Biology Lab Report - 840 Words

Screening and Cloning Of Bacterial ÃŽ ²-Glucosidase Gene That Can Degrade Salicin from NIF and Virulent Bacteria Hanan H. Ahmed Microbial Biotechnology Department, Genetic Engineering Biotechnology Institute, Minufiya University Sadat City, Egypt Abstract Two ÃŽ ²- glucosidase genes in Rhizobium leguminosarum bv. Trifloii able to utilize Salicin. SamI fragments (2 and 3 kbp) from Rhizobium leguminosarum bv. Trifloii were expressed in E. coli HC1061. Transformed clones with ÃŽ ²- glucosidase activity were selected by using Congo Red stain plate assay. Restriction enzyme analysis of recombinant plasmid indicated that the positive clones were contained the 2 and 3 kbp DNA inserts. The E. coliHC1061 transformed with 2 or 3 kbp fragment†¦show more content†¦Two types of pattern were obtained, the relative ability of each gene to cleave coniferin was assessed. Clones containing Agrobacterium tumefaciens B3/73 DNA rapidly and completely hydrolyzed coniferin to coniferyl alcohol. Over the same period, type 2 clones were completely inactive. The different substrate specificities of clones were also evident from their ability to grow on cellobiose Agrobacterium tumefaciens B3/73 was able to use cellobiose as the sole carbon source . Escherichia coli DH5ÃŽ ± and type 1 clones were not able to grow on cellobiose. Other clones were able to utilize cellobiose, but grew very slowly (Linda et al., 1992). The 5.7-kb HindIlI fragment common to all type 1 clones was purified and ligated into pBR322. Clones with inserts in either orientation were able to cleave X-glucose, indicating that the entire ÃŽ ²-glucosidase gene was probably located within this insert. An EcoRI, BamHI, BglII, and PstI restriction map of the insert showed that a 3.5-kb BamHI-PstI fragment with an internal PstI site was found to have the activity to cleave X-glucose when cloned into pUC19. The sequence surrounding the EcoRI site in the pUC19: 3.5-kb BamHI-PstI clone and the sequence were done. 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