The BioBuilder Lab Experience:What a Colorful World!

Present Prepare Perform

PRESENTThe Big Idea: Examine the role of cellular chassis through transformation

Objectives:

Explain the role of chassis in synthetic biology.

Conduct and interpret results from a bacterial transformation.

Properly use molecular genetics terms (operon, gene expression, transformation).

Properly use synthetic biology terms (chassis, system, device).

Where can it fit?

Molecular Genetics

An alternative to pGLOTM

Microbiology

Extension/Enrichment

University of Cambridge iGEM 2009

BioBuilder Emphasis:An Engineering Paradigm

Design Build

TestThe focus The focus

of thisof this lablab

The focus The focus of thisof this lablab

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

They moved these devices into E. coli because, well, everyone loves working with E. coli…

GREENpGRN

PURPLEpPRL

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

Consider This.... Do different chassises behave the

same?

The Chassis: “A frame where you hang the internal workings”

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

We insert the GREEN color generating device into each strain of bacteria?

Will both strains express the color in the same way?

What if....We insert the PURPLE color generating device into each strain of bacteria?

Will both strains express the color in the same way?

4-1

4-2

4-1

4-2

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.

PREPARATIONTo investigate our question we will

TRANSFORM.... the strains with each plasmid.

Wrong Transformation!

PURPLEpPRL

GREENpGRN

Simple Procedure:

1. Resuspend cells2. Mix the cells with the plasmid3. Label agar plates4. Heat shock cells mixed with plasmids5. Add LB to cells 6. Plate cells7. Incubate overnight

PERFORMWork Flow

Patch of 4-1 in CaCl2 no DNA

Add 100µl of 4-1 to

5µl pPRL tube

Add500µl LB

4242

Heat shock 90s

Add 100µl of 4-1 to

5µl pGRN tube

Add500µl LB

LB + amp4-1 pPRL

LB + amp4-1 pGRN

LB or LB+ampoptional (?)

Repeat for Strain 4-2

Plate 200µl Plate 200µl

Tomorrow...

1. Observe purple and green colonies. Any guesses as to what we will see?

Our

Data

4-1 4-1 pPRLpPRLLB + LB + ampamp

4-2 4-2 pPRLpPRLLB + LB + ampamp

4-2 4-2 pGRNpGRNLB + LB + ampamp

4-1 4-1 pGRNpGRNLB + LB + ampamp

2. Record data.

3. Calculate transformation efficiency.

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