LAB 5: Soil Microbial Diversity &Function Part 1: Culture-
Independent Identification of Soil Bacteria Your instructor will
return your frozen cleaned-up pcr products containing amplified
fragments of 16s rRNA gene from many of the species of soil
bacteria in your soil sample. Today you will insert your
bacterial 16s rRNA gene fragments into a patented cloning vector
(pCR-BluntII TOPO®) and then transform that vector into a
special genetically engineered strain of Escherichia coli
bacteria that will express a vector gene for kanamycin
resistance, allowing us to select for transformants on media
containing kanamycin. The principle behind TOPO® cloning is the
enzyme DNA topoisomerase I, which will function in this system
both as a restriction enzyme and as a ligase. Its biological
role is to cleave and rejoin DNA during replication. Vaccinia
virus topoisomerase I specifically recognizes the pentameric
sequence 5´-(C/T)CCTT-3´ and forms a covalent bond with the
phosphate group attached to the 3´ thymidine. It cle