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Phylogenetic Study of Beta‐Galactosidase in Model Organisms Using Bioinformatics and Western Blot Techniques
Author(s) -
Wood Lance Phelan
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.06495
Subject(s) - phylogenetic tree , biology , biochemistry , polyclonal antibodies , enzyme , western blot , microbiology and biotechnology , antibody , chemistry , genetics , gene
Beta‐galactosidase is a highly conserved enzyme found in many organisms, existing as a tetramer of four identical subunits of 1023 amino acids each; its substrate is lactose, although it can bind to other similar molecules as well (Jeurs, D et. Al). It can cleave the lactose molecule to form allolactose, which is involved in the lac operon of E. coli , and glucose, which is used in glycolysis in many other organisms (Jeurs, D. et al). This experiment uses published protein sequences for the Beta‐galactosidase enzyme in four different model organisms to construct a phylogenetic tree using E. coli , S. cerevisiae , D. melanogaster , and C. elegans proteomes. Using Western Blot techniques, the same enzyme from all four species will be used to create a phylogenetic tree based on the experimental data. Beta Galactosidase Antibody (NBP2‐42819) is a polyclonal rabbit antibody from Novus Biologicals will be used as the primary antibody for binding to the extracted enzyme. Rabbit IgG Horseradish Peroxidase conjugated Antibody, catalog number HAF008, also from Novus Biologicals will be used as the secondary antibody to bind to the primary. The in‐silico model will then be compared with the in‐vitro model to compare antibody binding to the bacterial, fungal, and animal forms of the enzyme. Preliminary bioinformatics work based on multiple sequence alignments and domain searches suggests that C. elegans and D. melanogaster forms of beta‐galactosidase are more closely related to each other than to any of the forms found in the other species in this study. The S. cerevisiae form of the protein was expected to be more like those belonging to C. elegans and D. melanogaster . However, the bioinformatics results do not support this, possibly due to poor literary documentation of the amino acid sequence of S. cerevisiae beta‐galactosidase. Support or Funding Information This work was made possible through the contributions of the Hillsborough Community College Brandon Campus Biotechnology and Microbiology Departments.