Lecture, chapter 10 -Gene regulation in prokaryotes

Today we stated chapter 10, on gene regulation in prokaryotes.

We discussed the importance of having gene regulation mechanisms in place for the correct functioning of prokaryotic cells, and why it is more common to find such mechanisms acting upon transcription than other cellular processes.

We also discussed the difference between positive and negative regulation, the role activators and repressors play in such processes, and how they are affected by the presence of inducers in the environment.

The basics of the operon model of gene regulation were also covered.

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Lab 10 - Protein purification(GFP) through Hydrophobic Interaction Chromatography (HIC)

Today we performed a Hydrophobic Interaction Chromatography (HIC) to separate the green fluorescent protein (GFP), produced in our bacterial cultures, from other proteins commonly found in bacteria.

A sample of bacteria was concentrated and then resuspended in a solution in which they were lysed. The high salt solution, containing all the proteins found in the bacteria, was then passed through a hydrophobic interaction column where molecules of the naturally hydrophobic GFP bound to the hydrophobic beads. The high salt solution increased the hydrophobicity of GFP by further exposing its hydrophobic residues.

A series of washes with buffers of decreasing salinity allows proteins with various levels of hydrophobicity to gradually unbind from the beads and be collected in a test tube. By switching collection tubes each time a buffer is added, different proteins can be collected. One of them was GFP and the tube in which it was collected should glow.

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Lab 07 - Ligation and transformation (GAPC gene from Arabidopsis and pJet1.2 plasmid)

Wednesday, January 20, 2010

Today we used the purified PCR product from the nested PCR lab (GAPC gene from Arabidopsis) to genetically transform E. coli.

The lab was divided in three main steps

• Preparation of competent cells
• Ligation
• Genetic transformation

During most of the lab students manipulated bacteria to make them competent (i.e. get them ready to uptake extracellular DNA). Once this was achieved, the GAPC gene from Arabidopsis, obtained via nested PCR, was ligated to the pJet1.2 plasmid.

The plasmid was then used to genetically transform E. coli that were spread on LB agar/Amp/IPTG plates and incubated.

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