Module 3, Lab 9 - Genetic transformation of E. coli with the pGLO plasmid

Aequorea victoria, original source of the green fluorescent protein (GFP)
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First lab in module 3. Today we used the pGLO plasmid to genetically transform E. coli.

pGLO is a plasmid that has been engineered to contain and express the Green Fluorescent Protein (GFP) gene, originally isolated from the jelly Aequorea victoria. GFP produces a green fluorescence when excited by blue or UV light.

In order to make the GFP gene a functional one it has been engineered so the sugar arabinose triggers the production of the protein. The genes in the arabinose operon (araB, araA, and araD) have been replaced by the GFP gene. Such genes encode proteins that break down arabinose when it is present in the environment, so they are expressed only if this is the case. The activating mechanism has been left intact, so in the engineered operon the presence of arabinose turns on the GFP gene and therefore GFP is produced.

Another feature of the pGLO plasmid is the presence of the beta-lactamase gene, which provides resistance against the antibiotic ampicillin.

The bacteria were transformed through the heat shock technique, and then plated on LB agar plates containing:

• Just LB (lysogeny broth)
• LB and ampicillin
• LB, ampicillin and arabinose

Plates are being incubated for 24 hours at 37ºC.

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