THE UNDERGRADUATE

GENOMIC SEQUENCING

PROJECT

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 reconstructionhttp://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

crucial for protein synthesisConserved structure and function across all

organismsUniversal in cellular organismsDiffering 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

http://www.scienceprofonline.org/science-image-libr/sci-image-libr-bacterial-growth-media.html

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

http://www.biotechlearn.org.nz/var/biotechlearn/storage/images/themes/dna_lab/images/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. Taq polymerase -

thermophilic Allows polymerase to

repeatedly amplify the regions that the primers specify

Confirmed on agarose gel

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

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

http://www.geneious.com/geneious_theme-theme/images/biomatters/screenshots/3.7_sequence_organize_sequence_visualize.png

Looking Forward

My sequenced organisms so farStaphylococcus pasteuriEnterobacter ?Bacillus amyloliquefaciens6 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