1 origin (4903-5358) Through the literature review, you found an information on green fluorescence protein (GFP), which can be isolated from the jellyfish Aequorea victoria (A. victoria). You wanted to clone the GFP gene into a PET-28a vector (Figure 1). The size of the vector is 5369 bp and the GFP gene size is 1250 bp. Some of the restriction enzymes that you consider for cloning, which are available at the Multiple Cloning Site (MCS) are EcoRI, BamHI and Sall. The recognition and cleavage sites for these enzyme are EcoRI = G'AATTC, BamHI G'GATCC, Sall= G’TCGAC. This genomic DNA of A. victoria has been digested by Sau3A enzyme ("GATC). Xho (15 Note Eagles Hind a Sall Sectre Drail) EcoR (12 BamH 10 Bpu1 102 103 We 2011 Nde12301 Nco 1296 Xbo 136 Bal 2013 SorA442 Sph) P1.4426 Saf 2426 Sma 45001 95-4807) Mu 1123 Bol 1137 Cla117) Nr403 PET-28a(+) (5369bp) acl (773-1852) BE1304 Apa 133) BH (154) ECOR (1573 Hpa 1629 E006716721 AN ori (3286) Psha Ess Baput 1204 Sap 10) Est1107 2005 Th111 2010 Bol 2107 FSO 2201 Papá 22:30) Figure 1
a) b) c) Decide the most suitable site to insert the GFP gene in PET-28a vector. Justify your (2) decision Decide the screening strategy to identify the positive clone that carries the correct (2) GFP gene insert and justify your decision. What is the expected size of the recombinant vector harboring the insert? Show your (2) calculation. You wanted to transform E.coli competent cells with this new recombinant vector by the heat-shock method. Discuss the 4 (FOUR) important steps during heat shock transformation procedures. d)