Please answer this question Thank you The GFP gene from the jellyfish Aequorea v
ID: 101415 • Letter: P
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The GFP gene from the jellyfish Aequorea victoria encodes the green fluorescent protein, which glows green upon UV stimulation. You lab advisor has taken advantage of this property by constructing a plasmid vector that contains the GFP gene with a BamHl site near the middle of its sequence, and an ampicillin resistance gene and origin of replication. After cutting this vector with the BamHl enzyme, you ligated it together with BamHl-digested rat genomic DNA, transformed the resultant molecules into ampicillin-sensitive E. coli cells, and plate these cells on petri plates containing ampicillin. When the plate is placed under UV light, some of the colonies growing on this plate glow green, while others do not glow. Why?Explanation / Answer
The plasmid vector in this case contains a GFP gene with BamH1 site in the middle, an ampicillin resistance gene and a promoter site. The experiment is done to clone rat genomic DNA into the plasmid vector. The presence of ampicillin resistance gene allows growth of transformed colonies (containing vector alone or vector with genomic DNA cloned) and prevents growth of non-transformed colonies. The GFP gene helps to distinguish the colonies which contain empty vector (GFP expressed – glows green) from colonies which contain rat genomic DNA cloned into the vector (GFP not expressed – no glow).
This is what happens-
When the vector is cut with BamH1 endonuclease, the vector gets cleaved in the middle of the GFP gene (BamH1 site). After this when the ligation is done with BamH1 digested rat genomic DNA, there are two possibilities in the reaction –
In case (1), self ligation allows the expression of GFP gene (digested site ligates back). The transformed colonies which have the self ligated empty vectors (no rat genomic DNA inserted) will glow green.
In case (2), GFP gene expression does not occur as the rat genomic DNA is inserted in digested site. These transformed colonies which contain rat genomic DNA will not glow green.