(Solved): 3. You want to clone the linear DNA fragment Illustrated below (1000 bp In size, Psti sticky ends, ...

3. You want to clone the linear DNA fragment Illustrated below (1000 bp In size, Psti sticky ends, a Ndel site at position 200) Into the Psti restriction site of the vector PBR 322, which contains both amplellin and tetracycline resistance genos. Ndel am 10 Psti Paul (3707 Ice Hindi 200 1000 Terwydate MAI PBR322 (4.36 kb) Xal 1145 1 NE (2009) a) List the antibiotics which you would include into the agor plates that would allow bacterial cells to grow that have been transformed with: 1) No plasmld. II) Non-recombinant PBR322. III) Recombinant PBR322 with the DNA fragment cloned into the Pst site. b) When all DNA molecules have the same sticky ends, an Insert can ligate into a vector in elther orientation (non-directional cloning). Draw the two recombinant plasmids that could result from folning this fragment into the plasmid in two different Insert orientations, Clearly indicate the vector DNA, the inserted fragment, and the restriction enzyme sites for Pstl, Ndel and Pull on each drawing. c) Predict the band sizes that will be generated by digestion of the non-recombinant PBR322 and either of the two recombinant with the enzymes listed below. Pst. Pvull Ndel Pvull Non recombinant PBR322 Recombinant PBR322 orientation 1) Recombinant PBR322 orientation 2) You want to modify plasmid PBR322 from a cloning plasmid into an expression vector, to specifically allow bacterial expression of genes cloned into the Pvull site. How can this be accomplished? Which will be the two most important sequences to include in your PBR322 modifications? Explain. ) d) U 2 14 110% + ay nent illustrated below (1000 bp in size, Psti sti che Psti restriction site of the vector PBR322, wh resistance genes. ECORI (4350) Hindili (30) BamHi (370) PLU (3600) Ampicillin Resistance Tetracycline Resistance PBR322 (4.36 kb) Xmalli (940) Origin Replication ? Pyull (2060) Nder Psti PstI 1 200 1000