Question

GFP purification by Hydrophobic Interaction Chromatography

1. Why are ampicillin and arabinose added to the LB broth when growing liquid cultures?

2. What would you see if a green pGLO colony from the LB/amp/ara agar plate was streaked onto LB/amp agar, incubated at 37 degrees C overnight, and viewed under UV light?

3. What would you see if a while pGLO colony from the LB/amp agar plate was streaked onto LB/amp/ara agar, incubated at 37 degrees C overnight and viewed under UV light?

Answer 1

1.The arabinose helps in the regulation of GFP gene as the pglo plasmid have arbinose operon and when of its gene in this operon is replaces by GFP and hence control of expression of GFP is in hands of arabinose i.e. if arabinose will be present in media then only the GFP gene will get expressed.

Ampicillin is added because the pglo plasmid also has beta lactmase gene that can degrade ampicillin.So only the transformed bacterial cells grow in media we need to add ampicillin as they will get resistant to it and resistance of ampicillin will be provided by pglo plasmid.So ensuring only transformed bacterial cells grow on media.

A2.The bacterial colony would be growing only because it has been transformed by pglo plasmid.So if we will see in uv light after incubation as arabinose is present in media the gfp gene will be turned on and colonies appears fluroescent green.

A3.The white pglo colony on lb/amp plate show the transformed colony with pglo plasmid but as the arabinose is not present in media the gfp gene is not turned on and hence no fluorescent green colour under UV.So of such a colony gets incubated to lb/amp/ara plate as arabinose is in media the gfp gene will get turned on and now we can observe fluorescent green colonies the next day of incubation under UV.