Bacterial Transformation Lab

WarmupDraw 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?

Bacterial TransformationStudent Training

By Quanina Quan and UCSD ScienceBridge

ObjectiveDemonstrate that changes in genotype causes changes in phenotype by transforming E.coli into fluorescent E.coli.

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.

BackgroundWhat is E.coli?

What is fluorescence?

A rod shaped bacterium found in our lower intestines

Absorbs light at one wavelength (UV) and re-emits light at a visible wavelength (color)

What is Fluorescence?

Fluorescence: the emission of light by something that has absorbed light at a different wavelength

NOTE: FLUORESCENCE IS

NOT LUMINESCENCE Fluorescence Bioluminescence

Why do we care?

Because it’s cool!

Frog CellC.E. Walczak

How will we make E. coli bacteria produce the red fluorescent protein?

E. coli bacterial cell

Bacterial genomeBacterial genome

E. coli bacterial cell

E. coli bacterial cell

Bacterial genomeBacterial genome

E. coli bacterial cell

I think we should alter the ________________ because _____________

DNARNA Protein

  Transcription   Translation ProteinRNA DNA

How We Make E.coli Glow

Bacteria now express cloned fluorescent protein…

Allow bacteria to grow for 1-3 days

Plasmid Uptake of foreign DNA, often a circular plasmid

DNA RNA Protein

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

E. coli bacterial cell

Bacterial genome

Origin

AmpR

(selectable

marker)

FP

Stop

promoter Multiple Cloning Site (MCS)

What is on the Plasmid?

What’s on the plasmid

AmpR

Ampicillinresistance gene

FP gene

Plasmid Mix 1

GFP

BFP

Grape

Plasmid Mix 2

YFP

Tangerine

Cherry

How We Make E.coli Glow

Bacteria now express cloned fluorescent protein…

Allow bacteria to grow for 1-3 days

Plasmid Uptake of foreign DNA, often a circular plasmid

DNA RNA Protein

How the Plasmid Gets in the Bacteria

Show Video

1. Add CaCl22. Heat/Shock

Why Ampicillin?

LB/AMP +

LB/AMP -

Why Ampicillin?

LB/No Amp

Why Care?

Frog CellC.E. Walczak Worm Embryo

I.M. Cheeseman