The BioBuilder Lab Experience: What a Colorful World! PresentPreparePerform

PRESENT 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). Where can it fit? Molecular Genetics An alternative to pGLO TM Microbiology Extension/Enrichment University of Cambridge iGEM 2009

BioBuilder Emphasis:An Engineering Paradigm DesignBuild Test The focus of this lab lab The focus of this lab 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 They moved these devices into E. coli because, well, everyone loves working with E. coli… GREEN pGRN PURPLE pPRL 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?

Advanced Prep View a video on how to prepare “patches” of 4-1 and 4-2 for transformation...Patching.Patching 2. Make sufficient quantities of LB and LB+amp agar plates. 3. Prepare 1% CaCl 2 solution. 4. Aliquot plasmids (2, 5µl of each plasmid) and transformation solutions (500µl) for student groups. PREPARATION To investigate our question we will TRANSFORM.... the strains with each plasmid. Wrong Transformation! PURPLE pPRL GREEN pGRN

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 PERFORM Work Flow Patch of 4-1 in CaCl 2 no DNA Add 100µl of 4-1 to 5µl pPRL tube Add 500µl LB 42 Heat shock 90s Add 100µl of 4-1 to 5µl pGRN tube Add 500µl LB LB + amp 4-1 pPRL LB + amp 4-1 pGRN LB or LB+amp optional (?) Repeat for Strain 4-2 Plate 200µl

Tomorrow Observe purple and green colonies. Any guesses as to what we will see? Our Data 4-1 pPRL LB + amp 4-2 pPRL LB + amp 4-2 pGRN LB + amp 4-1 pGRN LB + amp 2. Record data. 3. Calculate transformation efficiency.

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