Transport Nucleus Cytoplasm Protein gene DNA mRNA The Cell:

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Presentation transcript:

Transport Nucleus Cytoplasm Protein gene DNA mRNA The Cell:

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 Green Fluorescent Protein Ampicillin resistance Green Fluorescent Protein Aequorea victoria jellyfish gene araC regulator protein Regulates GFP transcription pGLO ori bla GFP araC

What is transformation? Uptake of foreign DNA often a circular plasmid GFP Amp Resistance

pGLO Transformation Cell wall Bacterial chromosomal DNA Beta lactamase GFP Bacterial chromosomal DNA Beta lactamase (ampicillin resistance) pGLO plasmids

pGLO Regulation RNA Polymerase araC ara GFP Operon GFP Gene ara Operon B A D Effector (Arabinose) Effector (Arabinose) araC B A D RNA Polymerase araC B A D

Transformation Procedure Suspend colonies in Transformation Solution Add 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 2. Heat-shock increases permeability of cell membrane 3. LB recovery allows beta lactamase expression Cell wall GFP Beta lactamase (ampicillin resistance)