| סיכום: | The aim of the research was to clone the protease gene from Xanthomonas campestris into Escherichia coli. This enzyme is active over a particulary broad pH range. Shotgun cloning was used to compare techniques using four different plasmids and vector. The results indicated that when (a) plasmid pUC19 in E. coli DH5a was used, 5 recombinant plasmids with a 0,5 - 7,5 kb EcoR1 insert were obtained, (b) plasmid pBluescript in E. coli XL-1-Blue was used, 1 recombinants plasmid with a 8 kb EcoR1 insert were obtained, (c) plasmid pRK415 in E. coli DH5a were used, 3 recombinant plasmids with a 1,5 - 10 kb EcoR1 insert were obtained, (d) plasmid pBBR1MCS2 in E. coli JM 107 was used, 47 recombinant plasmids with a 1,4 - 8 kb EcoR1 insert were obtained. Of these recombinants, two produced a typical clearing zone, when analysed using Skim Milk Agar. However, the typical clearing zone was not clear or stable. When the E. coli (pMB8) recombinant was transferred back into a protease negative mutant of Xanthomonas campestris, using a triparental method, a typical clearing zone in the Skim Milk Agar media was observed, confirming that the protease gene had succesfully cloned. A recombinant of E.coli (pMB.8) was grown in nutrient broth containing glycerol, the protease activity was detected after 20-24 hours. The crude protease showed optimum pH at 8,5 - 10, optinmm temperature 60°C and molecular sizes approximately 49 and 45 kD. The enzyme was inhibited by EDTA and also by PMSF indicating a serine protease function.
Keywords: cloning � protease � Xanthomonas � recomb
|
|---|