Recombinant DNA Plasmids and Bacteria Transformation

How are plasmids engineered?

Host DNA fragments (i.e. coral or jellyfish FP coding DNA) DNA Plasmid Vector

Ligate (paste) fragments into cut DNA vector Cut genomic DNA into fragments

Cut plasmids open with restriction enzymes

Screen for and select plasmid containing FP gene

Bioluminescence vs. Fluorescence

Bioluminescence

Fluorescence
http://fireflyforest.net/firefly/2006/11/13/fluorescent-scorpion-in-uv-light/

Natural Light

Scorpion- Natural Light

In the Dark

Scorpion- UV Light

Bioluminescent organism produces its own light.

A fluorescent organism absorbs light at one wavelength (UV) and a reemits the light at a visible wavelength= color

What is a plasmid?
A small circular piece of DNA Naturally occurring

Can be altered in lab to express protein of interest

What is Transformation?
Bacterial chromosome Plasmid

Uptake of foreign DNA, often a circular plasmid

What is Transformation?
Bacterial chromosome Plasmid

Uptake of foreign DNA, often a circular plasmid

Bacterial chromosome

Allow bacteria to grow for 1-3 days on plate with ampicillin.

What is Transformation?
Bacterial chromosome Plasmid

Uptake of foreign DNA, often a circular plasmid

Bacterial chromosome

Allow bacteria to grow for 1-3 days on plate with ampicillin.

Bacteria now express cloned fluorescent protein

Bacterial Transformation Procedure

Shielding the charge

CaCl2 Positive charge of Ca++ ions shields negative charge of DNA phosphates

Ca++
Ca++
O

O P O

Base

CH2

O
Sugar

Ca++

P O

Base

CH2

O
Sugar

OH

Stress through heat

Incubate on ice slows fluid cell membrane

Heat-shock increases permeability of membranes

Leave in heat 45 seconds!!! Too short, and bacteria won't let in plasmid. Too long, and the bacteria will die.

Why Ampicillin?

Ampicillin inhibits cell growth. Only cells that can inactivate the ampicillin around them will grow. Ampicillin resistance protein gene is tied to (expressed with) the fluorescent

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