Transcript Transformation of E.coli with pGlow

Transformation of E.coli with
pGLO
Dan Prochaska and Jeff Winslow
Talawanda/Butler Tech
Purpose
 Challenge students to participate in cutting
edge laboratory activities that emphasize
important skills carried out in a structured
environment.
 To make connections to modern
commercially viable protocols, i.e. human
insulin production.
Lesson Plan
 Three day lesson.
 Day 1: Powerpoint introduction, handouts with
protocol, streaking starter plates, and preassessment
 Day 2: Complete transformation protocol, student
groups make predictions about results
 Day 3: Observe plates using U.V. pen lights and
record observations. Make connections to real
world applications. Complete post assessments.
pGLO lab. Objectives
 Transform E. coli host cells with plasmid
DNA.
 Predict the results on selective agar plates.
 Observe and record actual plate data.
 Draw conclusions from the experiment and
list possible sources of error.
Introduction
What is genetic transformation?
 When host bacteria acquire novel traits that are
encoded in genes contained on plasmid, (pGLO)
DNA.
What new traits will E.coli acquire from the
transformation?
Antibiotic resistance (ampicillin)
Green Fluorescence: Where did this trait come from?
What is the source of green
flourescence?
 The marine jellyfish Aequorea victoria
Plasmid DNA
 Very small (103bp ) piece of circular DNA
 Replicates autonomously, (ori site).
 Is in addition to the large (106bp) E. coli
chromosome.
 Can be exchanged between cells.
 Carries important genes for traits such as
antibiotic resistance.
 In this laboratory we are investigating ampicillin
resistance.
Plasmid DNA
A small circular piece of DNA that is naturally
occurring in some bacteria. It can be exchanged
between cells and often carries antibiotic resistance
genes
pGLO an engineered plasmid
pGLO plasmid genes
 GFP gene encodes a green fluorescent protein
(GFP)
 Bla gene encodes the enzyme beta lactamase that
degrades the antibiotic ampicillin thus, protecting
the cell from its harmful effects.
 AraC is a regulatory gene that encodes a protein
that acts like a switch that turns on the expression
of the GFP gene.
Transformation- Terms
 Competence: cells are able to take up a
plasmid from their surroundings
 How do we make our E.coli cells
competent?
 Gene expression: cells replicate, transcribe,
and translate genes located on the pGLO
plasmid
Transformation
Cell wall
GFP
Bacterial
chromosomal
DNA
Beta lactamase
(ampicillin resistance)
pGLO plasmids