Transcript pGLO Origin of plasmid replication
pGLO Transformation
What is transformation?
• Uptake of foreign DNA -- often a circular plasmid
A plasmid used as a vector must have: a. An origin of replication
Origin of plasmid replication
b. A gene conferring resistance to an antibiotic for survival of the transformed organism.
Beta-lactamase to break down ampicillin
pGLO
Transformation with a plasmid
• Uptake of foreign DNA … that a. self replicates b. codes for a protein that confers antibiotic resistance to the host organism
Amp Resistance
A plasmid used as a vector must have: a. An origin of replication b. A gene conferring resistance to an antibiotic for survival of the transformed organism.
In addition … pGLO contains c. The gene to produce Green Fluorescent Protein - the protein of interest d. An ON/OFF switch that controls synthesis of GFP by the presence or absence of the sugar, arabinose .
Origin of plasmid replication
Beta-lactamase to break down ampicillin pGL O ON/OFF switch Green Fluorescent Protein
What is pGLO
• Plasmid that contains an extra gene from a jellyfish -- gene codes for GFP …in the presence of arabinose
GFP Amp Resistance
p
GLO Regulation
araC ara “BAD” Operon B A D Effector (Arabinose) araC B A D araC ara GFP Operon GFP Gene Effector (Arabinose) araC GFP Gene araC B RNA Polymerase A D araC RNA Polymerase GFP Gene
However, after synthesis, the GFP requires UV light in order to fluoresce ...
GFP Amp Resistance
Transformation Procedure
• Suspend colony in CaCl 2 Transformation Solution • Add pGLO plasmid DNA to “+” tube • Water to “ - ” control tube • Incubate tubes 10-15 minutes on
ice
• Heat shock 50 seconds at 42 o C • Incubate 2 minutes on
ice
• Add LB broth for “recovery” period • Plate on appropriate “flavored” media
Why cold - heat shock - cold?
• To affect membrane so cells take in plasmid
Why add LB broth?
• So cells can start replicating plasmid and begin expressing new proteins
Amp Resistance
What do you expect to grow on the various plates?
- pGLO Plain LB agar + pGLO LB agar + ampicillin (before arabinose) LB agar + ampicillin (after arabinose)
What color to you expect the colonies to be, in room light?
- pGLO + pGLO Plain LB agar LB agar + ampicillin (before arabinose) LB agar + ampicillin (after arabinose)
What color to you expect the colonies to be, in UV light ?
- pGLO + pGLO Plain LB agar LB agar + ampicillin (before arabinose) LB agar + ampicillin (after arabinose)