Question

The features of two plasmids, pkan-GFP and PBS, are illustrated below. poperon MCS LacZ enc (a-fragment) pKan-GFP 10,000 bp PBS 5,500 bp Ori pkan-GFP has a 6 kbp promoterless gfp operon that encodes a green-fluorescent protein, which glows green. Kan" is a gene (500 bp) that provides resistance to the antibiotic kanamycin. PBS contains the lacZ' gene (which has its own promoter) and a 100 bp multiple cloning site (MCS) located within the lacZ'gene. Tet" is a gene (1,500 bp) that provides resistance to the antibiotic tetracycline. You are asked to subclone the gfp operon, contained within pkan-GFP, into PBS to make tetracycline resistant bacteria that glow green.

Answer 1

Question:

Answer:

Bam HI digestion Grad student's digostion with KpnI 1 kbp Ladder PBS pkan" GFP Supervisore plasmid Lanes 3 4 5 - 8 kbp 7 kbp 6 kbp 5 kbp 4 kbp 3 kbp E III III 2 kbp 1 kbp 0.1 kbp

Explanation:

pKanR-GFP contains two restriction sites for Kpn I at both end of the gfp operon where pBS contain single restriction site for Kpn I. when plasmid will have formed then pBS consist of two Kpn I restriction sites. When plasmid is digested by Kpn I then only two bands will be formed: one as linear form of pBS which is 5,500 bp long while other band is fragment which contains gfp operon which is 6000 bp but Kpn I restriction site in pKanR-GFP is slightly far from gfp operon gene sequence. Finally, two band will be formed: 5500 bp and ~6000 bp.