The Cell DNA gene Nucleus m RNA Transport
- Slides: 8
The Cell: DNA gene Nucleus m. RNA Transport Cytoplasm Protein
What is a plasmid? • Circular piece of autonomously replicating DNA ori bla Originally evolved by bacteria n May express antibiotic resistance or be modified to express proteins of interest n
p. GLO Plasmid – Beta Lactamase • Ampicillin resistance – Green Fluorescent Protein • Aequorea victoria jellyfish gene – ara. C regulator protein • Regulates GFP transcription ara. C ori p. GLO bla GFP
What is transformation? • Uptake of foreign DNA often a circular plasmid GFP Amp Resistance
p. GLO Transformation Cell wall GFP Bacterial chromosomal DNA p. GLO plasmids Beta lactamase (ampicillin resistance)
p. GLO Regulation ara GFP Operon ara. C B A D ara. C Effector (Arabinose) ara. C B A D ara. C RNA Polymerase ara. C B A D GFP Gene RNA Polymerase ara. C GFP Gene
Transformation Procedure • Suspend colonies in Transformation • • • Solution Add p. GLO plasmid DNA to “+” tube Incubate tubes 10 minutes on ice Heat shock 50 seconds at 42 o. C 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)