Zachary Bendiks
Jonathan Eisen
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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
http://www.ucdmc.ucdavis.edu/medmicro/jeisen.html
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
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 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
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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
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Culturing Bacteria
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Place environmental
sample in liquid
overnight culture
Grow liquid culture
on nutrient plate
Use dilution
streaking to isolate
individual colonies,
and thus, individual
species
http://www.scienceprofonline.org/science-image-libr/sci-image-libr-bacterial-growth-media.html
Genomic Preparations
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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
http://www.biotechlearn.org.nz/var/biotechlearn/storage/images/themes/dna_lab/ima
ges/dna_precipitate/174993-1-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.
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 Taq polymerase -
thermophilic
Allows polymerase to
repeatedly amplify the
regions that the primers
specify
 Confirmed on agarose
gel
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http://upload.wikimedia.org/wikipedia/en/a/a9/Amit_Yadav_16S_PCR_Phytoplasma.JPG
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
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http://www.favorgen.com/jpg/FAEPK-02.jpg
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
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http://www.geneious.com/geneious_theme-theme/images/biomatters/screenshots/3.7_sequence_organize_sequence_visualize.png
Looking Forward
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My sequenced organisms so far
 Staphylococcus pasteuri
 Enterobacter ?
 Bacillus amyloliquefaciens
 6 more currently in sequencing facility
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Undergraduates will be assigned an
organism and write a summary report of
it and publish the full genome to a
database