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Measuring Zone of Inhibition

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.

BackgroundWhat is E.coli?

What is fluorescence?

A rod shaped bacterium found in our lower intestines

A substance that absorbs light at one wavelength (UV) and re-emits light at a visible wavelength (color)

Background: What is GFP?

Green Fluorescent Protein

Chromophore

How does fluorescence work?

Green light(Lower energy)

Excited state

Ground state

Blue light(High energy)

NOTE: FLUORESCENCE

IS NOT LUMINESCENCE Fluorescence: Absorbs

light at one wave length and emits it at another

Bioluminescence: Produces its own light

A Metaphor

Fluorescence: Absorbs light at one wave length and emits it at another

Bioluminescence: Produces its own light

Fluorescence: Absorbs light at one wave length and emits it at another

Fluorescent Organisms

GFP

RFP

Roger Tsien and Rainbow Proteins

Uses for FPs

Human CellJ. Waters

FAQsCan I make myself or someone I know glow?

Can I get a glowing pet?

Can we turn it on or off?

• A small circular piece of DNA

• Naturally occurring

• Can be altered in lab to express protein of interest

What is a plasmid?

E. coli bacterial cell

Bacterial genome

Plasmid

Cell produces proteincoded by plasmid DNA.

What is Transformation?DNA RNA Protein

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

What’s on the plasmid

AmpR

Ampicillinresistance gene

FP gene

Plasmid Mix 1

GFP

BFP

Grape

Plasmid Mix 2

YFP

Tangerine

Cherry

How the Plasmid Gets in the Bacteria

Show Video

1. Add CaCl22. Heat/Shock

Step 0: Label Plates

Step 1: Put CaCl2 in Tube

Step 2: Collect bacterial colonies“Like scraping whipped cream off of Jello.”

Ca++Ca++

OCH2

OP OO

O Base

CH2

OPO

O

OBase

OH

Sugar

Sugar

OCa++

Step 3: Put bacteria in CaCl2

Positive charge of Ca2+ ionsshields negative charge ofDNA phosphates

Step 4: Add Plasmid to (+) Tube

AmpR

Ampicillinresistance gene

FP gene

Plasmid Mix 1

GFP

BFP

Grape

Plasmid Mix 2

YFP

Tangerine

Cherry

• Incubate on ice to slow fluid cell membrane

• Heat-shock increases permeability of membranes

Step 5: Heat Shock!

Leave in heat 45 seconds!!!

- Too short, and bacteria won't let in plasmid.

- Too long, and the bacteria will die.

ICE – HEAT – ICE

Step 6: Plate on Ampicillin

• Ampicillin inhibits cell wall growth.

• Only cells that can inactivate the ampicillin around them will grow.

• Ampicillin resistance is tied to (expressed with) the fluorescent protein gene.

• Ampicillin is a selection mechanism that only allows transformed bacteria to grow on the plate.

We have two controlsLB only (-) = Control 1: check for viable cells

LB amp (-) = Control 2: check for ampicillin function

LB amp (+) = Experimental plate: grow transformants

Why Ampicillin?

LB/AMP +

LB/AMP -

Why Ampicillin?

LB/No Amp

Tricky Parts of Lab

Labeling Plates

Getting Bacteria

Pipetting the right amount

Plating

FAQWhy don’t I see anything glowing right now?