Biology Lab Report

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Screening and Cloning Of Bacterial β-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
Two β- 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 β- 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
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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 DH5α 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 β-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. (Linda et al., 1992). Woodward and Wiseman (1982) reported that there are two constitutive, β-glucosidase genes in Agrobacterium
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