transcription and translation (how a gene works) alison kraigsley january 18 th, 2011
TRANSCRIPT
From DNA to People
• DNA is your genetic blueprint• Too valuable to risk damaging• mRNA (messenger RNA) used to transfer
the genetic code into protein (specific trait)– DNA codes for hair colour– Protein is the actual hair with colour
DNA mRNA ProteinTranscription Translation
From DNA to People
• DNA is DNA = same for all living things• Genetic code is different
– 20,000-25,000 genes in humans (99.5% similar)– 32,000 -56,000 genes in rice (Oryza sativa) – 19,000 genes in earth worm (Caenorhabditis
elegans), – 25,000 gene in a plant (Arabidopsis thaliana )
DNA mRNA ProteinTranscription Translation
Green Fluorescent Protein (GFP)
• Revolutionized biology• GFP tagged genes, cells, proteins• Can tell where/when/how biology is
happening– But what is it exactly?
Green Fluorescent Protein (GFP)
• GFP comes from the Jellyfish Aequorea victoria.
• The gene was cloned (copied) and transferred to other organisms
• 2008 Nobel Prize in Chemistry
GFP Stem Cells
• Inner glow. Transplanted motor neurons (green) spread out from the spinal cord of an embryonic chick.
http://cmbi.bjmu.edu.cn/news/0208/44.htm, Wichterle et al., Directed Differentiation of Embryonic Stem Cells into Motor Neurons, Cell, 2002, 110, 385-397
GFP Reporter
• GFP reporter gene expression in central nervous system neurons that innervate the hindgut of Drosophila melanogaster
http://www.neuroscience.cam.ac.uk/directory/profile.php?gal26
GFP Reporter
• Spliced the right way, fru establishes a “courtship” circuit of neurons (green) in the male fly brain.
http://www.sciencemag.org/content/308/5727/1392.full
ME!!
• Education– B.Sc. Chemistry/Physic: Furman University, Greenville SC– M.S. Aerospace Engineering: University of Southern
California, Los Angeles CA– Ph.D. Molecular Biology: University of Southern California,
Los Angeles CA
• Research– M.S. : Polymers– Ph.D: Biofilms, evolution– NIST: Biofilm-material interactions
What about long term?
• What happens when a biofilm is present for long periods of time
• Can we observe evolutionary change in a biofilm?– Does some kind of GASP-like phenotype occur
in biofilms?
The GASP Phenotype
• 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.
• Growth Advantage in Stationary Phase.
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l o g C F U / m l129630
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1-day-old
10-day-old
a
**
Day
Log
CFU
/mL
Finkel and Kolter, 1999
The GASP PhenotypeThe GASP Phenotype
• Aged cells outcompete Aged cells outcompete younger, initially isogenic younger, initially isogenic cells when mixed.cells when mixed.
• Advantageous mutations Advantageous mutations are selected during are selected during incubation in stationary incubation in stationary phase.phase.
• To date, all experiments To date, all experiments performed on planktonic performed on planktonic cells or in stab cultures.cells or in stab cultures.
• GGrowth rowth AAdvantage in dvantage in SStationary tationary PPhase.hase.
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l o g C F U / m l129630
0
2
4
6
8
10
1-day-old
10-day-old
a
**
Day
Lo
g C
FU
/mL
Lo
g C
FU
/mL
Finkel and Kolter, Finkel and Kolter, 19991999
Biofilm GASP?
Competition-Invasion Assay
• Day 1= 12 of 23 trials significant in favour of 22-day-old cells
• Day 2= 21 of 24 trials significant in favour of 22-day-old cells• Box indicates titer error of 3 fold
0.1
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Trial #
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film
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22P
PB
BRatio
NIST Research
• How do biofilms respond to their substrate?
Modified from O’Toole et al., 2000
Does substrate matter?
Results: Decrease in Metabolic Activity at Low DC
•Decrease in metabolic activity between 4 and 24 hrs• Greater decrease at 24 hrs on low DC polymers• Unpublished dad
pGlo: GFP plasmid
• pGlo is a plasmid– Circular DNA– Can be transformed into bacteria– Independently replicating
• pGlo has Ampicillin Resistance• GFP on the plasmid is inducible by
arabinose
Transformation
• Def: inserting a plasmid into a bacterial cell• Two methods
– Heat Shock– Electroporation
• Mechanism unknown• Bacteria must have a reason to keep the
plasmids (ex. Drug resistance = benefit)
Genes at work
• pGlo DNA is NOT fluorescent• Only when the plasmid is transformed into
the bacteria can fluorescence be observed– Bacteria’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 ProteinTranscription Translation
Inducible Gene Expression
• When you want total control• Turn genes on or off with an external
control (ex. Arabinose)– Arabinose is a sugar
• GFP is under the control of a tightly regulated system on the plasmid. GFP will only be turned on when arabinose is present.
Procedure
• Walk through general procedure• The full manual has a lot of good
information and discussion points
Results
No growth Lawn of cellsAmpR = Cells have plasmid
Positive Control
NegativeControl
Have GFP gene, but not turned on
Plasmid sequence with GFP• 5-
AGATTGCAGCATTACACGTCTTGAGCGATTGTGTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCATTTAAATGGCGCGCCTTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTATTCATTAAGCATCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGTAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGACAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTAGGCGCGCCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTAAGGAGGATATTCATATGGTAAGTTACTGAAGAATTCGTTGACACTCTATCATTGATAGAGTTATTTTACCACTCCCCGGGTACCTAGAATTAAAGAGGAGAAATTAAGCGCTCATATGCGGAATTCGCTAGTTCTCATATGGACCATGGCTAATTCCCATGTCAGCCGTTAAGTGTTCCTGTGTCACTGAAAATTGCTTTGAGAGGCTCTAAGGGCTTCTCAGTGCGTTACATCCCTGGCTTGTTGTCCACAACCGTTAAACCTTAAAAGCTTTAAAAGCCTTATATATTCTTTTTTTTCTTATAAAACTTAAAACCTTAGAGGCTATTTAAGTTGCTGATTTATATTAATTTTATTGTTCAAACATGAGAGCTTAGTACGTGAAACATGAGAGCTTAGTACGTTAGCCATGAGAGCTTAGTACGTTAGCCATGAGGGTTTAGTTCGTTAAACATGAGAGCTTAGTACGTTAAACATGAGAGCTTAGTACGTGAAACATGAGAGCTTAGTACGTACTATCAACAGGTTGAACTGCGGATCTTGCGGCCGCAAAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGCATCGATGGCCCCCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTGGCCAGTGCCAAGCTTGCATGC