Welcome! to the “Modern Lab” section Graduate 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

Aequoria victoria jellyfish Dr. Shimomura in 1961 Green Fluorescent Protein “GFP” GFP successfully expressed in E.coli in 1994 http://3.bp.blogspot.com http://www.animalpicturesarchive.com http://www.nature.com

Cloning GFP Characterized the GFP protein Sequenced the protein’s DNA Cloned GFP DNA Attach it to any other protein in the cell that one wants to study Whatever you want to study is now visibly fluorescing and can be seen under a microscope THE MICROSCOPIC WORLD IS OPENED TO OUR EYES! http://www.olympusfluoview.com/applications/images/fpcolorpalettefigure2.jpg

Significance & Application Light Microscopy GFP-proteins using fluorescence microscopy http://content.answers.com/main/content/img/McGrawHill/Encyclopedia/images/CE428300FG0010.gif http://www.bioelcomind.de/gallery/MDCK_cell.jpg

Significance & Application 2 Days : GFP labeling the circulatory system 4 Days : GFP labeling proteins in nuerons (central nervous system) www.ucl.ac.uk/.../research/neuroanatomy.php www.exploratorium.edu/imaging_station/gallery...

Day1: Replicate the DNA Polymerase 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: Ligation Inserting your DNA into a plasmid cell How? How? GFP (protein)

Day2: Ligation Inserting your DNA into a plasmid cell The answer is a vector ! GFP (protein)

Day2: Ligation Inserting your DNA into a plasmid Piece of DNA… …in a circle vector

Day2: Ligation Inserting your DNA into a plasmid GFP DNA Need to insert GFP DNA into vector DNA vector

Day2: Ligation Inserting your DNA into a plasmid GFP DNA cut cut vector Restriction Enzyme

Day2: Ligation Inserting your DNA into a plasmid G A A T T C C T T A A G

Day2: Ligation Inserting your DNA into a plasmid vector

Day3: Transformation Inserting your GFP-plasmid into a bacterial cell Vector + GFP DNA GFP (protein)

Day3: Transformation Inserting your GFP-plasmid into a bacterial cell 42oCelsius Heat shock Bacterium Bacterial chromosome

Day3: Transformation Inserting your GFP-plasmid into a bacterial cell Origin of replication Basic plasmid Signal to read DNA Selection marker Resistance gene

Day3: Transformation Inserting your GFP-plasmid into a bacterial cell = DNA polymerase Origin of replication DNA polymerase recognizes the origin of replication Begins to replicate the plasmid (plus your inserted GFP sequence!) Basic plasmid Selection marker

Day3: Transformation Inserting your GFP-plasmid into a bacterial cell Result is a cell with multiple copies of the plasmid carrying your GFP DNA insert

Day3: Transformation Inserting your GFP-plasmid into a bacterial cell Streak onto agar plate Petri dish with agar

Day3: Transformation Inserting your GFP-plasmid into a bacterial cell Food source + ampicillan Selection marker

Day3: Transformation Inserting your GFP-plasmid into a bacterial cell Food source + ampicillan 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 Selection marker

Day4: Expression Transcribing DNA  RNA Translating RNA  green fluorescent protein Peyer movie

Day5: Fluorescence cell