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The Undergraduate Genomic Sequencing Project

Zachary Bendiks. The Undergraduate Genomic Sequencing Project. 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 ).”

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The Undergraduate Genomic Sequencing Project

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  1. Zachary Bendiks The Undergraduate Genomic Sequencing Project

  2. 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 http://www.ucdmc.ucdavis.edu/medmicro/jeisen.html

  3. 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)

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

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

  11. 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

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