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The Cell:. DNA. gene. Nucleus. mRNA. Transport. Cytoplasm. Protein. ori. bla. What is a plasmid?. Circular piece of autonomously replicating DNA. Originally evolved by bacteria May express antibiotic resistance or be modified to express proteins of interest. araC. ori. pGLO.

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Presentation Transcript
slide1

The Cell:

DNA

gene

Nucleus

mRNA

Transport

Cytoplasm

Protein

what is a plasmid

ori

bla

What is a plasmid?
  • Circular piece of autonomously replicating DNA
  • Originally evolved by bacteria
  • May express antibiotic resistance
  • or be modified to express proteins of interest
pglo plasmid

araC

ori

pGLO

GFP

bla

pGLO Plasmid
  • Beta Lactamase
    • Ampicillin resistance
  • Green Fluorescent Protein
    • Aequorea victoria jellyfish gene
  • araC regulator protein
    • Regulates GFP transcription
what is transformation
What is transformation?
  • Uptake of foreign DNAoften a circular plasmid

GFP

Amp Resistance

pglo transformation
pGLO Transformation

Cell wall

GFP

Bacterial

chromosomal DNA

Beta lactamase

(ampicillin resistance)

pGLO plasmids

p glo regulation

ara GFP Operon

araC

GFP Gene

araC

GFP Gene

RNA Polymerase

araC

GFP Gene

pGLO Regulation

ara Operon

araC

B

A

D

Effector (Arabinose)

Effector (Arabinose)

araC

B

A

D

RNA Polymerase

araC

B

A

D

transformation procedure
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 withLB broth
  • Plate
why perform each transformation step
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)