For a practical at Uni we used pGLO plasmid to transform competent E. coli cells. We placed DNA inset ALB fragment with HindIII ends in the GFP gene. Would the insert still be present in this recombinant plasmid if the transformed cells were plated on agar plates containing ampicillin only, no arabinose?
Dear Michelle Bird
,pGLO contains three genes:
- Bla, which codes for the enzyme beta-lactamase giving the transformed bacteria resistance to the beta-lactam family of antibiotics (such as of ampicillin). It provides the resistance for growth on a selection medium.
- araC, a promoter region that regulates the expression of GFP (specifically, the GFP gene will be expressed only in the presence of arabinose). It does not affect the primary metabolism.
- GFP, encoding a green fluorescent protein, which fluoresce in the green under UV light. It is the reporter for the transformation.
The presence of arabinose doesn't affect the transformed cells survival. It affects only the reporter. If I understand correctly, you simply silenced the reporter by inserting an ALB fragment in the GFP gene.
The cells that lose the plasmid, loose the resistance to the ampicillin in the medium and die. Thus, evolutionary pressure ensure that the plasmid is maintained. Loss of an entire insert from the plasmid is extremely unlikely in growth conditions.
Hi there,
Ampicilline is selecting clones containing beta lactamase encoding gene. Arabinose is an inducer. In your case, arabinose will allow GFP expression in ampicilline resistant clones if GFP gene is intact and no GFP expression if GFP gene has been disrupted by insertion.
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