Transcription and Translation (How a Gene Works) Alison Kraigsley January 18 th, 2011.

Transcription and Translation (How a Gene Works)

Alison Kraigsley

January 18th, 2011

Overview

1. Brief Introduction

2. My background

3. Research at NIST

4. Today’s experiment

Overview

1. Brief Introduction

2. My background

3. Research at NIST

4. Today’s experiment

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

Overview

1. Brief Introduction

2. My background

3. Research at NIST

4. Today’s experiment

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

PhD Work:Biofilm Life Cycle

Modified from O’Toole et al., 2000

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

0

2

4

6

8

10

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

1

10

100

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10000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Trial #

Bio

film

In

de

x

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22

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22P

PB

BRatio

Overview

1. Brief Introduction

2. My background

3. Research at NIST

4. Today’s experiment

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

Live/Dead- Confocal 24hr

UV treated

Overview

1. Brief Introduction

2. My background

3. Research at NIST

4. Today’s experiment

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

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