1. GFP Transformation Lab
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2. Overall Goal of Lab Experiment
Use genetic engineering techniques to insert the GFP gene into E. coli
Plasmid containing gene of interest
Protein we want to produce

3. GFP (Green Fluorescent Protein)
Naturally produced in Jellyfish– Aequorea victoria
Discovered in 1960’s
Source of bioluminescence when exposed to UV light
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4. Structure of the GFP Protein
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5. Why Is Bioluminescence Useful in Nature?
Attract Mates
See Food
Defense
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6. How GFP is being used Tag Cells (to detect specific cells)
Act as a reporter gene
- link it to another gene to show if it is expressed
Expressed in entire animals
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Top right– cells that have been tagged with GFP; allows researchers to distinguish certain cells
Bottom right- transgenic mice; mouse embryos were injected with GFP gene then implanted into a surrogate mouse. When born, the mice should express the GFP gene. The lab that works with these mice is trying to use these mice to help them study human disease
Bottom left- Alba the GFP bunny; partnership with an artist and a French lab. Supposed to be part of living art exhibit but lab decided at the last minute to keep the bunny in the lab.
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7. Nobel Prize 2008
In 2008, 3 scientists were awarded the 2008 Nobel Prize in Chemistry for their work on GFP

8. pGLO plasmid The plasmid we’re using in the lab 3 genes of interest:
GFP gene
Codes for the GFP protein
Bla gene
Codes for the enzyme b-lactamase
b-lactamase destroys the antibiotic ampicillin
araC gene
Codes for the araC protein
Ori = origin of replication. Students don’t have to worry about this.

9. Arabinose operon
The arabinose operon in bacteria consists of the following:
Usually, the araC protein binds to the arabinose operon operator  prevents transcription
When arabinose is present, it binds to the araC protein -> can’t bind to operator  RNA polymerase can continue

10. Modified arabinose operon
Scientists modified the arabinose operon in the pGLO plasmid to express the GFP gene.
araC protein binds to the operator  prevents transcription
When arabinose binds to araC it can no longer bind to operator  GFP gene is transcribed and translated

11. Selecting for Transformed Cells
Selection = process to determine which E. coli successfully took in a plasmid
Achieved through the use of selectable markers
Selectable markers = traits that help identify a cell with the plasmid in it (compared to one without it)
In our experiment, the bla gene will serve as the selectable marker
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12. Review Question… What protein does the bla gene code for?
b-lactamase protein
What does this protein do?
Digests the antibiotic ampicillin
How could the bla gene serve as a selectable marker?
Only cells with the pGLO plasmid will make b-lactamase  are resistant to ampicillin

13. Growing E. coli Bacteria is grown on LB agar
LB agar contains all of the nutrients and amino acids E. coli need to survive
Other substances such as antibiotics can also be added to the LB agar.

14. Selection Process All E. coli grow (transformed and untransformed)
E. coli cells that have gone through the tranformation process
grown on plain LB agar
grown on LB agar with ampicillin
All E. coli grow (transformed and untransformed)
Only transformed cells grow
Need to add controls slide

15. Controls Grow E. coli without plasmid (- DNA) on plain LB agar
Make sure E. coli can grow
Grow E. coli without plasmid (- DNA) on LB/amp
Make sure E. coli aren’t already resistant to ampicillin
Grow E. coli with plasmid (+ DNA) on LB/amp
Make sure transformation didn’t kill E. coli

16. Transformation Efficiency
Quantitative measurement of how well the transformation worked.
Measures the number of bacteria transformed by 1 mg of plasmid DNA.
= total # of cells growing
amount of DNA spread on plate