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Join the BioBuilder Lab and explore the role of cellular chassis in synthetic biology through a bacterial transformation experiment. Learn about molecular genetics, microbiology, and synthetic biology terms while conducting experiments with E. coli strains.
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PRESENT • Where can it fit? • Microbiology • Molecular Genetics • An alternative to pGLOTM • The 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). • Extension/Enrichment University of Cambridge iGEM 2009
Design Build Test BioBuilder Emphasis:An Engineering Paradigm The focus of this lab The focus of this lab
DEFINING THE PROBLEM... The Vision The Pathway • Cambridge iGEM Team 2009 wanted todevelop a marketable product that REPORTS the concentration of an inducer by color. • Promising candidate for natural violet pigment (violacein) production: Chromobacteriumviolaceum • 5 enzymes involved in pathway PURPLE pPRL • Manipulated C. violaceum operon to also produce green pigment • ABCDE construct expresses violet ABDE construct expresses a dark green pigment GREEN pGRN They moved these devices into E. coli because, well, everyone loves working with E. coli…
The Chassis: “A frame where you hang the internal workings” 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
We insert the PURPLE color generating deviceinto each strain of bacteria? • Will both strains express the color in the same way? OurSystem What if.... • We insert the GREEN color generating device into each strain of bacteria? • Will both strains express the color in the same way? 4-1 4-1 4-2 4-2
Wrong Transformation! 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. PURPLE pPRL GREEN pGRN
PERFORM Work Flow Simple Procedure: 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 Patch of 4-1 in CaCl2 no DNA Add 100µl of 4-1 to 5µl pPRL tube Add 100µl of 4-1 to 5µl pGRN tube Heat shock 90s 42 Add 500µl LB Add 500µl LB Plate 200µl Plate 200µl LB + amp 4-1 pPRL LB or LB+amp optional (?) LB + amp 4-1 pGRN Repeat for Strain 4-2
OurData 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 4-1 pGRN LB + amp 4-2 pGRN LB + amp 2. Record data. 3. Calculate transformation efficiency.
