Welcome!to the “Modern Lab”

sectionGraduate students: Francis Raycroft,

Aprell Carr, & Aranda Slabbekoorn

Why are you running a modern lab experiment?

This is a treat to yourself as a scientist!!!

Cloning a Fluorescent Gene

Detailed lab manual (Peyer Laboratory Systems, LLC)

Week-long lab

Work in pairs

WHAT YOU WILL DO

PCR, bacterial transformation, protein expression…

Will clone the protein GFP into E.coli cells

http://www.nature.comhttp://www.animalpicturesarchive.com

http://3.bp.blogspot.com

Aequoria victoria jellyfish

•Dr. Shimomura in 1961•Green Fluorescent Protein “GFP”•GFP successfully expressed in E.coli in 1994

Cloning GFPCharacterized the GFP protein

Sequenced the protein’s DNA

Cloned GFP DNA Attach it to any other protein in the cell that one

wants to studyWhatever you want to study is now visibly

fluorescing and can be seen under a microscopeTHE MICROSCOPIC WORLD IS OPENED TO OUR

EYES!

http://www.olympusfluoview.com/applications/images/fpcolorpalettefigure2.jpg

http://content.answers.com/main/content/img/McGrawHill/Encyclopedia/images/CE428300FG0010.gif http://www.bioelcomind.de/gallery/MDCK_cell.jpg

Significance & Application

Light Microscopy GFP-proteins using fluorescence microscopy

Significance & Application

www.ucl.ac.uk/.../research/neuroanatomy.php

www.exploratorium.edu/imaging_station/gallery...

2 Days : GFP labeling the circulatory system

4 Days : GFP labeling proteins in nuerons (central nervous system)

Day1: Replicate the DNAPolymerase Chain Reaction (PCR)

•Takes a piece of DNA and duplicates it over and over

•Couple examples of importance:

• Forensics: a very tiny DNA sample can be amplified by PCR. There is then more DNA to work with and run different tests on.

• Biochemistry: can use cloned DNA to transform and express protein in large amounts. Circumvents having to use live tissue samples.

Day2: LigationInserting your DNA into a plasmid

cellDNA

GFP (protein)

How?

How?

Day2: LigationInserting your DNA into a plasmid

cellDNA

GFP (protein)

The answer is a vector !

Day2: LigationInserting your DNA into a plasmid

Piece of DNA…

…in a circle

vector

Day2: LigationInserting your DNA into a plasmid

vector

GFP DNA

Need to insert GFP DNA into vector DNA

Day2: LigationInserting your DNA into a plasmid

vector

GFP DNA

Restriction Enzyme

cut

cut

Day2: LigationInserting your DNA into a plasmid

G A A T T C

C T T A A G

Day2: LigationInserting your DNA into a plasmid

vector

insert“Ligation”

Day3: TransformationInserting your GFP-plasmid into a bacterial cell

cell

Vector + GFP DNA

GFP (protein)

plasmid

42oCelsiusHeat shock

Bacterial chromosome

Bacterium

Day3: TransformationInserting your GFP-plasmid into a bacterial cell

Day3: TransformationInserting your GFP-plasmid into a bacterial cell

Basic plasmid

Selection marker

Origin of replication

Signal to read DNA

Resistance gene

Day3: TransformationInserting your GFP-plasmid into a bacterial cell

Basic plasmid

Selection marker

Origin of replication

= DNA polymerase

•DNA polymerase recognizes the origin of replication

•Begins to replicate the plasmid (plus your inserted GFP sequence!)

Day3: TransformationInserting your GFP-plasmid into a bacterial cell

Result is a cell with multiple copies of the plasmid carrying your GFP DNA insert

Day3: TransformationInserting your GFP-plasmid into a bacterial cell

Petri dish with agar

Streak onto agar plate

Day3: TransformationInserting your GFP-plasmid into a bacterial cell

Selection marker

Food source + ampicillan

Day3: TransformationInserting your GFP-plasmid into a bacterial cell

Selection marker

•Drug in the agar•Antibiotic resistance in the plasmid•All cells without the plasmid die•All cells that took up your GFP-plasmid will survive

Food source + ampicillan

Day4: ExpressionTranscribing DNA RNATranslating RNA green fluorescent protein

Peyer movie

Day5: Fluorescence

cell