You will be given two different plasmids, pLIT-GFP andpET28-bgal (Figure 2). pLITGFP has an ampicillin resistance (ampR)gene and pET28b-gal has a kanamycin resistance (kanR) gene. Youwill cleave both plasmids with restriction enzymes NcoI and XhoI,to release the fragment encoding green fluorescent protein (GFP)from pLITGFP, and ligate it with the vector pET28 cleaved with thesame enzymes. E. coli strain BL21(DE3) cells are transformed withthe recombinant plasmid mixture. Selecting for cells that containthe desired recombinant plasmid, pET-GFP, is done by platingtransformants on LB-agar that contains 50 µg/ml of kanamycin. Asantibiotics are heat sensitive, a sterile solution with theantibiotics is added to the agar after autoclaving, when the agarhas cooled down to about 55°C. pLIT-GFP (AmpR, ampicillinresistance marker) contains the GFP coding sequence between NcoIand XhoI sites. Ligation of the NcoI-XhoI digests of pLIT-GFP andpET28-bgal (KanR, kanamycin resistance marker) yields severaldifferent products, one of which is pET28-GFP, in which GFP gene issubstituted for beta-galactosidase. The T7 promoter drivestranscription of the adjacent gene when T7 RNA polymerase ispresent.
Considering the components of the ligation reaction forExperiment 1, Week 1, list all of the possible ligation productsthat could occur. Which of these products will confer kanamycinresistance in transformed bacteria?
You will be given two different plasmids, pLIT-GFP andpET28-bgal (Figure 2). pLITGFP has an ampicillin resistance (ampR)gene and pET28b-gal has a kanamycin resistance (kanR) gene. Youwill cleave both plasmids with restriction enzymes NcoI and XhoI,to release the fragment encoding green fluorescent protein (GFP)from pLITGFP, and ligate it with the vector pET28 cleaved with thesame enzymes. E. coli strain BL21(DE3) cells are transformed withthe recombinant plasmid mixture. Selecting for cells that containthe desired recombinant plasmid, pET-GFP, is done by platingtransformants on LB-agar that contains 50 µg/ml of kanamycin. Asantibiotics are heat sensitive, a sterile solution with theantibiotics is added to the agar after autoclaving, when the agarhas cooled down to about 55°C. pLIT-GFP (AmpR, ampicillinresistance marker) contains the GFP coding sequence between NcoIand XhoI sites. Ligation of the NcoI-XhoI digests of pLIT-GFP andpET28-bgal (KanR, kanamycin resistance marker) yields severaldifferent products, one of which is pET28-GFP, in which GFP gene issubstituted for beta-galactosidase. The T7 promoter drivestranscription of the adjacent gene when T7 RNA polymerase ispresent.
Considering the components of the ligation reaction forExperiment 1, Week 1, list all of the possible ligation productsthat could occur. Which of these products will confer kanamycinresistance in transformed bacteria?
