Transcript - BioBuilder

The BioBuilder Lab Experience:
What a Colorful World!
Present
Prepare
Perform
Where can it fit?
PRESENT
The Big Idea: Examine the
role of cellular chassis through
transformation
Microbiology
Molecular Genetics
Objectives:
Explain the role of chassis in
synthetic biology.
Conduct and interpret results from a
bacterial transformation.
An alternative to
pGLOTM
Extension/Enrichment
Properly use molecular genetics
terms (operon, gene expression,
transformation).
Properly use synthetic biology terms
(chassis, system, device).
University of Cambridge iGEM 2009
BioBuilder Emphasis:An
Engineering Paradigm
The focus of
this
lab
Design
Build
Test
The focus of
this
lab
DEFINING THE PROBLEM...
Cambridge iGEM Team 2009 wanted to
develop a marketable product that
REPORTS the concentration of an inducer
by color.
The Vision
The Pathway
Promising candidate for natural violet
pigment
(violacein)
production:
Chromobacterium violaceum
5 enzymes involved in pathway
Manipulated C. violaceum operon to also
produce green pigment
ABCDE construct expresses violet ABDE
construct expresses a dark green
pigment
PURPLE
pPRL
GREEN
pGRN
They moved these devices into E. coli because, well, everyone loves
working with E. coli…
Consider This....
Do different chassises behave the same?
Decisions, Decisions....
What chassis do YOU prefer?
There are two major laboratory strains
of E. coli:
K12 Strain (4-1)
B Strain (4-2)
Both strains have lost ability to thrive
outside the lab
Our
System
What if....
We insert the GREEN color
generating device into each
strain of bacteria?
We insert the PURPLE color
generating device into each
strain of bacteria?
Will both strains express the
color in the same way?
Will both strains express the
color in the same way?
4-1
4-1
4-2
4-2
PREPARATION
To investigate our question we will
TRANSFORM....
the strains with each plasmid.
Advanced Prep...
1. View a video on how to prepare “patches” of 4-1 and 4-2 for
transformation...Patching.
2. Make sufficient quantities of LB and LB+amp agar plates.
3. Prepare 1% CaCl2 solution.
4. Aliquot plasmids (2, 5µl of each plasmid) and transformation
solutions (500µl) for student groups.
GREEN
pGRN
PURPLE
pPRL
PERFORM
Work Flow
Simple Procedure:
Patch of 4-1 in CaCl2
no DNA
1. Resuspend cells
2. Mix the cells with the
plasmid
3. Label agar plates
4. Heat shock cells mixed
with plasmids
5. Add LB to cells
6. Plate cells
7. Incubate overnight
Add 100µl
of 4-1 to
5µl pGRN tube
Add 100µl
of 4-1 to
5µl pPRL tube
Heat shock 90s
Add
500µl LB
Add
500µl LB
Plate 200µl
LB + amp
4-1 pPRL
LB or LB+amp
optional (?)
Plate 200µl
LB + amp
4-1 pGRN
Repeat for Strain 4-2
Our
Data
Tomorrow...
1. Observe purple and green colonies. Any guesses as to what we
will see?
4-1
pPRL
LB +
amp
4-2
pPRL
LB +
amp
2. Record data.
3. Calculate transformation efficiency.
4-1
pGRN
LB +
amp
4-2
pGRN
LB +
amp
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