The document summarizes the process of transforming E. coli bacteria to produce fluorescent proteins. It will be done by inserting a plasmid containing a gene for a fluorescent protein into E. coli. The plasmid also contains an antibiotic resistance gene so that transformed bacteria can be selected by growing them in media containing antibiotics. The foreign DNA from the plasmid will be transcribed and translated within the bacterial cells, causing them to produce and glow with the fluorescent protein. This demonstrates how altering an organism's genotype through molecular biology techniques can change its phenotype.

1. Warmup
Draw or explain in words in the simplest way you can the
process of Protein synthesis (aka Central Dogma aka
Transcription/Translation)
Think of an awesome trait you would want to have. For
example, synesthesia, being taller, curly hair etc. Explain
how would you use the process of protein synthesis to get
that trait?
For Quanina’s Graduate Studies: Have you taken a
comprehensive health course in your educational career?
If so, what grade were you in and what did it cover (ex:
sex ed, family planning, etc. )? What school were you at
when you took this course?

2. Bacterial Transformation
Student Training
By Quanina Quan and UCSD ScienceBridge

3. Objective
Demonstrate that changes in genotype causes changes
in phenotype by transforming E.coli into fluorescent
E.coli.

4. The Purpose of the Labs
The purpose of this labs are to “transform” (change) a
normal E. coli bacterial cell into an E.coli bacterial cell
that produces fluorescent protein (FP) using what we
know about the central dogma. Finally, we will isolate
the FP.

5. Background
What is E.coli?
A rod shaped bacterium found in our lower intestines
What is fluorescence?
Absorbs light at one wavelength (UV) and re-emits light
at a visible wavelength (color)

6. What is Fluorescence?
Fluorescence: the emission of light by something that
has absorbed light at a different wavelength

7. NOTE: FLUORESCENCE
IS NOT LUMINESCENCE
Fluorescence Bioluminescence

8. Why do we
care?
Because it’s cool!
Frog Cell
C.E. Walczak

9. How will we make E. coli bacteria produce the red
fluorescent protein?
E. coli bacterial cell E. coli bacterial cell
Bacterial genome
Bacterial genome

10. DNA Transcription RNA Translation
Protein
E. coli bacterial cell E. coli bacterial cell
Bacterial genome
Bacterial genome
I think we should alter the ________________ because _____________
DNA
RNA
Protein

11. How We Make E.coli Glow
Plasmid Uptake of foreign DNA,
often a circular plasmid
Allow bacteria to grow for 1-3
days
DNA  RNA Protein
Bacteria now express cloned fluorescent protein…

12. Characteristics of a Plasmid:
Circular Piece of DNA
Occurs Naturally
Can be engineered (made) in the lab and then inserted
into bacteria to make a desired protein.
Can contain antibiotic resistance genes and/or other genes
Not incorporated to the bacterial genome but can
replicate on its own

13. What is on the Plasmid?
E. coli bacterial cell
FP
Bacterial genome
promoter Multiple
Cloning
Site
Origin (MCS)
Stop
AmpR
(selectable
marker)

14. What’s on the plasmid
Plasmid Mix 1 Plasmid Mix 2
GFP Cherry
FP gene
BFP Tangerine
Grape YFP
AmpR
Ampicillin
resistance gene

15. How We Make E.coli Glow
Plasmid Uptake of foreign DNA,
often a circular plasmid
Allow bacteria to grow for 1-3
days
DNA  RNA Protein
Bacteria now express cloned fluorescent protein…

16. How the Plasmid Gets in the
Bacteria
Show Video
1. Add CaCl2
2. Heat/Shock

17. Why Ampicillin?
LB/AMP +
LB/AMP -

18. Why Ampicillin?
LB/No Amp

19. Why Care?
Frog Cell
C.E. Walczak Worm Embryo
I.M. Cheeseman