the more likely thetherapy will work3.) CELLULAR PROTEINS- Proteins are small- Cell has many diverse proteins- Isolating proteins involves two basic steps:o Concentration of proteins (separates from non-proteins)o Purification of an interesting protein (to separate interesting protein from unwanted boring proteins)4.) PROTOCOL TO ISOLATE AND PURIFY A PROTEINPhase 1: release the proteins- Concentrate the bacterial cells- Lyse the cellsPhase 2: separate the proteins- Remove non-protein stuffPhase 3: select the protein of interest- Purify the protein5.) WAYS TO SEPARATE MOLECULES- Adsorption- Centrifugation- Distillation- Electrophoresis- Evaporation- Extraction- Flotation- Filtration6.) COLUMN CHROMATOGRAPHY- Separation of proteins based on their PHYSICAL properties- This lab involves interactions between a GFP and hydrophobic beads which are packed in a tube as a matrix and thus making a column- Cell extract is then filtered through the column7.) LAB PROTOCOLStep 1: Concentrate bacteria- Centrifugation (FIRST)- Separation by density- Bacteria in suspension- Results in pellet of bacteria which is separated from suspended particles in solution- Test the pellet for appearance of GFPStep 2: Rupture Bacterial Cells- Resuspend bacterial pellet- Add lysozyme (an enzyme that degrades cell walls but cell membrane will stay intact (protoplast))- Freeze/thaw to induce lysis of cell membraneStep 3: Remove cellular debris- Second centrifugation - Pellet fragments of membranes, walls and cells because some proteins may be bound to membranes- Supernatant (water soluble proteins including GFP)Step 4: Column