Biotechnology Explorer Program Serious About Science Education Aequorea victoria Central Framework of Molecular Biology DNA RNA Protein Trait Links to real-world research study of biological processes : localization of gene expression cell movement cell fate during development formation of different organs ** screenable marker **to identify transgenic organisms (glowing trait and antibiotic resistance) BioRad Kit Protocol • Transform bacteria with pGLO plasmid • Select for ampicillin resistance • Induce expression of GFP with arabinose • Grow transformed colony • Create glowing art with E. coli “paint” What is a plasmid? • Circular piece of autonomously replicating DNA ori bla Originally evolved by bacteria May express antibiotic resistance or be modified to express proteins of interest pGLO Plasmid –Beta Lactamase • Ampicillin resistance –Green Fluorescent Protein • Aequorea victoria jellyfish gene –araC regulator protein • Regulates GFP transcription araC ori pGLO bla GFP What is transformation? • Uptake of foreign DNA often a circular plasmid GFP Amp Resistance pGLO Transformation Cell wall GFP Bacterial chromosomal DNA pGLO plasmids Beta lactamase (ampicillin resistance) pGLO Regulation (AP Bio only) ara GFP Operon ara Operon araC B A D araC Effector (Arabinose) Effector (Arabinose) araC B A D araC RNA Polymerase araC B A D GFP Gene GFP Gene RNA Polymerase araC GFP Gene Transformation Procedure • Suspend colonies in Transformation Solution • Add loop of pGLO plasmid DNA to “+” tube • Incubate tubes 10 minutes on ice • Heat shock 50 seconds at 42oC • Incubate 2 minutes on ice • Incubate 10 minutes with LB broth • Plate Why perform each transformation step? 1. Incubation on ice slows fluid cell membranes Cell wall GFP 2. Heat-shock increases permeability of cell membrane 3. LB recovery allows beta lactamase expression Beta lactamase (ampicillin resistance) Volume Measurement (know these units and measuring points)