Exam 1

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Module 3, lab 08 (section 1)Genetic transformation of E. coli with the pGLO plasmid

We followed the same process we followed with section 2.

For more information click here.

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Module 2, lab 5 (section 1)GAPC gene nested PCRGel electrophoresis (PCR round 2) and PCR purification

Today we ran the agarose gel electrophoresis for the second round of the nested PCR of the Arabiodopsis GAPC gene (which we will clone, and eventually sequence), and also purified the reaction products, in order to eliminate leftover reagents and keep only the DNA.

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Lecture, chapter 6 - Transcription

Today we started chapter 6, on transcription, covering the basics of the process.

We discussed how to decide which are the template (sense) and coding (anti-sense) strands in DNA, the reasons for which some genes are expressed all the time (housekeeping genes) and some others are expressed only some times, and included some transcription-specific terminology.

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Module 3, Lab 08 (section 2)Genetic transformation of bacteria with the pGLO plasmid

Aequorea victoria, original source of the green fluorescent protein (GFP)

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Today we used the pGLO plasmid to genetically transform Escherichia coli.

pGLO is a plasmid that has been engineered to contain 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 The genes in the arabinose operon (araB, araA, and araD) have been replaced by the GFP gene.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 regulatory sequence 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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