Presentation on theme: "Transcription and Translation (How a Gene Works)"— Presentation transcript:
1 Transcription and Translation (How a Gene Works) Alison KraigsleyJanuary 18th, 2011
2 Overview Brief Introduction My background Research at NIST Today’s experiment
3 Overview Brief Introduction My background Research at NIST Today’s experiment
4 From DNA to People DNA is your genetic blueprint Too valuable to risk damagingmRNA (messenger RNA) used to transfer the genetic code into protein (specific trait)DNA codes for hair colourProtein is the actual hair with colourDNA mRNA ProteinTranscriptionTranslation
5 From DNA to People DNA is DNA = same for all living things Genetic code is different20,000-25,000 genes in humans (99.5% similar)32, ,000 genes in rice (Oryza sativa)19,000 genes in earth worm (Caenorhabditis elegans),25,000 gene in a plant (Arabidopsis thaliana )DNA mRNA ProteinTranscriptionTranslation
6 Green Fluorescent Protein (GFP) Revolutionized biologyGFP tagged genes, cells, proteinsCan tell where/when/how biology is happeningBut what is it exactly?
7 Green Fluorescent Protein (GFP) GFP comes from the Jellyfish Aequorea victoria.The gene was cloned (copied) and transferred to other organisms2008 Nobel Prize in Chemistry
8 GFP Stem CellsInner glow. Transplanted motor neurons (green) spread out from the spinal cord of an embryonic chick.Wichterle et al., Directed Differentiation of Embryonic Stem Cells into Motor Neurons, Cell, 2002, 110,
9 GFP ReporterGFP reporter gene expression in central nervous system neurons that innervate the hindgut of Drosophila melanogaster
10 GFP ReporterSpliced the right way, fru establishes a “courtship” circuit of neurons (green) in the male fly brain.
11 Overview Brief Introduction My background Research at NIST Today’s experiment
12 ME!!EducationB.Sc. Chemistry/Physic: Furman University, Greenville SCM.S. Aerospace Engineering: University of Southern California, Los Angeles CAPh.D. Molecular Biology: University of Southern California, Los Angeles CAResearchM.S. : PolymersPh.D: Biofilms, evolutionNIST: Biofilm-material interactions
13 PhD Work: Biofilm Life Cycle Modified from O’Toole et al., 2000
14 What about long term?What happens when a biofilm is present for long periods of timeCan we observe evolutionary change in a biofilm?Does some kind of GASP-like phenotype occur in biofilms?
15 The GASP Phenotype Growth Advantage in Stationary Phase. Aged cells outcompete younger, initially isogenic cells when mixed.Advantageous mutations are selected during incubation in stationary phase.To date, all experiments performed on planktonic cells or in stab cultures.Log CFU/mLFinkel and Kolter, 1999
16 The GASP Phenotype Growth Advantage in Stationary Phase. Biofilm GASP? Aged cells outcompete younger, initially isogenic cells when mixed.Advantageous mutations are selected during incubation in stationary phase.To date, all experiments performed on planktonic cells or in stab cultures.Log CFU/mLBiofilm GASP?Finkel and Kolter, 1999
17 Competition-Invasion Assay Day 1= 12 of 23 trials significant in favour of 22-day-old cellsDay 2= 21 of 24 trials significant in favour of 22-day-old cellsBox indicates titer error of 3 fold
18 Overview Brief Introduction My background Research at NIST Today’s experiment
19 NIST Research How do biofilms respond to their substrate? Modified from O’Toole et al., 2000Does substrate matter?
20 Results: Decrease in Metabolic Activity at Low DC Decrease in metabolic activity between 4 and 24 hrsGreater decrease at 24 hrs on low DC polymersUnpublished dad
22 Overview Brief Introduction My background Research at NIST Today’s experiment
23 pGlo: GFP plasmid pGlo is a plasmid pGlo has Ampicillin Resistance Circular DNACan be transformed into bacteriaIndependently replicatingpGlo has Ampicillin ResistanceGFP on the plasmid is inducible by arabinose
24 Transformation Def: inserting a plasmid into a bacterial cell Two methodsHeat ShockElectroporationMechanism unknownBacteria must have a reason to keep the plasmids (ex. Drug resistance = benefit)
25 Genes at work pGlo DNA is NOT fluorescent Only when the plasmid is transformed into the bacteria can fluorescence be observedBacteria’s cellular machinery takes the DNA coding for GFP, makes mRNA, then the Green Fluorescent Protein.The GENE is NOT fluorescent, the PROTEIN IS fluorescent.DNA mRNA ProteinTranscriptionTranslation
26 Inducible Gene Expression When you want total controlTurn genes on or off with an external control (ex. Arabinose)Arabinose is a sugarGFP is under the control of a tightly regulated system on the plasmid. GFP will only be turned on when arabinose is present.
27 Procedure Walk through general procedure The full manual has a lot of good information and discussion points