Transcript Transformation of bacteria with pGLO

Transformation of bacteria
with pGLO
Lab 4
pGLO gene
• Bioluminescent jelly fish – Aequorea
victoria – GFP causes fish to glow in dark
• Transformed E.coli with GFP will glow a
brilliant green color under ultraviolet light
Plasmid
• Circular pieces of DNA – plasmid
• Contain genes for one or more traits –
beneficial to bacterial survival
• Transfer plasmids – share these beneficial
genes – adapts to new environment –
evolution
- Bacterial resistance to antibiotics
pGLO plasmid
• Gene for GFP
• Gene for resistance to ampicillin
• Operon system – gene on/off regulation
Operon in Prokaryotes
Section that codes for mRNA
which later get translated to
proteins
Operon in Prokaryotes
• Operator – turn off operon (by regulatory molecule)
– Operon get “turned off” b/c RNA polymerase is blocked from
continuing down the strand to the gene
– No protein is produced
– Blocking and unblocking is how bacteria make certain
proteins certain times
– Example: Lactose
Some steps
• Transformation solution – Ca2+ cation of
transformation solution neutralizes the repulsive
negative charges of phosphate backbone of
DNA and phospholipids of cell membrane
• Heat shock – increases permeability of cell
membrane to DNA
• Recovery – add LB nutrient broth allows cells to
grow and express beta-lactamase – ampicillin
resistance protein
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This project is funded by a grant awarded under the President’s Community Based Job Training Grant as
implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60).
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