Zachary Bendiks

Jonathan Eisen  UC Davis Genome Center  Lab focus: “Our work focuses on genomic basis for the origin of novelty in microorganisms (how new processes and functions evolve).”  Metagenomics – assessing the microbial makeup of environmental samples  Bioinformatics - applying computer technology to phylogeny reconstruction

16S rDNA  Present in all prokaryotes.  The 16S gene codes for the small rRNA subunit. ~1.5 kb in length  rRNA can be phylogenetically significant crucial for protein synthesis Conserved structure and function across all organisms Universal in cellular organisms Differing rates of evolution throughout the gene can help identify species  Carl Woese (1970)

The Undergraduate Genomic Sequencing Project  First attempt by Eisen lab to incorporate undergraduates  Attempts to strengthen mechanical lab skills by taking undergraduates through the process of isolating genomic DNA.  Learn simple genetic analysis techniques and methods used to identify organisms

Culturing Bacteria  Place environmental sample in liquid overnight culture  Grow liquid culture on nutrient plate  Use dilution streaking to isolate individual colonies, and thus, individual species

Genomic Preparations  Extraction of genomic DNA from the cell  Lysis of the membrane  Cellular proteins and other impurities are dissolved  Genomic DNA is suspended in rehydration solution  Presence of DNA is confirmed on agarose gel ges/dna_precipitate/ eng-AU/dna_precipitate_large.jpg

16S PCR  DNA polymerase, forward and reverse primers, and buffers are combined with the DNA  Solution heated through cycles for several hours. Taq polymerase - thermophilic  Allows polymerase to repeatedly amplify the regions that the primers specify  Confirmed on agarose gel

PCR Purification  Free-floating primers, buffers, and other impurities are still in the solution. Need pure DNA for sequencing  Use a series of solutions and a filtering tube to remove contaminants  Pure DNA solution can then be diluted to the proper concentration and sent for sequencing

Genetic Analysis  Geneious – creates alignments between fragments and generates consensus sequence  BLAST – compare sequence to a database of organisms  GOLD – lists the number of in progress or future projects for organisms

Looking Forward  My sequenced organisms so far Staphylococcus pasteuri Enterobacter ? Bacillus amyloliquefaciens 6 more currently in sequencing facility  Undergraduates will be assigned an organism and write a summary report of it and publish the full genome to a database