pGLO TM Bacterial Transformation Courtesy BioRad Corporation 4/29/2020 1

pGLO/ Green Fluorescent Protein Isolation

A single bacteria might be worth 5 billion dollars to the right buyer

.

DNA

Central Framework of Molecular Biology

RNA Protein Trait

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What is transformation?



Uptake of foreign DNA

, often a circular plasmid, from the surroundings with the expression of these genes.

GFP Amp Resistance 4/29/2020 4

What is a plasmid?

 A circular piece of autonomously replicating DNA

ori bla

 Originally evolved by bacteria  May express antibiotic resistance gene or be modified to express proteins of interest 4/29/2020 5

Uses of Genetic Transformation

 Agriculture  Introduction of drought, pest, or frost resistance genes “BT Corn” 6    Bioremediation  Introduction of genes into bacteria giving them the ability to degrade hydrocarbons, “Oil Eating Bacteria” Medicine  Gene therapy Production of human proteins  Insulin, clotting factors 4/29/2020

pGLO Plasmid Draw this!!

 Beta Lactamase • Ampicillin resistance  Green Fluorescent Protein •

Aequorea victoria

gene jellyfish

ori

 araC regulator protein • Regulates GFP transcription 7

bla pGLO araC GFP

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8 Purpose of pGLO Transformation  To transform

E. coli

K-12 with a plasmid that contains a gene that codes for Green Fluorescent Pigment ( GFP )  Transformed cells will produce GFP and glow a brilliant green under UV light.

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Work of the pGLO Plasmid

Arabinose Operon

RNA Polymerase Regulatory Gene--araC

Binds RNA polymerase

10 RNA Polymerase Gene products 4/29/2020

araC araC

Transcriptional Regulation

B ara Operon A D araC ara GFP Operon GFP Gene Effector (Arabinose) Effector (Arabinose) B A D araC GFP Gene araC B RNA Polymerase A D araC RNA Polymerase GFP Gene 4/29/2020 11

Transformation Procedure

 Suspend bacterial colonies in

Transformation Solution

 Add

pGLO plasmid DNA

 Place tubes on

ice



Heat shock

at 42 o C and place on

ice

 Incubate with

nutrient broth



Streak

plates 4/29/2020 12

Transformation Procedures

Day 1 Day 2 4/29/2020 13

Reasons for performing each transformation step?

Ca ++ Ca ++

O P  Transformation solution = CaCl 2 O O CH 2 O

O Base Sugar

Positive charge of Ca

+2

ions shields negative charge of DNA phosphates 14

Ca ++

O O P O O CH 2

O Sugar Base

OH 4/29/2020

Why perform each

transformation

step?



Incubation on ice slows fluid cell membranes



Heat-shock increases permeability of cell membrane



Incubation in LB nutrient broth allows beta lactamase expression

15

Cell wall GFP Beta lactamase (ampicillin resistance)

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16

Inducible, Repressible or Both??

http://faculty.clintoncc.suny.edu/faculty/Mic hael.Gregory/files/bio%20101/Bio%20101 %20Lectures/Gene%20Regulation/gene.ht

m 4/29/2020

17

The Gel Analysis

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Graphing Migration Distance 18 4/29/2020